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Click Here. Short Trick to learn Classification of Fungi. Short Trick to learn Hypothalamus Control. Short Trick to learn Pituitary Hormone. Short Trick to learn Facial Bones. Short Trick to learn Carpal Bones. Short Trick to learn Bacteria Types.

Short Trick to learn Function of Large Intestine. Short Trick to learn Bacteria Disease. Such membranes form cellular and organellar boundaries and are selectively permeable to uncharged molecules. The precise lipid composition varies from cell to cell and from organelle to organelle. Proteins are also a major component of cell membranes Fig. Peripheral membrane proteins are loosely bound to the outer surface or are anchored via a lipid or glycosyl phosphatidylinositol anchor and are relatively easy to remove.

Integral membrane proteins are embedded in the membrane and cannot be removed without destroying the membrane. Some protrude from the outer or inner surface of the membrane while transmembrane proteins span the bilayer completely and have both extracellular and intracellular domains see Topic B2. The transmembrane regions of these proteins contain predominantly hydrophobic amino acids. Schematic diagram of a plasma membrane showing the major macromolecular components.

Noncovalent interactions Most macromolecular assemblies are held together by a large number of different noncovalent interactions. Charge´┐Żcharge interactions salt bridges operate between ionizable groups of opposite charge at physiological pH, for example between the negative phosphates of DNA and the positive lysine and arginine side chains of DNA-binding proteins such as histones see Topic D2.

Charge´┐Żdipole and dipole´┐Żdipole interactions are weaker and form when either or both of the participants is a dipole due to the asymmetric distribution of charge in the molecule Fig.

Even uncharged groups like methyl groups 14 Section A ´┐Ż Cells and macromolecules Fig. Examples of a van der Waals forces and b a hydrogen bond. Noncovalent associations between electrically neutral molecules are known collectively as van der Waals forces. Hydrogen bonds are of great importance. They form between a covalently bonded hydrogen atom on a donor group e. The presence of uncharged and nonpolar substances, for example lipids, in an aqueous environment tends to force a highly ordered structure on the surrounding water molecules.

This is energetically unfavorable as it reduces the entropy of the system. Hence, nonpolar molecules tend to clump together, reducing the overall surface area exposed to water. This attraction is termed a hydrophobic water-hating interaction and is a major stabilizing force in protein´┐Żprotein and protein´┐Żlipid interactions and in nucleic acids. They behave as zwitterions in solution.

Glutamic acid and aspartic acid have additional carboxyl groups and usually impart a negative charge to proteins. These three basic amino acids generally impart a positive charge to proteins.

Polar uncharged side chains Serine and threonine have hydroxyl groups, asparagine and glutamine have amide groups and cysteine has a thiol group. Nonpolar aliphatic side chains Glycine is the simplest amino acid with no side chain. Proline is a secondary amino acid imino acid.

Alanine, valine, leucine and isoleucine have hydrophobic alkyl groups. Methionine has a thioether sulfur atom. Aromatic side chains Related topics Phenylalanine, tyrosine and tryptophan have bulky aromatic side chains which absorb ultraviolet light. Thus, amino acids can exist as pairs of optically active stereoisomers D- and L-. However, only the L-isomers are found in proteins. Amino acids are dipolar ions zwitterions in aqueous solution and behave as both acids and bases they are amphoteric.

The side chains differ in size, shape, charge and chemical reactivity, and are responsible for the differences in the properties of different proteins Fig. A few proteins contain nonstandard amino acids, such as 4-hydroxyproline and 5-hydroxylysine in collagen. Charged side chains Taking pH 7 as a reference point, several amino acids have ionizable groups in their side chains which provide an extra positive or negative charge at this 16 Section B ´┐Ż Protein structure Fig. General structure of an L-amino acid.

The R group is the side chain. The imidazole group of histidine has a pKa near neutrality. Reversible protonation of this group under physiological conditions contributes to the catalytic mechanism of many enzymes.

Together, acidic and basic amino acids can form important salt bridges in proteins see Topic A4. Side chains R of the 20 common amino acids. The standard three-letter abbreviations and one-letter code are shown in brackets. Polar uncharged side chains These contain groups that form hydrogen bonds with water. Serine and threonine have hydroxyl groups, while asparagine and glutamine are the amide derivatives of aspartic and glutamic acids.

B1 ´┐Ż Amino acids 17 Nonpolar aliphatic Glycine has a hydrogen atom in place of a side chain and is optically inactive. Methionine has a sulfur atom in a thioether link within its alkyl side chain. Aromatic side chains Phenylalanine, tyrosine and tryptophan have bulky hydrophobic side chains.

Their aromatic structure accounts for most of the ultraviolet UV absorbance of proteins, which absorb maximally at nm. The phenolic hydroxyl group of tyrosine can also form hydrogen bonds.

Sizes range from a few thousand to several million Daltons. Some proteins have associated nonproteinaceous material, for example lipid or carbohydrate or small co-factors. The resulting polypeptide sequence has an N terminus and a C terminus. Polypeptides commonly have between and amino acids linked in this way.

Secondary structure Polypeptides can fold into a number of regular structures. Denaturation leads to loss of secondary and tertiary structure. Quaternary structure Many proteins have more than one polypeptide subunit. Large complexes such as microtubules are constructed from the quaternary association of individual polypeptide chains.

Allosteric effects usually depend on subunit interactions. Prosthetic groups Conjugated proteins have associated nonprotein molecules which provide additional chemical functions to the protein. What do proteins do? Proteins have a wide variety of functions. Enzymes catalyze most biochemical reactions. For example, hemoglobin transports oxygen in the blood and ferritin stores iron in the liver. Domains, motifs, families and evolution Related topics Domains form semi-independent structural and functional units within a single polypeptide chain.

Domains are often encoded by individual exons within a gene. New proteins may have evolved through new combinations of exons and, hence, protein domains. Motifs are groupings of secondary structural elements or amino acid sequences often found in related members of protein families. Similar structural motifs are also found in proteins which have no sequence similarity. Protein families arise through gene duplication and subsequent divergent evolution of the new genes.

Globular proteins are folded compactly and behave in solution more or less as spherical particles; most enzymes are globular in nature. Molecular masses can range from a few thousand Daltons Da e. Some proteins contain bound nonprotein material, either in the form of small prosthetic groups, which may act as co-factors in enzyme reactions, or as large associations e. When two amino acid residues are linked in this way the product is a dipeptide.

Many amino acids linked by peptide bonds form a polypeptide Fig. Hence, polypeptides are directional, with an N terminus and a C terminus. Sometimes the N terminus is blocked with, for example, an acetyl group.

The sequence of amino acids from the N to the C terminus is the primary structure of the polypeptide. Typical sizes for single polypeptide chains are within the range ´┐Ż amino acids, though longer and shorter ones exist.

Section of a polypeptide chain. The peptide bond is boxed. This makes the peptide bond unit rigid and planar, though there is free rotation between adjacent peptide bonds. This polarity also favors hydrogen bond formation between appropriately spaced and oriented peptide bond units.

Thus, polypeptide chains are able to fold into a number of regular structures which are held together by these hydrogen bonds. The polypeptide backbone forms a right-handed helix with 3. Several sections of polypeptide chain may be involved side-byside, giving a sheet structure with the side chains R projecting alternately above and below the sheet.

If these sections run in the same direction e. The connective tissue protein collagen has an unusual triple helix secondary structure in which three polypeptide chains are intertwined, making it very strong. The nature of the tertiary structure is inherent in the primary structure and, given the right conditions, most polypeptides will fold spontaneously into the correct tertiary structure as it is generally the lowest energy conformation for that sequence.

However, in vivo, correct folding is often assisted by proteins called chaperones which help prevent misfolding of new polypeptides before their synthesis and primary structure is complete. Folding is such that amino acids with hydrophilic side chains locate mainly on the exterior of the protein where they can interact with water or solvent ions, while the hydrophobic amino acids become buried in the interior from which water is excluded. This gives overall stability to the structure.

Various types of noncovalent interaction between side chains hold the tertiary structure together: van der Waals forces, hydrogen bonds, electrostatic salt bridges between oppositely charged groups e. Disruption of secondary and B2 ´┐Ż Protein structure and function 21 Fig. Schematic diagram of a section of protein tertiary structure. Quaternary structure Many proteins are composed of two or more polypeptide chains subunits. These may be identical or different.

This level of organization is known as the quaternary structure and has certain consequences. First, it allows very large protein molecules to 22 Section B ´┐Ż Protein structure be made. These are the microtubules of the cytoskeleton. Secondly, it can provide greater functionality to a protein by combining different activities into a single entity, as in the fatty acid synthase complex.

Prosthetic groups Many conjugated proteins contain covalently or noncovalently attached small molecules called prosthetic groups which give chemical functionality to the protein that the amino acid side chains cannot provide. Many of these are co-factors in enzyme-catalyzed reactions. A protein without its prosthetic group is known as an apoprotein.

These can enhance the rate of biochemical reactions by several orders of magnitude. Side chains can also be directly involved in catalysis, for example by acting as nucleophiles, or proton donors or abstractors. Receptor proteins in cell membranes can bind ligands e. Ligand binding is similar to substrate binding but the ligand usually remains unchanged.

Some hormones are themselves small proteins, such as insulin and growth hormone. Transport and storage. Hemoglobin transports oxygen in the red blood cells while transferrin transports iron to the liver. Once in the liver, iron is stored bound to the protein ferritin. Dietary fats are carried in the blood by lipoproteins. Many other molecules and ions are transported and stored in a protein-bound form. This can enhance solubility and reduce reactivity until they are required.

Structure and movement. Collagen is the major protein in skin, bone and connective tissue, while hair is made mainly from keratin. There are also many structural proteins within the cell, for example in the cytoskeleton. Casein and ovalbumin are the major proteins of milk and eggs, respectively, and are used to provide the amino acids for growth of developing offspring.

Seed proteins also provide nutrition for germinating plant embryos. Antibodies, which recognize and bind to bacteria, viruses and other foreign material the antigen are proteins. Transcription factors bind to and modulate the function of DNA. Many other proteins modify the functions of other molecules by binding to them. Many proteins are composed of structurally independent units, or domains, that are connected by sections with limited higher order structure within the same polypeptide.

