Nucleic bases

The Nucleic Bases

Purines and pyrimidines are nitrogen-containing heterocycles, cyclic compounds whose rings contain both carbon and other elements(nucleic bases) . The smaller pyrimidine has the longer name and the larger purine the shorter name and that their six-atom rings are numbered in opposite directions . The oxo and amino groups of purines and pyrimidines exhibit keto-enol and amine-imine tautomerism , but physiologic conditions strongly favor the amino and oxo forms.

Tautomerism of the oxo and amino functional groups of purines and pyrimidines
Nucleosides & Nucleotides
Nucleosides are derivatives of purines and pyrimidines that have a sugar linked to a ring nitrogen of a heterocyclic "base," . Numerals with a prime (eg, 2' or 3') distinguish atoms of the sugar from those of the heterocyclic base. The sugar in ribonucleosides is D-ribose, and in deoxyribonucleosides it is 2-deoxy-D-ribose. The sugar is linked to the heterocyclic base via a -N-glycosidic bond, almost always to N-1 of a pyrimidine or to N-9 of a purine .



Mononucleotides are nucleosides with a phosphoryl group esterified to a hydroxyl group of the sugar. The 3'- and 5'-nucleotides are nucleosides with a phosphoryl group on the 3'- or 5'-hydroxyl group of the sugar, respectively. Since most nucleotides are 5'-, the prefix "5'-" is usually omitted when naming them. UMP and dAMP thus represent nucleotides with a phosphoryl group on C-5 of the pentose. Additional phosphoryl groups linked by acid anhydride bonds to the phosphoryl group of a mononucleotide form nucleoside diphosphates and triphosphates .





The following table lists the major purines and pyrimidines and their nucleoside and nucleotide derivatives. Single-letter abbreviations are used to identify adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), whether free or present in nucleosides or nucleotides. The prefix "d" (deoxy) indicates that the sugar is 2'-deoxy-D-ribose (eg, dGTP) .



Small quantities of additional purines and pyrimidines occur in DNA and RNAs. Examples include 5-methylcytosine of bacterial and human DNA, .Free nucleotides include hypoxanthine, xanthine, and uric acid , intermediates in the catabolism of adenine and guanine . Methylated heterocyclic bases of plants include the xanthine derivatives caffeine of coffee, theophylline of tea, and theobromine of cocoa .
Nucleosides or free purine or pyrimidine bases are uncharged at physiologic pH. By contrast, the pKa values of the primary and secondary phosphoryl groups of nucleotides are about 1.0 and 6.2, respectively. Nucleotides therefore bear a negative charge at physiologic pH. The conjugated double bonds of purine and pyrimidine derivatives absorb ultraviolet light. The mutagenic effect of ultraviolet light results from its absorption by nucleotides in DNA with accompanying chemical changes. At pH 7.0 all the common nucleotides absorb light at a wavelength close to 260 nm. The concentration of nucleotides and nucleic acids thus often is expressed in terms of "absorbance at 260 nm."
Synthetic Nucleotide Analogs
Synthetic analogs of purines, pyrimidines, nucleosides, and nucleotides altered in either the heterocyclic ring or the sugar moiety have numerous applications in clinical medicine. Their toxic effects reflect either inhibition of enzymes essential for nucleic acid synthesis or their incorporation into nucleic acids with resulting disruption of base-pairing. Oncologists employ 5-fluoro- or 5-iodouracil, 3-deoxyuridine, 6-thioguanine and 6-mercaptopurine, 5- or 6-azauridine, 5- or 6-azacytidine, and 8-azaguanine , which are incorporated into DNA prior to cell division. The purine analog allopurinol, used in treatment of hyperuricemia and gout, inhibits purine biosynthesis and xanthine oxidase activity. Cytarabine is used in chemotherapy of cancer. Finally, azathioprine, which is catabolized to 6-mercaptopurine, is employed during organ transplantation to suppress immunologic rejection.
Polynucleotides
Phosphodiester bonds link the 3'- and 5'-carbons of adjacent monomers. Each end of a nucleotide polymer thus is distinct. We therefore refer to the "5'- end" or the "3'- end" of polynucleotides, the 5'- end being the one with a free or phosphorylated 5'-hydroxyl.
The base sequence or primary structure of a polynucleotide can be represented as shown below. The phosphodiester bond is represented by P or p, bases by a single letter, and pentoses by a vertical line.



Where all the phosphodiester bonds are 5' --3'

The Nucleic acids : The DNA

The genetic information is coded along the length of a polymeric molecule composed of only four types of monomeric units . This polymeric molecule, deoxyribonucleic acid (DNA), is the chemical basis of heredity and is organized into genes, the fundamental units of genetic information. The basic information pathwayie, DNA directs the synthesis of RNA, which in turn directs protein synthesishas been elucidated. Genes do not function autonomously; their replication and function are controlled by various gene products, often in collaboration with components of various signal transduction pathways.
Chemical structure :
The chemical nature of the monomeric deoxynucleotide units of DNA are deoxyadenylate, deoxyguanylate, deoxycytidylate, and thymidylate. These monomeric units of DNA are held in polymeric form by 3',5'-phosphodiester bridges constituting a single strand, . The informational content of DNA (the genetic code) resides in the sequence in which these monomerspurine and pyrimidine deoxyribonucleotidesare ordered. The polymer possesses a polarity; one end has a 5'-hydroxyl or phosphate terminal while the other has a 3'-phosphate or hydroxyl terminal.





Since the genetic information resides in the order of the monomeric units within the polymers, there must exist a mechanism of reproducing or replicating this specific information with a high degree of fidelity. That requirement, together with x-ray diffraction data from the DNA molecule and the observation of Chargaff that in DNA molecules the concentration of deoxyadenosine (A) nucleotides equals that of thymidine (T) nucleotides (A = T), while the concentration of deoxyguanosine (G) nucleotides equals that of deoxycytidine (C) nucleotides (G = C), led Watson, Crick, and Wilkins to propose in the early 1950s a model of a double-stranded DNA molecule as shown in the model below. The two strands of this double-stranded helix are held in register by both hydrogen bonds between the purine and pyrimidine bases of the respective linear molecules and by van der Waals and hydrophobic interactions between the stacked adjacent base pairs. The pairings between the purine and pyrimidine nucleotides on the opposite strands are very specific and are dependent upon hydrogen bonding of A with T and G with C



This common form of DNA is said to be right-handed because as one looks down the double helix, the base residues form a spiral in a clockwise direction. In the double-stranded molecule, restrictions imposed by the rotation about the phosphodiester bond, of the four bases (A, G, T, and C) allow A to pair only with T and G only with C. This base-pairing restriction explains the earlier observation that in a double-stranded DNA molecule the content of A equals that of T and the content of G equals that of C. The two strands of the double-helical molecule, each of which possesses a polarity, are antiparallel; ie, one strand runs in the 5' to 3' direction and the other in the 3' to 5' direction. This is analogous to two parallel streets, each running one way but carrying traffic in opposite directions. In the double-stranded DNA molecules, the genetic information resides in the sequence of nucleotides on one strand, the template strand. This is the strand of DNA that is copied during ribonucleic acid (RNA) synthesis. It is sometimes referred to as the noncoding strand. The opposite strand is considered the coding strand because it matches the sequence of the RNA transcript (but containing uracil in place of thymine) that encodes the protein. The two strands, in which opposing bases are held together by interstrand hydrogen bonds, wind around a central axis in the form of a double helix. The distance spanned by one turn of B-DNA is 3.4 nm (34 ). The width (helical diameter) of the double helix in B-DNA is 2 nm (20 ).
As shown below three hydrogen bonds hold the deoxyguanosine nucleotide to the deoxycytidine nucleotide, whereas the other pair, the AT pair, is held together by two hydrogen bonds. Thus, the GC bonds are much more resistant to denaturation, or "melting," than AT-rich regions.