The connections can act as hinges to permit the individual domains to move in relation to each other, and breakage of these connections by limited proteolysis can often separate the domains, which can then behave like independent globular proteins. The active site of an enzyme is sometimes formed in a groove between two domains, which wrap around the substrate.

When such a function is required in many different proteins, the same domain structure is often found. In eukaryotes, domains are often encoded by discrete parts of genes called exons see Topic O3.

Therefore, it has been suggested that, during evolution, new proteins were created by the duplication and rearrangement of domain-encoding exons in the genome to produce new combinations of binding sites, catalytic sites and structural elements in the resulting new polypeptides. In this way, the rate of evolution of new functional proteins may have been greatly increased.

Structural motifs also known as supersecondary structures are groupings of secondary structural elements that frequently occur in globular proteins. Alternatively, they may represent the best solution to a structural´┐Żfunctional requirement that has been arrived at independently in unrelated proteins. Sequence motifs consist of only a few conserved, functionally important amino acids rather than supersecondary structures.

Protein families arise through successive duplications and subsequent divergent evolution of an ancestral gene. Their 24 Section B ´┐Ż Protein structure Fig. Evolution of globins from an ancestral globin gene. Family members in different species that have retained the same function and carry out the same biochemical role e.

The degree of similarity between the amino acid sequences of orthologous members of a protein family in different organisms depends on how long ago the two organisms diverged from their common ancestor and on how important conservation of the sequence is for the function of the protein.

The function of a protein, whether structural or catalytic, is inherently related to its structure. As indicated above, similar structures and functions can also be achieved by convergent evolution whereby unrelated genes evolve to produce proteins with similar structures or catalytic activities. A good example is provided by the proteolytic enzymes subtilisin bacterial and chymotrypsin animal.

Even though their amino acid sequences are very different and they are composed of different structural motifs, they have evolved the same spatial orientation of the catalytic triad of active site amino acids ´┐Ż serine, histidine and aspartic acid ´┐Ż and use exactly the same catalytic mechanism to hydrolyse peptide bonds.

Such proteins are termed functional analogs. Where similar structural motifs have evolved independently, the resulting proteins are structural analogs. Ion-exchange chromatography, isoelectric focusing and electrophoresis take advantage of the different ionic charges on proteins.

Hydrophobic interaction chromatography exploits differences in hydrophobicity. Antibodies Antibodies are proteins produced by the immune system of vertebrates in response to a foreign agent the antigen , usually a protein on the surface of a virus or bacterium. They are an important natural defense against infection.

X-ray crystallography and NMR Many proteins can be crystallized and their three-dimensional structures determined by X-ray diffraction. The structures of small proteins in solution can also be determined by multi-dimensional nuclear magnetic resonance, particularly if the normal 12C and 14N are substituted by 13C and 15N.

Functional analysis Functional analysis of a protein involves its isolation and study in vitro combined with a study of the behavior of a mutant organism in which the protein has been rendered nonfunctional by mutation or deletion of its gene.

The function of a new protein can sometimes be predicted by comparing its sequence and structure to those of known proteins. When applied to the top of the column, protein molecules larger than the pores elute at the bottom relatively quickly, as they are excluded from the beads and so have a relatively small volume of buffer available through which to travel. Molecules smaller than the pore size can enter the beads and so have a larger volume of buffer through which they can diffuse.

Thus, smaller proteins elute from the column after larger ones. Ultracentrifugation can be used to separate proteins but is also a powerful analytical tool for studying protein structure. Charge Because of the presence of ionizable side chains on their surface, proteins carry a net charge. As these side chains are all titratable, there will exist for each protein a pH at which its net surface charge is zero ´┐Ż the isoelectric point pI. Proteins are least soluble at their pI.

Depending on their net charge, proteins will travel at different rates towards the anode or cathode and can then be recovered from the gel after separation. In ion-exchange chromatography, ions that are electrostatically bound to an insoluble support the ion exchanger packed in a column are reversibly replaced with charged proteins from solution.

Usually, a salt gradient of increasing ionic strength is passed through the column and the bound proteins elute separately. Proteins migrate to positions corresponding to their isoelectric points in this gradient and form tight, focused bands as their net charge becomes zero and they stop moving Fig.

Isoelectric focusing of a mixture of proteins with pI values 4, 5, 6 and 7. B3 ´┐Ż Protein analysis 27 Hydrophobicity Hydrophobic interactions between proteins and a column material containing aromatic or aliphatic alkyl groups are promoted in solutions of high ionic strength. By applying a gradient of decreasing ionic strength, proteins elute at different stages.

The insulin receptor is a low abundance cell-surface protein responsible for transmitting the biological activity of insulin to the cell interior. For example, if the gene for the protein is inserted in a phage or plasmid expression vector under the control of a strong promoter see Topic K3 and the vector is then transformed into E.

The tag often has little effect on the function of the protein. Protein sequencing An essential requirement for understanding how a protein works is a knowledge of its primary structure. This indicates how many glycines and serines, etc. For example, trypsin cleaves only after lysine K or arginine R and V8 protease only after glutamic acid E. Cyanogen bromide cleaves polypeptides only after methionine residues.

Each peptide is then subjected to sequential Edman degradation in an automated protein sequencer. Phenylisothiocyanate reacts with the N-terminal amino acid which, after acid treatment, is released as the phenylthiohydantoin PTH derivative, leaving a new N terminus. Example of polypeptide sequence determination. Mass spectrometry MS offers an extremely accurate method. For small molecule analysis, samples are usually vaporized and ionized by a beam of Xe or Ar atoms.

However, such methods have an upper mass limit of only a few kDa and are B3 ´┐Ż Protein analysis 29 too destructive for protein analysis. Recent, non-destructive ionization techniques have greatly extended this mass range. These ion sources can be coupled to a variety of different mass analyzers and detectors, each suited to a different purpose.

For example, an ESI ion source may be attached to a quadrupole detector. ESI-quadrupole systems allow the masses of proteins smaller than kDa to be measured within 0. The detector then counts the different ions as they arrive. Antibodies Antibodies are useful molecular tools for investigating protein structure and function. Antibodies are themselves proteins immunoglobulins, Ig and are generated by the immune system of higher animals when they are injected with a macromolecule the antigen such as a protein that is not native to the animal.

Antibodies fall into various classes, e. The most useful are the IgG class, produced as soluble proteins by B lymphocytes. Structure of an antibody molecule. This region recognizes and binds to a short sequence of 5´┐Ż8 amino acids on the surface of the antigen an epitope.

Usually, a single antigen elicits the production of several different antibodies by different B cell clones, each of which recognizes a different epitope on the antigen a polyclonal antibody mixture. They can also be used to detect proteins after separation of cell extracts by SDS gel electrophoresis see above.

After separation, the proteins are transferred from the gel to a membrane in a procedure similar to Southern blotting see Topic J1. The second antibody has previously been linked covalently to an enzyme such as peroxidase or alkaline phosphatase which catalyzes a color-generating reaction when incubated with the appropriate substrate.

Thus, a colored band appears on the blot in the position of the protein of interest. This procedure is semiquantitative and is similar in principle to the enzyme-linked immunosorbent assay ELISA commonly used in clinical diagnostics. The 3-D structure can then be determined by X-ray crystallography. X-rays interact with the electrons in the matter through which they pass. By measuring the pattern of diffraction of a beam of X-rays as it passes through a crystal, the positions of the atoms in the crystal can be calculated.

By crystallizing an enzyme in the presence of its substrate, the precise intermolecular interactions responsible for binding and catalysis can be seen.

The structures of small globular proteins in solution can also be determined by two- or three-dimensional nuclear magnetic resonance NMR spectroscopy.

The properties of this relaxation depend on the relative positions of the protons in the molecule. The multi-dimensional approach is required for proteins to spread out and resolve the overlapping data produced by the large number of protons.

Substituting 13C and 15N for the normal isotopes 12C and 14N in the protein also greatly improves data resolution by eliminating unwanted resonances. In this way, the structures of proteins up to about 30 kDa in size can be deduced. Where both X-ray and NMR methods have been used to determine the structure of a protein, the results usually agree well.

This suggests that the measured structures are the true in vivo structures. Functional analysis The three-dimensional structures of many proteins have now been determined. However, tertiary and quaternary structural determination is still a relatively costly and laborious procedure and lags well behind the availability of new protein primary structures predicted from genomic sequencing projects see Topic J2. Thus, there is now great interest in computational methods that will allow the prediction of both structure and possible function from simple amino acid sequence information.

These methods are based largely on the fact that there are only a limited number of supersecondary structures in nature and involve mapping of new protein sequences on to the known three dimensional structures of proteins with related amino acid sequences. However, caution must still be exerted in interpreting such results. It is not an enzyme, as might be predicted on the basis of sequence similarity. Thus, at least for the moment, understanding of the true function of a protein still requires its isolation and biochemical and structural characterization.

Isolation is greatly aided by recombinant techniques. Knowledge of the biochemical properties of an isolated protein does not necessarily tell you what that protein does in the cell. Additional genetic analysis is usually required. If the gene for the protein can be inactivated by mutagenesis or deleted by recombinant DNA techniques, then the phenotype of the resulting mutant can be studied.

In conjunction with the biochemical information, the altered behavior of the mutant cell can help to pinpoint the function of the protein in vivo. In RNA, thymine is replaced by the structurally very similar pyrimidine, uracil U.

The repeat unit is a nucleotide. Nucleic acids are highly charged polymers with a negative charge on each phosphate. Two separate and antiparallel chains of DNA are wound around each other in a right-handed helical coiled path, with the sugar´┐Żphosphate backbones on the outside and the bases, paired by hydrogen bonding and stacked on each other, on the inside.

Adenine pairs with thymine; guanine pairs with cytosine. RNA secondary structure Most RNA molecules occur as a single strand, which may be folded into a complex conformation, involving local regions of intramolecular base pairing and other hydrogen bonding interactions.

Adenine A and guanine G are purines, bicyclic structures two fused rings , whereas cytosine C , thymine T and uracil U are monocyclic pyrimidines. In RNA, the thymine base is replaced by uracil. Thymine differs from uracil only in having a methyl group at the 5-position, that is thymine is 5-methyluracil. Nucleic acid bases. The point of attachment to the base is the 1-position N-1 of the pyrimidines and the 9-position N-9 of the purines Fig.

The bond between the bases and the sugars is the glycosylic or glycosidic bond. If the sugar is ribose, the nucleosides technically ribonucleosides are adenosine, guanosine, cytidine and uridine. Thymidine and deoxythymidine may be used interchangeably. If the sugar is deoxyribose, then the compounds are termed deoxynucleotides Fig. Chemically, the compounds are phosphate esters. In the course of DNA or RNA synthesis, two phosphates are split off as pyrophosphate to leave one phosphate per nucleotide incorporated into the nucleic acid chain see Topics E1 and K1.

This kind of bond or linkage is called a phosphodiester bond, since the phosphate is chemically in the form of a diester. A nucleic acid chain can hence be seen to have a direction.

At neutral pH, each phosphate group has a single negative charge. This is why nucleic acids are termed acids; they are the anions of strong acids. Nucleic acids are thus highly charged polymers. The basic features of this structure were deduced by James Watson and Francis Crick in Two separate chains of DNA are wound around each other, each following a helical coiling path, resulting in a right-handed double helix Fig.

The negatively charged sugar´┐Żphosphate backbones of the molecules are on the outside, and the planar bases of each strand stack one above the 36 Section C ´┐Ż Properties of nucleic acids Fig.

Phosphodiester bonds and the covalent structure of a DNA strand. Between the backbone strands run the major and minor grooves, which also follow a helical path. The strands are joined noncovalently by hydrogen bonding between the bases on opposite strands, to form base pairs. There are around 10 base pairs per turn in the DNA double helix. This last feature arises because the structures of the bases and the constraints of the DNA backbone dictate that the bases hydrogen-bond to each other as purine´┐Żpyrimidine pairs which have very similar geometry and dimensions Fig.

Guanine pairs with cytosine three H-bonds and adenine pairs with thymine two H-bonds. Hence, any sequence can be accommodated within a regular double-stranded DNA structure. A, B and Z helices In fact, a number of different forms of nucleic acid double helix have been observed and studied, all having the basic pattern of two helically-wound antiparallel strands. C1 ´┐Ż Nucleic acid structure 37 Fig. The DNA double helix.

The DNA base pairs. DNA can be induced to form an alternative helix, known as the A-form Fig. The A-form is right-handed, like the Bform, but has a wider, more compressed structure in which the base pairs are tilted with respect to the helix axis, and actually lie off the axis seen end-on, the A-helix has a hole down the middle.

This is because in this structure, the pyrimidine and the purine nucleotides adopt very different conformations, unlike in A- and B-form, where each nucleotide has essentially the same conformation and immediate environment. The pyrimidine nucleotides, and all nucleotides in the A- and B- forms adopt the anti conformation. Table 1. RNA instead forms relatively globular conformations, in which local regions of helical structure are formed by intramolecular hydrogen bonding and base stacking within the single nucleic acid chain.

RNA structures range from short small nuclear RNAs, which help to mediate the splicing of pre-mRNAs in eukaryotic cells see Topic O3 , to large rRNA molecules, which form the structural backbone of the ribosomes and participate in the chemistry of protein synthesis see Topic Q2.

The alternative helical forms of the DNA double helix. These are considered in more detail in Topics O3 and P2. Effect of acid Highly acidic conditions may hydrolyze nucleic acids to their components: bases, sugar and phosphate. Moderate acid causes the hydrolysis of the purine base glycosylic bonds to yield apurinic acid. More complex chemistry has been developed to remove particular bases, and is the basis of chemical DNA sequencing.

Some chemicals, such as urea and formamide, can denature DNA and RNA at neutral pH by disrupting the hydrophobic forces between the stacked bases.

DNA has a density of around 1. In fact this is not the case. As in proteins see Topic A4 , the presence of H-bonds within a structure does not normally confer stability. This is because one must consider the difference in energy between, in the case of DNA, the single- C2 ´┐Ż Chemical and physical properties of nucleic acids 41 stranded random coil state, and the double-stranded conformation.

H-bonds between base pairs in double-stranded DNA merely replace what would be equally strong and energetically favorable H-bonds with water molecules in free solution, if the DNA were single-stranded.

The root of this stability lies elsewhere, in the stacking interactions between the base pairs see Topic C1, Fig. The hydrogen bonding network of bulk water becomes destabilized in the vicinity of a hydrophobic surface, since not all the water molecules can participate in full hydrogen bonding interactions, and they become more ordered. Hence it is energetically favorable to exclude water altogether from pairs of such surfaces by stacking them together; more water ends up in the bulk hydrogen-bonded network.

This also maximizes the interaction between charge dipoles on the bases see Topic A4. Even in single-stranded DNA, the bases have a tendency to stack on top of each other, but this stacking is maximized in double-stranded DNA, and the hydrophobic effect ensures that this is the most energetically favorable arrangement.

In more dilute mineral acid, for example at pH 3´┐Ż4, the most easily hydrolyzed bonds are selectively broken. These are the glycosylic bonds attaching the purine bases to the ribose ring, and hence the nucleic acid becomes apurinic see Topic F2.

The effect of alkali is to change the tautomeric state of the bases. This effect can be seen with reference to the model compound, cyclohexanone Fig. The molecule is in equilibrium between the tautomeric keto and enol forms 1 and 2. At neutral pH, the compound is predominantly in the keto form 1. Increasing the pH causes a shift to the enolate form 3 when the molecule loses a proton, since the negative charge is most stably accommodated on the electronegative oxygen atom.

In the same way, the structure of guanine Fig. The denaturation of DNA at high pH. Intramolecular cleavage of RNA phosphodiester bonds in alkali. A relatively high concentration of these agents several molar has the effect of disrupting the hydrogen bonding of the bulk water solution.

This means that the energetic stabilization of the nucleic acid secondary structure, caused by the exclusion of water from between the stacked hydrophobic bases, is lessened and the strands become denatured. Viscosity Cellular DNA is very long and thin; technically, it has a high axial ratio. DNA is around 2 nm in diameter, and may have a length of micrometers, millimeters or even several centimeters in the case of eukaryotic chromosomes.

In addition, DNA is a relatively stiff molecule; its stiffness would be similar to that of partly cooked spaghetti, using the same analogy.

A consequence of this is that DNA solutions have a high viscosity. Furthermore, long DNA molecules can easily be damaged by shearing forces, or by sonication high-intensity ultrasound , with a concomitant reduction in viscosity.

Note that neither shearing nor sonication denatures the DNA; they merely reduce the length of the double-stranded molecules in the solution. In solutions containing high concentrations of a high molecular weight salt, for example 8 M cesium chloride CsCl , DNA has a similar density to the bulk solution, around 1.

If the solution is centrifuged at very high speed, the dense cesium salt tends to migrate down the tube, setting up a density gradient Fig. Eventually the DNA sample will migrate to a sharp band at a position in the gradient corresponding to its own buoyant density. Equilibrium density gradient centrifugation of DNA.

Quantitation of nucleic acids The absorbance at nm is used to determine the concentration of nucleic acids. For pure DNA, the value is 1. Values above 1. Denaturation may be detected by the change in A Related topics UV absorption Nucleic acid structure C1 Chemical and physical properties of nucleic acids C2 Genome complexity D4 Nucleic acids absorb UV light due to the conjugated aromatic nature of the bases; the sugar´┐Żphosphate backbone does not contribute appreciably to absorption.

The absorption properties of nucleic acids can be used for detection, quantitation and assessment of purity. Double-stranded DNA has equal numbers of purines and pyrimidines, and so does not show this effect. The absorbance values also depend on the amount of secondary structure double-stranded regions in a given molecule, due to hypochromicity.

The shape of the absorption spectrum Fig. The process of denaturation can be observed conveniently by the increase in absorbance as double-stranded nucleic acids are converted to single strands Fig. As the temperature is increased, the absorbance of an RNA sample gradually and erratically increases as the stacking of the bases in double-stranded regions is reduced. Shorter regions of base pairing will denature before longer regions, since they are more thermally mobile.

In contrast, the thermal denaturation, or melting, of dsDNA is co-operative. Renaturation The thermal denaturation of DNA may be reversed by cooling the solution. Rapid cooling allows only the formation of local regions of dsDNA, formed by the base pairing or annealing of short regions of complementarity within or between DNA strands; the decrease in A is hence rather small Fig.

The renaturation of regions of complementarity between different nucleic acid strands is known as hybridization. The number of links is known as the linking number Lk. Most natural DNA is negatively supercoiled, that is the DNA is deformed in the direction of unwinding of the double helix. Twist and writhe Supercoiling is partitioned geometrically into a change in twist, the local winding up or unwinding of the double helix, and a change in writhe, the coiling of the helix axis upon itself.

Intercalators Intercalators, such as ethidium bromide, bind to DNA by inserting themselves between the base pairs intercalation , resulting in the local untwisting of the DNA helix. If the DNA is closed-circular, then there will be a corresponding increase in writhe.

Energy of supercoiling Negatively supercoiled DNA has a high torsional energy, which facilitates the untwisting of the DNA helix and can drive processes which require the DNA to be unwound. DNA gyrase introduces negative supercoiling using the energy derived from ATP hydrolysis, and topoisomerase IV unlinks decatenates daughter chromosomes. The molecule has no free ends, and the two single strands are linked together a number of times corresponding to the number of double-helical turns in the molecule.

This number is known as the linking number Lk. A good way to imagine these is to consider the DNA double helix as a piece of rubber tubing, with a line drawn along its length to enable us to follow its twisting. The tubing may be joined by a connector into a closed circle Fig. If we imagine a twisting of the DNA helix tubing followed by the joining of the ends, then the deformation so formed is locked into the system Fig.

This deformation is known as supercoiling, since it manifests itself as a coiling of the DNA axis around itself in a higher-order coil, and corresponds to a change in linking number from the simple circular situation. If the twisting of the DNA is in the same direction as that of the double helix, that is the helix is twisted up before closure, then the supercoiling formed is positive; if the helix is untwisted, then the supercoiling is negative.

Almost all DNA molecules in cells are on average negatively supercoiled. This is true even for linear DNAs such as eukaryotic chromosomes, which are constrained into large loops by interaction with a protein scaffold see Topics D2 and E3.

The changes in linking number are indicated see text for details. Topoisomer The linking number of a closed-circular DNA is a topological property, that is one which cannot be changed without breaking one or both of the DNA backbones. A molecule of a given linking number is known as a topoisomer. Topoisomers differ from each other only in their linking number. Twist and writhe The conformation geometry of the DNA can be altered while the linking number remains constant.