The Denaturation (Melting) of DNA
The double-stranded structure of DNA can be separated into two component strands (melted) in solution by increasing the temperature or decreasing the salt concentration. Not only do the two stacks of bases pull apart but the bases themselves unstack while still connected in the polymer by the phosphodiester backbone. Concomitant with this denaturation of the DNA molecule is an increase in the optical absorbance of the purine and pyrimidine basesa phenomenon referred to as hyperchromicity of denaturation. Because of the stacking of the bases and the hydrogen bonding between the stacks, the double-stranded DNA molecule exhibits properties of a rigid rod and in solution is a viscous material that loses its viscosity upon denaturation. Separated strands of DNA renature or reassociate when appropriate physiologic temperature and salt conditions are achieved, a process often referred to as hybridization. At a given temperature and salt concentration, a particular nucleic acid strand will associate tightly only with a complementary strand. Hybrid molecules will also form under appropriate conditions.

The grooves in the DNA molecule:

A major groove and a minor groove winding along the molecule parallel to the phosphodiester backbones is present in the DNA . In these grooves, proteins can interact specifically with exposed atoms of the nucleotides (via specific hydrophobic and ionic interactions) thereby recognizing and binding to specific nucleotide sequences without disrupting the base pairing of the double-helical DNA molecule. These are regulatory proteins control the expression of specific genes via such interactions.


Supercoiled DNA
In some organisms such as bacteria, bacteriophages, many DNA-containing animal viruses, as well as organelles such as mitochondria , the ends of the DNA molecules are joined to create a closed circle with no covalently free ends. This of course does not destroy the polarity of the molecules, but it eliminates all free 3' and 5' hydroxyl and phosphoryl groups. Closed circles exist in relaxed or supercoiled forms. Supercoils are introduced when a closed circle is twisted around its own axis or when a linear piece of duplex DNA, whose ends are fixed, is twisted.


Replication & Transcription
The genetic information stored in the nucleotide sequence of DNA serves two purposes. It is the source of information for the synthesis of all protein molecules of the cell and organism, and it provides the information inherited by daughter cells or offspring. Both of these functions require that the DNA molecule serve as a templatein the first case for the transcription of the information into RNA and in the second case for the replication of the information into daughter DNA molecules.
The complementarity of the Watson and Crick double-stranded model of DNA strongly suggests that replication of the DNA molecule occurs in a semiconservative manner. Thus, when each strand of the double-stranded parental DNA molecule separates from its complement during replication, each serves as a template on which a new complementary strand is synthesized. The two newly formed double-stranded daughter DNA molecules, each containing one strand (but complementary rather than identical) from the parent double-stranded DNA molecule, are then sorted between the two daughter cells . Each daughter cell contains DNA molecules with information identical to that which the parent possessed; yet in each daughter cell the DNA molecule of the parent cell has been only semiconserved.

The RNA's

Ribonucleic acid (RNA) is a polymer of purine and pyrimidine ribonucleotides linked together by 3',5'-phosphodiester bridges analogous to those in DNA . Although sharing many features with DNA, RNA possesses several specific differences :
(1) In RNA, the sugar moiety to which the phosphates and purine and pyrimidine bases are attached is ribose rather than the 2'-deoxyribose of DNA.
(2) The pyrimidine components of RNA differ from those of DNA. Although RNA contains the ribonucleotides of adenine, guanine, and cytosine, it does not possess thymine except in the rare case mentioned below. Instead of thymine, RNA contains the ribonucleotide of uracil.
(3) RNA exists as a single strand, whereas DNA exists as a double-stranded helical molecule. However, given the proper complementary base sequence with opposite polarity, the single strand of RNA is capable of folding back on itself and thus acquiring double-stranded characteristics.
(4) Since the RNA molecule is a single strand complementary to only one of the two strands of a gene, its guanine content does not necessarily equal its cytosine content, nor does its adenine content necessarily equal its uracil content.
(5) RNA can be hydrolyzed by alkali to 2',3' cyclic diesters of the mononucleotides, compounds that cannot be formed from alkali-treated DNA because of the absence of a 2'-hydroxyl group. The alkali lability of RNA is useful both diagnostically and analytically.
Information within the single strand of RNA is contained in its sequence ("primary structure") of purine and pyrimidine nucleotides within the polymer. The sequence is complementary to the template strand of the gene from which it was transcribed. Because of this complementarity, an RNA molecule can bind specifically via the base-pairing rules to its template DNA strand; it will not bind ("hybridize") with the other (coding) strand of its gene. The sequence of the RNA molecule (except for U replacing T) is the same as that of the coding strand of the gene.