Two extreme conformations of a supercoiled DNA topoisomer may be envisaged Fig. The line on the rubber tubing model helps to keep track of local twisting of the DNA axis. The equilibrium situation lies between these two extremes Fig. The changes in twist and writhe at constant linking number are shown see text for details. Intercalators The geometry of a supercoiled molecule may be altered by any factor which affects the intrinsic twisting of the DNA helix.

For example, an increase in temperature reduces the twist, and an increase in ionic strength may increase the twist. One important factor is the presence of an intercalator. The bestknown example of an intercalator is ethidium bromide Fig.

This is a positively charged polycyclic aromatic compound, which binds to DNA by inserting itself between the base pairs intercalation; Fig. This is a reduction in twist, and hence results in a corresponding increase in writhe in a closed-circular molecule.

Energy of supercoiling Supercoiling involves the introduction of torsional stress into DNA molecules. For negative supercoiling, this energy makes it easier for the DNA helix to be locally untwisted, or unwound. Negative supercoiling may thus facilitate processes which require the unwinding of the helix, such as transcription initiation or replication see Topics E1 and K1.

Topoisomerases Enzymes exist which regulate the level of supercoiling of DNA molecules; these are termed topoisomerases. To alter the linking number of DNA, the enzymes must transiently break one or both DNA strands, which they achieve by the attack of a tyrosine residue on a backbone phosphate, resulting in a temporary covalent attachment of the enzyme to one of the DNA ends via a phosphotyrosine bond.

There are two classes of topoisomerase. Most topoisomerases reduce the level of positive or negative supercoiling, that is they operate in the energetically favorable direction.

Since their mechanism involves the passing of one double strand through another, type II topoisomerases are also able to unlink DNA molecules, such as daughter molecules produced in replication, which are linked catenated together see Topic E2. In bacteria, this function is carried out by topoisomerase IV. Topoisomerases are essential enzymes in all organisms, being involved in replication, recombination and transcription see Topics E1, F4 and K1.

DNA gyrase and topoisomerase IV are the targets of anti-bacterial drugs in bacteria, and both type I and type II enzymes are the target of anti-tumor agents in humans. The mechanisms of a type I and b type II topoisomerases see text for details. In normal growth, the DNA is being replicated continuously.

DNA domains The genome is organized into 50´┐Ż large loops or domains of 50´┐Ż kb in length, which are constrained by binding to a membrane´┐Żprotein complex.

Supercoiling of the genome The genome is negatively supercoiled. Individual domains may be topologically independent, that is they may be able to support different levels of supercoiling. These proteins constrain about half of the supercoiling of the DNA.

The bulk of the DNA in E. The DNA is packaged into a region of the cell known as the nucleoid. This region has a very high DNA concentration, perhaps 30´┐Ż50 mg ml´┐Ż1, as well as containing all the proteins associated with DNA, such as polymerases, repressors and others see below.

A fairly high DNA concentration in the test tube would be 1 mg ml´┐Ż1. In normal growth, the DNA is being replicated continuously and there may be on average around two copies of the genome per cell, when growth is at the maximal rate see Topic E2. The DNA consists of 50´┐Ż domains or loops, the ends of which are constrained by binding to a structure which probably consists of proteins attached to part of the cell membrane Fig.

The loops are about 50´┐Ż kb in size. It is not known whether the loops are static or dynamic, but one model suggests that the DNA may spool through sites of polymerase or other enzymic action at the base of the loops.

A schematic view of the structure of the E. The thin line is the DNA double helix. Supercoiling of the genome The E. Electron micrographs indicate that some domains may not be supercoiled, perhaps because the DNA has become broken in one strand see Topic C4 , where other domains clearly do contain supercoils Fig. The attachment of the DNA to the protein´┐Żmembrane scaffold may act as a barrier to rotation of the DNA, such that the domains may be topologically independent.

There is, however, no real biochemical evidence for major differences in the level of supercoiling in different regions of the chromosome in vivo. These proteins are sometimes known as histone-like proteins see Topic D2 , and have the effect of compacting the DNA, which is essential for the packaging of the DNA into the nucleoid, and of stabilizing and constraining the supercoiling of the chromosome. Only about half the supercoiling is unconstrained in the sense of being able to adopt the twisting and writhing conformations described in Topic C4.

It may be that the organization of the nucleoid is fairly complex, although highly ordered DNA´┐Żprotein complexes such as nucleosomes see Topic D2 have not been detected. The name chromatin is given to the highly ordered DNA´┐Żprotein complex which makes up the eukaryotic chromosomes. The chromatin structure serves to package and organize the chromosomal DNA, and is able to alter its level of packing at different stages of the cell cycle.

The major protein components of chromatin are the histones; small, basic positively charged proteins which bind tightly to DNA. There are four families of core histone, H2A, H2B, H3, H4, and a further family, H1, which has some different properties, and a distinct role. Individual species have a number of variants of the different histone proteins. Nucleosomes The nucleosome core is the basic unit of chromosome structure, consisting of a protein octamer containing two each of the core histones, with bp of DNA wrapped 1.

A nucleosome core plus H1 is known as a chromatosome. In some cases, H1 is replaced by a variant, H5, which binds more tightly, and is associated with DNA which is inactive in transcription. The nucleosomal repeat unit is hence around bp. Most chromatin exists in this form. The overall structure somewhat resembles that of the organizational domains of prokaryotic DNA. DNA supercoiling C4 Prokaryotic chromosome structure D1 Eukaryotic chromosome structure D3 Genome complexity D4 54 Section D ´┐Ż Prokaryotic and eukaryotic chromosome structure Chromatin The total length of DNA in a eukaryotic cell depends on the species, but it can be thousands of times as much as in a prokaryotic genome, and is made up of a number of discrete bodies called chromosomes 46 in humans.

The DNA in each chromosome is believed to be a single linear molecule, which can be up to several centimeters long see Topic D3. All this DNA must be packaged into the nucleus see Topic A2 , a space of approximately the same volume as a bacterial cell; in fact, in their most highly condensed forms, the chromosomes have an enormously high DNA concentration of perhaps mg ml´┐Ż1 see Topic D1. This feat of packing is accomplished by the formation of a highly organized complex of DNA and protein, known as chromatin, a nucleoprotein complex see Topic A4.

Chromosomes greatly alter their level of compactness as cells progress through the cell cycle see Topics D3 and E3 , varying between highly condensed chromosomes at metaphase just before cell division , and very much more diffuse structures in interphase.

This implies the existence of different levels of organization of chromatin. The core histones are small proteins, with masses between 10 and 20 kDa, and H1 histones are a little larger at around 23 kDa. This means that histones will bind very strongly to the negatively charged DNA in forming chromatin.

Within a given species, there are normally a number of closely similar variants of a particular class, which may be expressed in different tissues, and at different stages in development. There is not much similarity in sequence between the different histone classes, but structural studies have shown that the classes do share a similar tertiary structure see Topic B2 , suggesting that all histones are ultimately evolutionarily related see Topic B3.

H1 histones are somewhat distinct from the other histone classes in a number of ways; in addition to their larger size, there is more variation in H1 sequences both between and within species than in the other classes. Histone H1 is more easily extracted from bulk chromatin, and seems to be present in roughly half the quantity of the other classes, of which there are very similar amounts.

Nucleosomes A number of studies in the s pointed to the existence of a basic unit of chromatin structure. Nucleases are enzymes which hydrolyze the phosphodiester bonds of nucleic acids. Exonucleases release single nucleotides from the ends of nucleic acid strands, whereas endonucleases cleave internal phosphodiester bonds.

Treatment of chromatin with micrococcal nuclease, an endonuclease which cleaves double-stranded DNA, led to the isolation of DNA fragments with discrete sizes, in multiples of approximately bp. It was discovered that each bp fragment is associated with an octamer core of histone proteins, H2A 2 H2B 2 H3 2 H4 2, which is why these are designated the core histones, and more loosely with one molecule of H1.

The proteins protect the DNA from the action of micrococcal nuclease. More D2 ´┐Ż Chromatin structure 55 prolonged digestion with nuclease leads to the loss of H1 and yields a very resistant structure consisting of bp of DNA associated very tightly with the histone octamer. This structure is known as the nucleosome core, and is structurally very similar whatever the source of the chromatin. The structure of the nucleosome core particle is now known in considerable detail, from structural studies culminating in X-ray crystallography.

The histone octamer forms a wedge-shaped disk, around which the bp of DNA is wrapped in 1. Figure 1 shows the basic features and the dimensions of the structure.

The left-handed wrapping of the DNA around the nucleosome corresponds to negative supercoiling, that is the turns are superhelical turns technically writhing; see Topic C4. Although eukaryotic DNA is negatively supercoiled to a similar level as that of prokaryotes, on average, virtually all the supercoiling is accounted for by wrapping in nucleosomes and there is no unconstrained supercoiling see Topic D1. A schematic view of the structure of a nucleosome core and a chromatosome. The role of H1 One molecule of histone H1 binds to the nucleosome, and acts to stabilize the point at which the DNA enters and leaves the nucleosome core Fig.

In the presence of H1, a further 20 bp of DNA is protected from nuclease digestion, making bp in all, corresponding to two full turns around the histone octamer.

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For example, hemoglobin transports oxygen in the blood and ferritin stores iron in the liver. Domains, motifs, families and evolution Related topics Domains form semi-independent structural and functional units within a single polypeptide chain. Domains are often encoded by individual exons within a gene.

New proteins may have evolved through new combinations of exons and, hence, protein domains. Motifs are groupings of secondary structural elements or amino acid sequences often found in related members of protein families. Similar structural motifs are also found in proteins which have no sequence similarity. Protein families arise through gene duplication and subsequent divergent evolution of the new genes. Globular proteins are folded compactly and behave in solution more or less as spherical particles; most enzymes are globular in nature.

Molecular masses can range from a few thousand Daltons Da e. Some proteins contain bound nonprotein material, either in the form of small prosthetic groups, which may act as co-factors in enzyme reactions, or as large associations e. When two amino acid residues are linked in this way the product is a dipeptide. Many amino acids linked by peptide bonds form a polypeptide Fig.

Hence, polypeptides are directional, with an N terminus and a C terminus. Sometimes the N terminus is blocked with, for example, an acetyl group. The sequence of amino acids from the N to the C terminus is the primary structure of the polypeptide.

Typical sizes for single polypeptide chains are within the range ´┐Ż amino acids, though longer and shorter ones exist. Section of a polypeptide chain. The peptide bond is boxed. This makes the peptide bond unit rigid and planar, though there is free rotation between adjacent peptide bonds.

This polarity also favors hydrogen bond formation between appropriately spaced and oriented peptide bond units. Thus, polypeptide chains are able to fold into a number of regular structures which are held together by these hydrogen bonds.

The polypeptide backbone forms a right-handed helix with 3. Several sections of polypeptide chain may be involved side-byside, giving a sheet structure with the side chains R projecting alternately above and below the sheet. If these sections run in the same direction e. The connective tissue protein collagen has an unusual triple helix secondary structure in which three polypeptide chains are intertwined, making it very strong.

The nature of the tertiary structure is inherent in the primary structure and, given the right conditions, most polypeptides will fold spontaneously into the correct tertiary structure as it is generally the lowest energy conformation for that sequence.

However, in vivo, correct folding is often assisted by proteins called chaperones which help prevent misfolding of new polypeptides before their synthesis and primary structure is complete.

Folding is such that amino acids with hydrophilic side chains locate mainly on the exterior of the protein where they can interact with water or solvent ions, while the hydrophobic amino acids become buried in the interior from which water is excluded. This gives overall stability to the structure. Various types of noncovalent interaction between side chains hold the tertiary structure together: van der Waals forces, hydrogen bonds, electrostatic salt bridges between oppositely charged groups e.

Disruption of secondary and B2 ´┐Ż Protein structure and function 21 Fig. Schematic diagram of a section of protein tertiary structure. Quaternary structure Many proteins are composed of two or more polypeptide chains subunits. These may be identical or different. This level of organization is known as the quaternary structure and has certain consequences.

First, it allows very large protein molecules to 22 Section B ´┐Ż Protein structure be made. These are the microtubules of the cytoskeleton. Secondly, it can provide greater functionality to a protein by combining different activities into a single entity, as in the fatty acid synthase complex.

Prosthetic groups Many conjugated proteins contain covalently or noncovalently attached small molecules called prosthetic groups which give chemical functionality to the protein that the amino acid side chains cannot provide. Many of these are co-factors in enzyme-catalyzed reactions. A protein without its prosthetic group is known as an apoprotein. These can enhance the rate of biochemical reactions by several orders of magnitude.

Side chains can also be directly involved in catalysis, for example by acting as nucleophiles, or proton donors or abstractors. Receptor proteins in cell membranes can bind ligands e. Ligand binding is similar to substrate binding but the ligand usually remains unchanged.

Some hormones are themselves small proteins, such as insulin and growth hormone. Transport and storage. Hemoglobin transports oxygen in the red blood cells while transferrin transports iron to the liver.

Once in the liver, iron is stored bound to the protein ferritin. Dietary fats are carried in the blood by lipoproteins. Many other molecules and ions are transported and stored in a protein-bound form.

This can enhance solubility and reduce reactivity until they are required. Structure and movement. Collagen is the major protein in skin, bone and connective tissue, while hair is made mainly from keratin. There are also many structural proteins within the cell, for example in the cytoskeleton. Casein and ovalbumin are the major proteins of milk and eggs, respectively, and are used to provide the amino acids for growth of developing offspring. Seed proteins also provide nutrition for germinating plant embryos.

Antibodies, which recognize and bind to bacteria, viruses and other foreign material the antigen are proteins. Transcription factors bind to and modulate the function of DNA. Many other proteins modify the functions of other molecules by binding to them. Many proteins are composed of structurally independent units, or domains, that are connected by sections with limited higher order structure within the same polypeptide.

The connections can act as hinges to permit the individual domains to move in relation to each other, and breakage of these connections by limited proteolysis can often separate the domains, which can then behave like independent globular proteins.

The active site of an enzyme is sometimes formed in a groove between two domains, which wrap around the substrate. When such a function is required in many different proteins, the same domain structure is often found. In eukaryotes, domains are often encoded by discrete parts of genes called exons see Topic O3. Therefore, it has been suggested that, during evolution, new proteins were created by the duplication and rearrangement of domain-encoding exons in the genome to produce new combinations of binding sites, catalytic sites and structural elements in the resulting new polypeptides.

In this way, the rate of evolution of new functional proteins may have been greatly increased. Structural motifs also known as supersecondary structures are groupings of secondary structural elements that frequently occur in globular proteins. Alternatively, they may represent the best solution to a structural´┐Żfunctional requirement that has been arrived at independently in unrelated proteins.

Sequence motifs consist of only a few conserved, functionally important amino acids rather than supersecondary structures. Protein families arise through successive duplications and subsequent divergent evolution of an ancestral gene. Their 24 Section B ´┐Ż Protein structure Fig. Evolution of globins from an ancestral globin gene. Family members in different species that have retained the same function and carry out the same biochemical role e.

The degree of similarity between the amino acid sequences of orthologous members of a protein family in different organisms depends on how long ago the two organisms diverged from their common ancestor and on how important conservation of the sequence is for the function of the protein. The function of a protein, whether structural or catalytic, is inherently related to its structure. As indicated above, similar structures and functions can also be achieved by convergent evolution whereby unrelated genes evolve to produce proteins with similar structures or catalytic activities.

A good example is provided by the proteolytic enzymes subtilisin bacterial and chymotrypsin animal. Even though their amino acid sequences are very different and they are composed of different structural motifs, they have evolved the same spatial orientation of the catalytic triad of active site amino acids ´┐Ż serine, histidine and aspartic acid ´┐Ż and use exactly the same catalytic mechanism to hydrolyse peptide bonds.

Such proteins are termed functional analogs. Where similar structural motifs have evolved independently, the resulting proteins are structural analogs. Ion-exchange chromatography, isoelectric focusing and electrophoresis take advantage of the different ionic charges on proteins. Hydrophobic interaction chromatography exploits differences in hydrophobicity. Antibodies Antibodies are proteins produced by the immune system of vertebrates in response to a foreign agent the antigen , usually a protein on the surface of a virus or bacterium.

They are an important natural defense against infection. X-ray crystallography and NMR Many proteins can be crystallized and their three-dimensional structures determined by X-ray diffraction. The structures of small proteins in solution can also be determined by multi-dimensional nuclear magnetic resonance, particularly if the normal 12C and 14N are substituted by 13C and 15N.

Functional analysis Functional analysis of a protein involves its isolation and study in vitro combined with a study of the behavior of a mutant organism in which the protein has been rendered nonfunctional by mutation or deletion of its gene. The function of a new protein can sometimes be predicted by comparing its sequence and structure to those of known proteins.

When applied to the top of the column, protein molecules larger than the pores elute at the bottom relatively quickly, as they are excluded from the beads and so have a relatively small volume of buffer available through which to travel. Molecules smaller than the pore size can enter the beads and so have a larger volume of buffer through which they can diffuse. Thus, smaller proteins elute from the column after larger ones.

Ultracentrifugation can be used to separate proteins but is also a powerful analytical tool for studying protein structure. Charge Because of the presence of ionizable side chains on their surface, proteins carry a net charge. As these side chains are all titratable, there will exist for each protein a pH at which its net surface charge is zero ´┐Ż the isoelectric point pI. Proteins are least soluble at their pI. Depending on their net charge, proteins will travel at different rates towards the anode or cathode and can then be recovered from the gel after separation.

In ion-exchange chromatography, ions that are electrostatically bound to an insoluble support the ion exchanger packed in a column are reversibly replaced with charged proteins from solution. Usually, a salt gradient of increasing ionic strength is passed through the column and the bound proteins elute separately. Proteins migrate to positions corresponding to their isoelectric points in this gradient and form tight, focused bands as their net charge becomes zero and they stop moving Fig.

Isoelectric focusing of a mixture of proteins with pI values 4, 5, 6 and 7. B3 ´┐Ż Protein analysis 27 Hydrophobicity Hydrophobic interactions between proteins and a column material containing aromatic or aliphatic alkyl groups are promoted in solutions of high ionic strength. By applying a gradient of decreasing ionic strength, proteins elute at different stages. The insulin receptor is a low abundance cell-surface protein responsible for transmitting the biological activity of insulin to the cell interior.

For example, if the gene for the protein is inserted in a phage or plasmid expression vector under the control of a strong promoter see Topic K3 and the vector is then transformed into E. The tag often has little effect on the function of the protein. Protein sequencing An essential requirement for understanding how a protein works is a knowledge of its primary structure. This indicates how many glycines and serines, etc. For example, trypsin cleaves only after lysine K or arginine R and V8 protease only after glutamic acid E.

Cyanogen bromide cleaves polypeptides only after methionine residues. Each peptide is then subjected to sequential Edman degradation in an automated protein sequencer. Phenylisothiocyanate reacts with the N-terminal amino acid which, after acid treatment, is released as the phenylthiohydantoin PTH derivative, leaving a new N terminus. Example of polypeptide sequence determination.

Mass spectrometry MS offers an extremely accurate method. For small molecule analysis, samples are usually vaporized and ionized by a beam of Xe or Ar atoms. However, such methods have an upper mass limit of only a few kDa and are B3 ´┐Ż Protein analysis 29 too destructive for protein analysis. Recent, non-destructive ionization techniques have greatly extended this mass range. These ion sources can be coupled to a variety of different mass analyzers and detectors, each suited to a different purpose.

For example, an ESI ion source may be attached to a quadrupole detector. ESI-quadrupole systems allow the masses of proteins smaller than kDa to be measured within 0. The detector then counts the different ions as they arrive. Antibodies Antibodies are useful molecular tools for investigating protein structure and function. Antibodies are themselves proteins immunoglobulins, Ig and are generated by the immune system of higher animals when they are injected with a macromolecule the antigen such as a protein that is not native to the animal.

Antibodies fall into various classes, e. The most useful are the IgG class, produced as soluble proteins by B lymphocytes. Structure of an antibody molecule. This region recognizes and binds to a short sequence of 5´┐Ż8 amino acids on the surface of the antigen an epitope.

Usually, a single antigen elicits the production of several different antibodies by different B cell clones, each of which recognizes a different epitope on the antigen a polyclonal antibody mixture. They can also be used to detect proteins after separation of cell extracts by SDS gel electrophoresis see above.