Types and functions of RNA's :
Those cytoplasmic RNA molecules that serve as templates for protein synthesis (ie, that transfer genetic information from DNA to the protein-synthesizing machinery) are designated messenger RNAs, or mRNAs. Many other cytoplasmic RNA molecules (ribosomal RNAs; rRNAs) have structural roles wherein they contribute to the formation and function of ribosomes (the organellar machinery for protein synthesis) or serve as adapter molecules (transfer RNAs; tRNAs) for the translation of RNA information into specific sequences of polymerized amino acids.
Much of the RNA synthesized from DNA templates in eukaryotic cells, including mammalian cells, is degraded within the nucleus, and it never serves as either a structural or an informational entity within the cellular cytoplasm.
In all eukaryotic cells there are small nuclear RNA (snRNA) species that are not directly involved in protein synthesis but play important roles in RNA processing. These relatively small molecules vary in size from 90 to about 300 nucleotides.
The genetic material for some animal and plant viruses is RNA rather than DNA. Although some RNA viruses never have their information transcribed into a DNA molecule, many animal RNA virusesspecifically, the retroviruses (the HIV virus, for example)are transcribed by an RNA-dependent DNA polymerase, the so-called reverse transcriptase, to produce a double-stranded DNA copy of their RNA genome. In many cases, the resulting double-stranded DNA transcript is integrated into the host genome and subsequently serves as a template for gene expression and from which new viral RNA genomes can be transcribed.
Messenger RNA
In all prokaryotic and eukaryotic organisms, three main classes of RNA molecules exist: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). Each differs from the others by size, function, and general stability.
mRNA class is the most heterogeneous in size and stability. All members of the class function as messengers conveying the information in a gene to the protein-synthesizing machinery, where each serves as a template on which a specific sequence of amino acids is polymerized to form a specific protein molecule, the ultimate gene product.

Messenger RNAs, particularly in eukaryotes, have some unique chemical characteristics. The 5' terminal of mRNA is "capped" by a 7-methylguanosine triphosphate that is linked to an adjacent 2'-O-methyl ribonucleoside at its 5'-hydroxyl through the three phosphates. The mRNA molecules frequently contain internal 6-methyladenylates and other 2'-O-ribose methylated nucleotides. The cap is involved in the recognition of mRNA by the translation machinery . The protein-synthesizing machinery begins translating the mRNA into proteins beginning downstream of the 5' or capped terminal. The other end of most mRNA molecules, the 3'-hydroxyl terminal, has an attached polymer of adenylate residues 20250 nucleotides in length. The specific function of the poly(A) "tail" at the 3'-hydroxyl terminal of mRNAs is not fully understood, but it seems that it maintains the intracellular stability of the specific mRNA by preventing the attack of 3'-exonucleases. Some mRNAs, including those for some histones, do not contain poly(A).


In mammalian cells, including cells of humans, the mRNA molecules present in the cytoplasm are not the RNA products immediately synthesized from the DNA template but must be formed by processing from a precursor molecule before entering the cytoplasm. Thus, in mammalian nuclei, the immediate products of gene transcription constitute a fourth class of RNA molecules. These nuclear RNA molecules, are very heterogeneous in size and are quite large. The heterogeneous nuclear RNA (hnRNA) molecules may have a molecular weight in excess of 107, whereas the molecular weight of mRNA molecules is generally less than 2 x 106. These hnRNA molecules are processed to generate the mRNA molecules which then enter the cytoplasm to serve as templates for protein synthesis.



Transfer RNA (tRNA)
tRNA molecules vary in length from 74 to 95 nucleotides. They also are generated by nuclear processing of a precursor molecule . The tRNA molecules serve as adapters for the translation of the information in the sequence of nucleotides of the mRNA into specific amino acids. There are at least 20 species of tRNA molecules in every cell, at least one (and often several) corresponding to each of the 20 amino acids required for protein synthesis. Although each specific tRNA differs from the others in its sequence of nucleotides, the tRNA molecules as a class have many features in common. The primary structureie, the nucleotide sequenceof all tRNA molecules allows extensive folding and intrastrand complementarity to generate a secondary structure that appears in two dimensions like a cloverleaf .


All tRNA molecules contain four main arms. The acceptor arm terminates in the nucleotides CpCpAOH. These three nucleotides are added posttranscriptionally by a specific nucleotidyl transferase enzyme. The tRNA-appropriate amino acid is attached, or "charged" onto, the 3'-OH group of the A moiety of the acceptor arm. The D, TψC, and extra arms help define a specific tRNA.

Ribosomal RNA (rRNA)

A ribosome is a cytoplasmic nucleoprotein structure that acts as the machinery for the synthesis of proteins from the mRNA templates. On the ribosomes, the mRNA and tRNA molecules interact to translate into a specific protein molecule information transcribed from the gene. In active protein synthesis, many ribosomes are associated with an mRNA molecule in an assembly called the polysome.
The components of the mammalian ribosome, which has a molecular weight of about 4.2 x 106 and a sedimentation velocity of 80S (Svedberg units),. The mammalian ribosome contains two major nucleoprotein subunitsa larger one with a molecular weight of 2.8 x 106 (60S) and a smaller subunit with a molecular weight of 1.4 x 106 (40S). The 60S subunit contains a 5S ribosomal RNA (rRNA), a 5.8S rRNA, and a 28S rRNA; there are also probably more than 50 specific polypeptides. The 40S subunit is smaller and contains a single 18S rRNA and approximately 30 distinct polypeptide chains.

Small RNA

A large number of small stable RNA species are found in eukaryotic cells. Most of these molecules are complexed with proteins to form ribonucleoproteins and are distributed in the nucleus, in the cytoplasm, or in both. They range in size from 20 to 300 nucleotides and are present in 100,0001,000,000 copies per cell.
1. Small Nuclear RNAs (snRNAs). snRNAs, a subset of the small RNAs, are significantly involved in mRNA processing and gene regulation. Of the several snRNAs, U1, U2, U4, U5, and U6 are involved in intron removal and the processing of hnRNA into mRNA. The U7 snRNA is involved in production of the correct 3' ends of histone mRNAwhich lacks a poly(A) tail.
2. Micro RNAs, miRNAs, and Small Interfering RNAs, siRNAs. These two classes of RNAs represent a subset of small RNAs; both play important roles in gene regulation. Presently, all known miRNAs and siRNAs cause inhibition of gene expression by decreasing specific protein production apparently via distinct mechanisms. miRNAs are typically 2125 nucleotides in length and are generated by nucleolytic processing of the products of distinct genes/transcription units .

Nucleic acid Metabolism

Nucleases are enzymes capable of degrading nucleic acids . These nucleases can be classified in several ways. Those which exhibit specificity for deoxyribonucleic acid are referred to as deoxyribonucleases. Those which specifically hydrolyze ribonucleic acids are ribonucleases. Within both of these classes are enzymes capable of cleaving internal phosphodiester bonds to produce either 3'-hydroxyl and 5'-phosphoryl terminals or 5'-hydroxyl and 3'-phosphoryl terminals. These are referred to as endonucleases. Some are capable of hydrolyzing both strands of a double-stranded molecule, whereas others can only cleave single strands of nucleic acids. Some nucleases can hydrolyze only unpaired single strands, while others are capable of hydrolyzing single strands participating in the formation of a double-stranded molecule. There exist classes of endonucleases that recognize specific sequences in DNA; the majority of these are the restriction endonucleases, which have in recent years become important tools in molecular genetics and medical sciences. Some nucleases are capable of hydrolyzing a nucleotide only when it is present at a terminal of a molecule; these are referred to as exonucleases. Exonucleases act in one direction (3' →5' or 5'→3') only.
Metabolism of Nucleotides :
Human tissues can synthesize purines and pyrimidines from amphibolic intermediates. Ingested nucleic acids and nucleotides, which therefore are dietarily nonessential, are degraded in the intestinal tract to mononucleotides, which may be absorbed or converted to purine and pyrimidine bases. The purine bases are then oxidized to uric acid, which may be absorbed and excreted in the urine. While little or no dietary purine or pyrimidine is incorporated into tissue nucleic acids.