After separation, the proteins are transferred from the gel to a membrane in a procedure similar to Southern blotting see Topic J1. The second antibody has previously been linked covalently to an enzyme such as peroxidase or alkaline phosphatase which catalyzes a color-generating reaction when incubated with the appropriate substrate.

Thus, a colored band appears on the blot in the position of the protein of interest. This procedure is semiquantitative and is similar in principle to the enzyme-linked immunosorbent assay ELISA commonly used in clinical diagnostics. The 3-D structure can then be determined by X-ray crystallography. X-rays interact with the electrons in the matter through which they pass. By measuring the pattern of diffraction of a beam of X-rays as it passes through a crystal, the positions of the atoms in the crystal can be calculated.

By crystallizing an enzyme in the presence of its substrate, the precise intermolecular interactions responsible for binding and catalysis can be seen. The structures of small globular proteins in solution can also be determined by two- or three-dimensional nuclear magnetic resonance NMR spectroscopy. The properties of this relaxation depend on the relative positions of the protons in the molecule.

The multi-dimensional approach is required for proteins to spread out and resolve the overlapping data produced by the large number of protons.

Substituting 13C and 15N for the normal isotopes 12C and 14N in the protein also greatly improves data resolution by eliminating unwanted resonances. In this way, the structures of proteins up to about 30 kDa in size can be deduced. Where both X-ray and NMR methods have been used to determine the structure of a protein, the results usually agree well. This suggests that the measured structures are the true in vivo structures.

Functional analysis The three-dimensional structures of many proteins have now been determined. However, tertiary and quaternary structural determination is still a relatively costly and laborious procedure and lags well behind the availability of new protein primary structures predicted from genomic sequencing projects see Topic J2. Thus, there is now great interest in computational methods that will allow the prediction of both structure and possible function from simple amino acid sequence information.

These methods are based largely on the fact that there are only a limited number of supersecondary structures in nature and involve mapping of new protein sequences on to the known three dimensional structures of proteins with related amino acid sequences.

However, caution must still be exerted in interpreting such results. It is not an enzyme, as might be predicted on the basis of sequence similarity. Thus, at least for the moment, understanding of the true function of a protein still requires its isolation and biochemical and structural characterization.

Isolation is greatly aided by recombinant techniques. Knowledge of the biochemical properties of an isolated protein does not necessarily tell you what that protein does in the cell. Additional genetic analysis is usually required. If the gene for the protein can be inactivated by mutagenesis or deleted by recombinant DNA techniques, then the phenotype of the resulting mutant can be studied. In conjunction with the biochemical information, the altered behavior of the mutant cell can help to pinpoint the function of the protein in vivo.

In RNA, thymine is replaced by the structurally very similar pyrimidine, uracil U. The repeat unit is a nucleotide. Nucleic acids are highly charged polymers with a negative charge on each phosphate. Two separate and antiparallel chains of DNA are wound around each other in a right-handed helical coiled path, with the sugar´┐Żphosphate backbones on the outside and the bases, paired by hydrogen bonding and stacked on each other, on the inside.

Adenine pairs with thymine; guanine pairs with cytosine. RNA secondary structure Most RNA molecules occur as a single strand, which may be folded into a complex conformation, involving local regions of intramolecular base pairing and other hydrogen bonding interactions.

Adenine A and guanine G are purines, bicyclic structures two fused rings , whereas cytosine C , thymine T and uracil U are monocyclic pyrimidines. In RNA, the thymine base is replaced by uracil. Thymine differs from uracil only in having a methyl group at the 5-position, that is thymine is 5-methyluracil.

Nucleic acid bases. The point of attachment to the base is the 1-position N-1 of the pyrimidines and the 9-position N-9 of the purines Fig. The bond between the bases and the sugars is the glycosylic or glycosidic bond.

If the sugar is ribose, the nucleosides technically ribonucleosides are adenosine, guanosine, cytidine and uridine. Thymidine and deoxythymidine may be used interchangeably. If the sugar is deoxyribose, then the compounds are termed deoxynucleotides Fig. Chemically, the compounds are phosphate esters. In the course of DNA or RNA synthesis, two phosphates are split off as pyrophosphate to leave one phosphate per nucleotide incorporated into the nucleic acid chain see Topics E1 and K1.

This kind of bond or linkage is called a phosphodiester bond, since the phosphate is chemically in the form of a diester. A nucleic acid chain can hence be seen to have a direction.

At neutral pH, each phosphate group has a single negative charge. This is why nucleic acids are termed acids; they are the anions of strong acids.

Nucleic acids are thus highly charged polymers. The basic features of this structure were deduced by James Watson and Francis Crick in Two separate chains of DNA are wound around each other, each following a helical coiling path, resulting in a right-handed double helix Fig.

The negatively charged sugar´┐Żphosphate backbones of the molecules are on the outside, and the planar bases of each strand stack one above the 36 Section C ´┐Ż Properties of nucleic acids Fig. Phosphodiester bonds and the covalent structure of a DNA strand. Between the backbone strands run the major and minor grooves, which also follow a helical path. The strands are joined noncovalently by hydrogen bonding between the bases on opposite strands, to form base pairs.

There are around 10 base pairs per turn in the DNA double helix. This last feature arises because the structures of the bases and the constraints of the DNA backbone dictate that the bases hydrogen-bond to each other as purine´┐Żpyrimidine pairs which have very similar geometry and dimensions Fig. Guanine pairs with cytosine three H-bonds and adenine pairs with thymine two H-bonds. Hence, any sequence can be accommodated within a regular double-stranded DNA structure.

A, B and Z helices In fact, a number of different forms of nucleic acid double helix have been observed and studied, all having the basic pattern of two helically-wound antiparallel strands.

C1 ´┐Ż Nucleic acid structure 37 Fig. The DNA double helix. The DNA base pairs. DNA can be induced to form an alternative helix, known as the A-form Fig. The A-form is right-handed, like the Bform, but has a wider, more compressed structure in which the base pairs are tilted with respect to the helix axis, and actually lie off the axis seen end-on, the A-helix has a hole down the middle. This is because in this structure, the pyrimidine and the purine nucleotides adopt very different conformations, unlike in A- and B-form, where each nucleotide has essentially the same conformation and immediate environment.

The pyrimidine nucleotides, and all nucleotides in the A- and B- forms adopt the anti conformation. Table 1. RNA instead forms relatively globular conformations, in which local regions of helical structure are formed by intramolecular hydrogen bonding and base stacking within the single nucleic acid chain.

RNA structures range from short small nuclear RNAs, which help to mediate the splicing of pre-mRNAs in eukaryotic cells see Topic O3 , to large rRNA molecules, which form the structural backbone of the ribosomes and participate in the chemistry of protein synthesis see Topic Q2.

The alternative helical forms of the DNA double helix. These are considered in more detail in Topics O3 and P2. Effect of acid Highly acidic conditions may hydrolyze nucleic acids to their components: bases, sugar and phosphate. Moderate acid causes the hydrolysis of the purine base glycosylic bonds to yield apurinic acid. More complex chemistry has been developed to remove particular bases, and is the basis of chemical DNA sequencing.

Some chemicals, such as urea and formamide, can denature DNA and RNA at neutral pH by disrupting the hydrophobic forces between the stacked bases.

DNA has a density of around 1. In fact this is not the case. As in proteins see Topic A4 , the presence of H-bonds within a structure does not normally confer stability.

This is because one must consider the difference in energy between, in the case of DNA, the single- C2 ´┐Ż Chemical and physical properties of nucleic acids 41 stranded random coil state, and the double-stranded conformation. H-bonds between base pairs in double-stranded DNA merely replace what would be equally strong and energetically favorable H-bonds with water molecules in free solution, if the DNA were single-stranded.

The root of this stability lies elsewhere, in the stacking interactions between the base pairs see Topic C1, Fig. The hydrogen bonding network of bulk water becomes destabilized in the vicinity of a hydrophobic surface, since not all the water molecules can participate in full hydrogen bonding interactions, and they become more ordered. Hence it is energetically favorable to exclude water altogether from pairs of such surfaces by stacking them together; more water ends up in the bulk hydrogen-bonded network.

This also maximizes the interaction between charge dipoles on the bases see Topic A4. Even in single-stranded DNA, the bases have a tendency to stack on top of each other, but this stacking is maximized in double-stranded DNA, and the hydrophobic effect ensures that this is the most energetically favorable arrangement. In more dilute mineral acid, for example at pH 3´┐Ż4, the most easily hydrolyzed bonds are selectively broken.

These are the glycosylic bonds attaching the purine bases to the ribose ring, and hence the nucleic acid becomes apurinic see Topic F2. The effect of alkali is to change the tautomeric state of the bases. This effect can be seen with reference to the model compound, cyclohexanone Fig. The molecule is in equilibrium between the tautomeric keto and enol forms 1 and 2. At neutral pH, the compound is predominantly in the keto form 1.

Increasing the pH causes a shift to the enolate form 3 when the molecule loses a proton, since the negative charge is most stably accommodated on the electronegative oxygen atom. In the same way, the structure of guanine Fig. The denaturation of DNA at high pH. Intramolecular cleavage of RNA phosphodiester bonds in alkali. A relatively high concentration of these agents several molar has the effect of disrupting the hydrogen bonding of the bulk water solution.

This means that the energetic stabilization of the nucleic acid secondary structure, caused by the exclusion of water from between the stacked hydrophobic bases, is lessened and the strands become denatured. Viscosity Cellular DNA is very long and thin; technically, it has a high axial ratio. DNA is around 2 nm in diameter, and may have a length of micrometers, millimeters or even several centimeters in the case of eukaryotic chromosomes.

In addition, DNA is a relatively stiff molecule; its stiffness would be similar to that of partly cooked spaghetti, using the same analogy. A consequence of this is that DNA solutions have a high viscosity. Furthermore, long DNA molecules can easily be damaged by shearing forces, or by sonication high-intensity ultrasound , with a concomitant reduction in viscosity. Note that neither shearing nor sonication denatures the DNA; they merely reduce the length of the double-stranded molecules in the solution.

In solutions containing high concentrations of a high molecular weight salt, for example 8 M cesium chloride CsCl , DNA has a similar density to the bulk solution, around 1. If the solution is centrifuged at very high speed, the dense cesium salt tends to migrate down the tube, setting up a density gradient Fig. Eventually the DNA sample will migrate to a sharp band at a position in the gradient corresponding to its own buoyant density.