Biosynthesis of Purine Nucleotides

Purine and pyrimidine nucleotides are synthesized in vivo at rates consistent with physiologic need. Intracellular mechanisms sense regulate the pool sizes of nucleotide triphosphates (NTPs), which rise during growth or tissue regeneration when cells are rapidly dividing.


Nucleotides biosynthesis :
Three processes contribute to purine nucleotide biosynthesis. These are, in order of decreasing importance: (1) synthesis from amphibolic intermediates (synthesis de novo), (2) phosphoribosylation of purines, and (3) phosphorylation of purine nucleosides.The carbons added in at positions 2 and 8 are contributed by derivatives of tetrahydrofolate. Purine deficiency states, while rare in humans, generally reflect a deficiency of folic acid. Compounds that inhibit formation of tetrahydrofolates and therefore block purine synthesis have been used in cancer chemotherapy.
The Salvage Reactions :
Conversion of purines, their ribonucleosides, and their deoxyribonucleosides to mononucleotides involves so-called "salvage reactions" that require far less energy than de novo synthesis. The more important mechanism involves phosphoribosylation by PRPP of a free purine (Pu) to form a purine 5'-mononucleotide (Pu-RP).







A second salvage mechanism involves phosphoryl transfer from ATP to a purine ribonucleoside (PuR):


Liver, the major site of purine nucleotide biosynthesis, provides purines and purine nucleosides for salvage and for utilization by tissues incapable of their biosynthesis. For example, human brain has a low level of PRPP glutamyl amidotransferase (involved in the biosynthesis of PRPP) and hence depends in part on exogenous purines.
Biosynthesis of Pyrimidine Nucleotides :
Pyrimidine are initially synthesized from :
CO2 , Glutamic acid , Aspartic acid and ATP and several enzymes in the cytosol .One of the reactions involve the utilization of tetrahydrofolate derivative . The methylene group of N5,N10-methylene-tetrahydrofolate is reduced to the methyl group that is transferred, and tetrahydrofolate is oxidized to dihydrofolate. For further pyrimidine synthesis to occur, dihydrofolate must be reduced back to tetrahydrofolate, a reaction catalyzed by dihydrofolate reductase. Dividing cells are especially sensitive to inhibitors of dihydrofolate reductase such as the anticancer drug methotrexate.
Catabolism of Purines :
Humans convert adenosine and guanosine to uric acid . Adenosine is first converted to inosine by adenosine deaminase. In mammals other than higher primates, uricase converts uric acid to the water-soluble product allantoin. However, since humans lack uricase, the end product of purine catabolism in humans is uric acid. Birds also lack uricase and excrete uric acid as end product. At physiological PH, some uric acid combine with Na forming mono Na-Urate salt which is more soluble than uric acid .At PH5 Urate are ten times less soluble than at PH 7 , therefore , alkalinazation of urine causes more urate to dissolve and prevent crystalization (and subsequent stone formation) of urate.