Equilibrium density gradient centrifugation of DNA. Quantitation of nucleic acids The absorbance at nm is used to determine the concentration of nucleic acids. For pure DNA, the value is 1. Values above 1. Denaturation may be detected by the change in A Related topics UV absorption Nucleic acid structure C1 Chemical and physical properties of nucleic acids C2 Genome complexity D4 Nucleic acids absorb UV light due to the conjugated aromatic nature of the bases; the sugar´┐Żphosphate backbone does not contribute appreciably to absorption.

The absorption properties of nucleic acids can be used for detection, quantitation and assessment of purity. Double-stranded DNA has equal numbers of purines and pyrimidines, and so does not show this effect.

The absorbance values also depend on the amount of secondary structure double-stranded regions in a given molecule, due to hypochromicity. The shape of the absorption spectrum Fig. The process of denaturation can be observed conveniently by the increase in absorbance as double-stranded nucleic acids are converted to single strands Fig.

As the temperature is increased, the absorbance of an RNA sample gradually and erratically increases as the stacking of the bases in double-stranded regions is reduced. Shorter regions of base pairing will denature before longer regions, since they are more thermally mobile. In contrast, the thermal denaturation, or melting, of dsDNA is co-operative. Renaturation The thermal denaturation of DNA may be reversed by cooling the solution.

Rapid cooling allows only the formation of local regions of dsDNA, formed by the base pairing or annealing of short regions of complementarity within or between DNA strands; the decrease in A is hence rather small Fig.

The renaturation of regions of complementarity between different nucleic acid strands is known as hybridization. The number of links is known as the linking number Lk. Most natural DNA is negatively supercoiled, that is the DNA is deformed in the direction of unwinding of the double helix. Twist and writhe Supercoiling is partitioned geometrically into a change in twist, the local winding up or unwinding of the double helix, and a change in writhe, the coiling of the helix axis upon itself.

Intercalators Intercalators, such as ethidium bromide, bind to DNA by inserting themselves between the base pairs intercalation , resulting in the local untwisting of the DNA helix. If the DNA is closed-circular, then there will be a corresponding increase in writhe. Energy of supercoiling Negatively supercoiled DNA has a high torsional energy, which facilitates the untwisting of the DNA helix and can drive processes which require the DNA to be unwound.

DNA gyrase introduces negative supercoiling using the energy derived from ATP hydrolysis, and topoisomerase IV unlinks decatenates daughter chromosomes. The molecule has no free ends, and the two single strands are linked together a number of times corresponding to the number of double-helical turns in the molecule.

This number is known as the linking number Lk. A good way to imagine these is to consider the DNA double helix as a piece of rubber tubing, with a line drawn along its length to enable us to follow its twisting.

The tubing may be joined by a connector into a closed circle Fig. If we imagine a twisting of the DNA helix tubing followed by the joining of the ends, then the deformation so formed is locked into the system Fig. This deformation is known as supercoiling, since it manifests itself as a coiling of the DNA axis around itself in a higher-order coil, and corresponds to a change in linking number from the simple circular situation.

If the twisting of the DNA is in the same direction as that of the double helix, that is the helix is twisted up before closure, then the supercoiling formed is positive; if the helix is untwisted, then the supercoiling is negative.

Almost all DNA molecules in cells are on average negatively supercoiled. This is true even for linear DNAs such as eukaryotic chromosomes, which are constrained into large loops by interaction with a protein scaffold see Topics D2 and E3.

The changes in linking number are indicated see text for details. Topoisomer The linking number of a closed-circular DNA is a topological property, that is one which cannot be changed without breaking one or both of the DNA backbones. A molecule of a given linking number is known as a topoisomer. Topoisomers differ from each other only in their linking number. Twist and writhe The conformation geometry of the DNA can be altered while the linking number remains constant.

Two extreme conformations of a supercoiled DNA topoisomer may be envisaged Fig. The line on the rubber tubing model helps to keep track of local twisting of the DNA axis. The equilibrium situation lies between these two extremes Fig. The changes in twist and writhe at constant linking number are shown see text for details. Intercalators The geometry of a supercoiled molecule may be altered by any factor which affects the intrinsic twisting of the DNA helix. For example, an increase in temperature reduces the twist, and an increase in ionic strength may increase the twist.

One important factor is the presence of an intercalator. The bestknown example of an intercalator is ethidium bromide Fig. This is a positively charged polycyclic aromatic compound, which binds to DNA by inserting itself between the base pairs intercalation; Fig. This is a reduction in twist, and hence results in a corresponding increase in writhe in a closed-circular molecule.

Energy of supercoiling Supercoiling involves the introduction of torsional stress into DNA molecules. For negative supercoiling, this energy makes it easier for the DNA helix to be locally untwisted, or unwound. Negative supercoiling may thus facilitate processes which require the unwinding of the helix, such as transcription initiation or replication see Topics E1 and K1. Topoisomerases Enzymes exist which regulate the level of supercoiling of DNA molecules; these are termed topoisomerases.

To alter the linking number of DNA, the enzymes must transiently break one or both DNA strands, which they achieve by the attack of a tyrosine residue on a backbone phosphate, resulting in a temporary covalent attachment of the enzyme to one of the DNA ends via a phosphotyrosine bond.

There are two classes of topoisomerase. Most topoisomerases reduce the level of positive or negative supercoiling, that is they operate in the energetically favorable direction.

Since their mechanism involves the passing of one double strand through another, type II topoisomerases are also able to unlink DNA molecules, such as daughter molecules produced in replication, which are linked catenated together see Topic E2. In bacteria, this function is carried out by topoisomerase IV. Topoisomerases are essential enzymes in all organisms, being involved in replication, recombination and transcription see Topics E1, F4 and K1. DNA gyrase and topoisomerase IV are the targets of anti-bacterial drugs in bacteria, and both type I and type II enzymes are the target of anti-tumor agents in humans.

The mechanisms of a type I and b type II topoisomerases see text for details. In normal growth, the DNA is being replicated continuously. DNA domains The genome is organized into 50´┐Ż large loops or domains of 50´┐Ż kb in length, which are constrained by binding to a membrane´┐Żprotein complex.

Supercoiling of the genome The genome is negatively supercoiled. Individual domains may be topologically independent, that is they may be able to support different levels of supercoiling. These proteins constrain about half of the supercoiling of the DNA. The bulk of the DNA in E. The DNA is packaged into a region of the cell known as the nucleoid.

This region has a very high DNA concentration, perhaps 30´┐Ż50 mg ml´┐Ż1, as well as containing all the proteins associated with DNA, such as polymerases, repressors and others see below. A fairly high DNA concentration in the test tube would be 1 mg ml´┐Ż1. In normal growth, the DNA is being replicated continuously and there may be on average around two copies of the genome per cell, when growth is at the maximal rate see Topic E2. The DNA consists of 50´┐Ż domains or loops, the ends of which are constrained by binding to a structure which probably consists of proteins attached to part of the cell membrane Fig.

The loops are about 50´┐Ż kb in size. It is not known whether the loops are static or dynamic, but one model suggests that the DNA may spool through sites of polymerase or other enzymic action at the base of the loops. A schematic view of the structure of the E. The thin line is the DNA double helix. Supercoiling of the genome The E. Electron micrographs indicate that some domains may not be supercoiled, perhaps because the DNA has become broken in one strand see Topic C4 , where other domains clearly do contain supercoils Fig.

The attachment of the DNA to the protein´┐Żmembrane scaffold may act as a barrier to rotation of the DNA, such that the domains may be topologically independent. There is, however, no real biochemical evidence for major differences in the level of supercoiling in different regions of the chromosome in vivo. These proteins are sometimes known as histone-like proteins see Topic D2 , and have the effect of compacting the DNA, which is essential for the packaging of the DNA into the nucleoid, and of stabilizing and constraining the supercoiling of the chromosome.

Only about half the supercoiling is unconstrained in the sense of being able to adopt the twisting and writhing conformations described in Topic C4.

It may be that the organization of the nucleoid is fairly complex, although highly ordered DNA´┐Żprotein complexes such as nucleosomes see Topic D2 have not been detected. The name chromatin is given to the highly ordered DNA´┐Żprotein complex which makes up the eukaryotic chromosomes. The chromatin structure serves to package and organize the chromosomal DNA, and is able to alter its level of packing at different stages of the cell cycle.

The major protein components of chromatin are the histones; small, basic positively charged proteins which bind tightly to DNA. There are four families of core histone, H2A, H2B, H3, H4, and a further family, H1, which has some different properties, and a distinct role. Individual species have a number of variants of the different histone proteins.

Nucleosomes The nucleosome core is the basic unit of chromosome structure, consisting of a protein octamer containing two each of the core histones, with bp of DNA wrapped 1.

A nucleosome core plus H1 is known as a chromatosome. In some cases, H1 is replaced by a variant, H5, which binds more tightly, and is associated with DNA which is inactive in transcription. The nucleosomal repeat unit is hence around bp. Most chromatin exists in this form. The overall structure somewhat resembles that of the organizational domains of prokaryotic DNA. DNA supercoiling C4 Prokaryotic chromosome structure D1 Eukaryotic chromosome structure D3 Genome complexity D4 54 Section D ´┐Ż Prokaryotic and eukaryotic chromosome structure Chromatin The total length of DNA in a eukaryotic cell depends on the species, but it can be thousands of times as much as in a prokaryotic genome, and is made up of a number of discrete bodies called chromosomes 46 in humans.

The DNA in each chromosome is believed to be a single linear molecule, which can be up to several centimeters long see Topic D3.

All this DNA must be packaged into the nucleus see Topic A2 , a space of approximately the same volume as a bacterial cell; in fact, in their most highly condensed forms, the chromosomes have an enormously high DNA concentration of perhaps mg ml´┐Ż1 see Topic D1.

This feat of packing is accomplished by the formation of a highly organized complex of DNA and protein, known as chromatin, a nucleoprotein complex see Topic A4. Chromosomes greatly alter their level of compactness as cells progress through the cell cycle see Topics D3 and E3 , varying between highly condensed chromosomes at metaphase just before cell division , and very much more diffuse structures in interphase.

This implies the existence of different levels of organization of chromatin. The core histones are small proteins, with masses between 10 and 20 kDa, and H1 histones are a little larger at around 23 kDa. This means that histones will bind very strongly to the negatively charged DNA in forming chromatin. Within a given species, there are normally a number of closely similar variants of a particular class, which may be expressed in different tissues, and at different stages in development.

There is not much similarity in sequence between the different histone classes, but structural studies have shown that the classes do share a similar tertiary structure see Topic B2 , suggesting that all histones are ultimately evolutionarily related see Topic B3. H1 histones are somewhat distinct from the other histone classes in a number of ways; in addition to their larger size, there is more variation in H1 sequences both between and within species than in the other classes.

Histone H1 is more easily extracted from bulk chromatin, and seems to be present in roughly half the quantity of the other classes, of which there are very similar amounts. Nucleosomes A number of studies in the s pointed to the existence of a basic unit of chromatin structure.

Nucleases are enzymes which hydrolyze the phosphodiester bonds of nucleic acids. Exonucleases release single nucleotides from the ends of nucleic acid strands, whereas endonucleases cleave internal phosphodiester bonds.

Treatment of chromatin with micrococcal nuclease, an endonuclease which cleaves double-stranded DNA, led to the isolation of DNA fragments with discrete sizes, in multiples of approximately bp. It was discovered that each bp fragment is associated with an octamer core of histone proteins, H2A 2 H2B 2 H3 2 H4 2, which is why these are designated the core histones, and more loosely with one molecule of H1.

The proteins protect the DNA from the action of micrococcal nuclease. More D2 ´┐Ż Chromatin structure 55 prolonged digestion with nuclease leads to the loss of H1 and yields a very resistant structure consisting of bp of DNA associated very tightly with the histone octamer. This structure is known as the nucleosome core, and is structurally very similar whatever the source of the chromatin.

The structure of the nucleosome core particle is now known in considerable detail, from structural studies culminating in X-ray crystallography. The histone octamer forms a wedge-shaped disk, around which the bp of DNA is wrapped in 1. Figure 1 shows the basic features and the dimensions of the structure. The left-handed wrapping of the DNA around the nucleosome corresponds to negative supercoiling, that is the turns are superhelical turns technically writhing; see Topic C4.

Although eukaryotic DNA is negatively supercoiled to a similar level as that of prokaryotes, on average, virtually all the supercoiling is accounted for by wrapping in nucleosomes and there is no unconstrained supercoiling see Topic D1. A schematic view of the structure of a nucleosome core and a chromatosome. The role of H1 One molecule of histone H1 binds to the nucleosome, and acts to stabilize the point at which the DNA enters and leaves the nucleosome core Fig.

In the presence of H1, a further 20 bp of DNA is protected from nuclease digestion, making bp in all, corresponding to two full turns around the histone octamer. The larger size of H1 compared with the core histones is due to the presence of an additional C-terminal tail, which serves to stabilize the DNA between the 56 Section D ´┐Ż Prokaryotic and eukaryotic chromosome structure nucleosome cores.

As stated above, H1 is more variable in sequence than the other histones and, in some cell types, it may be replaced by an extreme variant called histone H5, which binds chromatin particularly tightly, and is associated with DNA which is not undergoing transcription see Topic D3. This comprises globular particles nucleosomes , connected by thin strands of DNA.

This linker DNA is the additional DNA required to make up the bp nucleosomal repeat apparent in the micrococcal nuclease experiments see above. The average length of linker DNA between core particles is 55 bp, but the length varies between species and tissues from almost nothing to more than bp Fig.

Detailed studies of this process have suggested that the D2 ´┐Ż Chromatin structure 57 nucleosomes are wound into a higher order left-handed helix, dubbed a solenoid, with around six nucleosomes per turn Fig. Higher order structure The organization of chromatin at the highest level seems rather similar to that of prokaryotic DNA see Topic D1.

Electron micrographs of chromosomes which have been stripped of their histone proteins show a looped domain structure, which is similar to that illustrated in Topic D1, Fig. Even the size of the loops is approximately the same, up to around kb of DNA, although there are many more loops in a eukaryotic chromosome.

The loops are constrained by interaction with a protein complex known as the nuclear matrix see Topic E4. The centromere The centromere is the region where the two chromatids are joined and is also the site of attachment, via the kinetochore, to the mitotic spindle, which pulls apart the sister chromatids at anaphase. Interphase chromosomes Heterochromatin Euchromatin In interphase, the chromosomes adopt a much more diffuse structure, although the chromosomal loops remain attached to the nuclear matrix.

Heterochromatin is a portion of the chromatin in interphase which remains relatively compacted and is transcriptionally inactive. As the daughter chromosomes are pulled apart by the mitotic spindle at cell division, the fragile centimeters-long chromosomal DNA would certainly be sheared by the forces generated, were it not in this highly compact state.

The structure in Fig. The tips of the chromosomes are the telomeres, which are also the ends of the DNA molecule; the DNA maps in a linear fashion along the length of the chromosome, albeit in a very convoluted path. The structure of the mitotic chromosome. The structure of a section of a mitotic chromatid is shown in Fig.

The chromosomal loops see Topic D2 fan out from a central scaffold or nuclear matrix region consisting of protein. One possibility is that consecutive loops may trace a helical path along the length of the chromosome. The centromere The centromere is the constricted region where the two sister chromatids are joined in the metaphase chromosome.

This is the site of assembly of the kinetochore, a protein complex which attaches to the microtubules see Topic A4 of the mitotic spindle. The microtubules act to separate the chromatids at anaphase. The telomeric DNA forms a special secondary structure, the function of which is to protect the ends of the chromosome proper from degradation. Independent synthesis of the telomere acts to counteract the gradual shortening of the chromosome resulting from the inability of normal replication to copy the very end of a linear DNA molecule see Topic E4.

Interphase chromosomes In interphase, the genes on the chromosomes are being transcribed and DNA replication takes place during S-phase; see Topics E3 and E4. During this time, which is most of the cell cycle, the chromosomes adopt a much more diffuse structure and cannot be visualized individually.

It is believed, however, that the chromosomal loops are still present, attached to the nuclear matrix see Topic D2, Fig. Heterochromatin Heterochromatin comprises a portion of the chromatin in interphase which remains highly compacted, although not so compacted as at metaphase. It has been shown more recently that heterochromatin is transcriptionally inactive. It is believed that much of the heterochromatin may consist of the repeated satellite DNA close to the centromeres of the chromosomes see Topic D4 , although in some cases entire chromosomes can remain as heterochromatin, for example one of the two X chromosomes in female mammals.

Euchromatin The rest of the chromatin, which is not visible as heterochromatin, is known historically by the catch-all name of euchromatin, and is the region where all transcription takes place. Parts of these regions may be depleted of nucleosomes altogether, particularly within promoters, to allow the binding of transcription factors and other proteins Fig. DNase I hypersensitivity The sensitivity of chromatin to the nuclease deoxyribonuclease I DNase I , which cuts the backbone of DNA unless the DNA is protected by bound protein, has been used to map the regions of transcriptionally active chromatin in cells.

Longer regions of sensitivity represent sequences where transcription is taking place. D3 ´┐Ż Eukaryotic chromosome structure 61 Fig. Euchromatin, showing active and inactive regions see text for details and sites of DNase I hypersensitivity indicated by large and small arrows. CpG sites are normally methylated in mammalian cells, and are relatively scarce throughout most of the genome.

This is because 5-methylcytosine spontaneously deaminates to thymine and, since this error is not always repaired, methylated CpG mutates fairly rapidly to TpG see Topics F1 and F2.

The methylation of CpG is associated with transcriptionally inactive regions of chromatin. These islands are commonly around bp long, and are coincident with regions of particular sensitivity to DNase I Fig. The CpG islands surround the promoter regions of genes which are expressed in almost all cell types, so called housekeeping genes, and may be largely free of nucleosomes. For example, actively transcribed chromatin is associated with the acetylation of lysine residues in the N-terminal regions see Topic B2 of the core histones see Topic D2 , whereas the condensation of chromosomes at mitosis is accompanied by the phosphorylation of histone H1.

These changes 62 Section D ´┐Ż Prokaryotic and eukaryotic chromosome structure Fig. CpG islands and the promoters of housekeeping genes. Longer term differences in chromatin condensation are associated with changes due to stages in development and different tissue types.

These changes are associated with the utilization of alternative histone variants see Topic D2 , which may also act by altering the stability of chromatin conformations. Histone H5 is an extreme example of this effect, replacing H1 in some very inactive chromatin, for example in avian red blood cells see Topic D2.

Much of this DNA consists of multiple repeats, sometimes thousands or hundreds of thousands of copies, of a few relatively short sequence elements. Unique sequence DNA The fraction of DNA which reassociates most slowly is unique sequence, essentially comprising single-copy genes, or those repeated a few times. Escherichia coli DNA is essentially all unique sequence.

Tandem gene clusters Regions of the genome where certain genes or gene clusters are tandemly repeated up to a few hundred times comprise part of the moderately repetitive DNA. Examples include rRNA genes and histone gene clusters. The most prominent examples in humans are the Alu and the L1 elements, which may be parasitic DNA sequences, replicating themselves by transposition.

Satellite DNA Satellite DNA, which occurs mostly near the centromeres of chromosomes, and may be involved in attachment of the mitotic spindle, consists of huge numbers of tandem repeats of short up to 30 bp sequences. Genetic polymorphism Base changes mutations in a gene or a chromosomal locus can create multiple forms polymorphs of that locus which is then said to show genetic polymorphism.

The term can describe different alleles of a single copy gene in a single individual as well as the different sequences present in different individuals in a population. Where SNPs create or destroy the sequence recognized by a restriction enzyme, restriction fragment length polymorphism RFLP will result. It is clear that much of this DNA does not code for protein, since there are not times as many proteins in humans as in E.

The coding regions of genes are interrupted by intron sequences see Topic O3 and genes may hence take up many kilobases of sequence but, despite this, genes are by no means contiguous along the genome; they are separated by long stretches of sequence for which the function, if any, is unknown. It has become apparent that much of this noncoding DNA consists of multiple repeats of similar or identical copies of a few different types of sequence.

These copies may follow one another directly tandemly repeated , for example satellite DNA, or they may occur as multiple copies scattered throughout the genome interspersed , such as the Alu elements.

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