Metabolic Disorder of Purine Catabolism

While purine deficiency states are rare in human subjects, there are numerous genetic disorders of purine catabolism. Hyperuricemias may be differentiated based on whether patients excrete normal or excessive quantities of total urates. Some hyperuricemias reflect specific enzyme defects. Others are secondary to diseases such as cancer or psoriasis that enhance tissue turnover.
Gout :
Various genetic defects in PRPP synthetase present clinically as gout. Each defect results in overproduction and overexcretion of purine catabolites. When serum urate levels exceed the solubility limit, sodium urate crystalizes in soft tissues and joints and causes an inflammatory reaction, gouty arthritis. However, most cases of gout reflect abnormalities in renal handling of uric acid.
Lesch-Nyhan Syndrome
Lesch-Nyhan syndrome, an overproduction hyperuricemia characterized by frequent episodes of uric acid lithiasis and a bizarre syndrome of self-mutilation, reflects a defect in hypoxanthine-guanine phosphoribosyl transferase, an enzyme of purine salvage reaction. The accompanying rise in intracellular PRPP results in purine overproduction. Mutations that decrease or abolish hypoxanthine-guanine phosphoribosyltransferase activity include deletions, frameshift mutations, base substitutions, and aberrant mRNA splicing.
Von Gierke's Disease
Purine overproduction and hyperuricemia in von Gierke's disease (glucose-6-phosphatase deficiency) occurs secondary to enhanced generation of the PRPP precursor ribose 5-phosphate. An associated lactic acidosis elevates the renal threshold for urate, elevating total body urates.
Hypouricemia
Hypouricemia and increased excretion of hypoxanthine and xanthine are associated with xanthine oxidase deficiency due to a genetic defect or to severe liver damage. Patients with a severe enzyme deficiency may exhibit xanthinuria and xanthine lithiasis.
Catabolism of Pyrimidines :
The end products of pyrimidine catabolism are highly water-soluble: CO2, NH3, β -alanine, and β-aminoisobutyrate . Excretion of β -aminoisobutyrate increases in leukemia and severe x-ray radiation exposure due to increased destruction of DNA. However, many persons of Chinese or Japanese ancestry routinely excrete B-aminoisobutyrate. Humans probably transaminate B-aminoisobutyrate to methylmalonate semialdehyde, which then forms succinyl-CoA .


DNA Organization , Replication and Repair
Chromatin is the chromosomal material extracted from nuclei of cells of eukaryotic organisms. Chromatin consists of very long double-stranded DNA molecules and a nearly equal mass of rather small basic proteins termed histones as well as a smaller amount of nonhistone proteins (most of which are acidic and larger than histones) and a small quantity of RNA. The nonhistone proteins include enzymes involved in DNA replication and proteins involved in transcription, such as the RNA polymerase complex. The double-stranded DNA helix in each chromosome has a length that is thousands of times the diameter of the cell nucleus. One purpose of the molecules that comprise chromatin, particularly the histones, is to condense the DNA. Electron microscopic studies of chromatin have demonstrated dense spherical particles called nucleosomes, which are approximately 10 nm in diameter and connected by DNA filaments (see figure below). Nucleosomes are composed of DNA wound around a collection of histone molecules.




Histones :
The histones are a small family of closely related basic proteins. The super-packing of nucleosomes in nuclei is seemingly dependent upon the interaction of the H1 histones with adjacent nucleosomes H1 histones are the ones least tightly bound to chromatin and are, therefore, easily removed with a salt solution, after which chromatin becomes soluble. The organizational unit of this soluble chromatin is the nucleosome. Nucleosomes contain four types of histones: H2A, H2B, H3, and H4. The structures of all four histonesH2A, H2B, H3, and H4, the so-called core histones forming the nucleosomehave been highly conserved between species. This extreme conservation implies that the function of histones is identical in all eukaryotes and that the entire molecule is involved quite specifically in carrying out this function. The histones interact with each other in very specific ways. H3 and H4 form a tetramer containing two molecules of each (H3/H4)2, while H2A and H2B form dimers (H2A-H2B). Under physiologic conditions, these histone oligomers associate to form the histone octamer of the composition (H3/H4)2-(H2A-H2B)2.
The Microstructure of Chromatin :

The DNA organization :

At metaphase, mammalian chromosomes possess a twofold symmetry, with the identical duplicated sister chromatids connected at a centromere, the relative position of which is characteristic for a given chromosome . The centromere is an adenine-thymine (AT) rich region ranging in size from 102 (brewers' yeast) to 106 (mammals) base pairs. It binds several proteins with high affinity. This complex, called the kinetochore, . It is an essential structure for chromosomal segregation during mitosis.


The ends of each chromosome contain structures called telomeres. Telomeres consist of short, repeat TG-rich sequences. Human telomeres have a variable number of repeats of the sequence 5'-TTAGGG-3', which can extend for several kilobases. Telomerase, a multisubunit RNA-containing complex related to viral RNA-dependent DNA polymerases (reverse transcriptases), is the enzyme responsible for telomere synthesis and thus for maintaining the length of the telomere. Since telomere shortening has been associated with both malignant transformation and aging, telomerase has become an attractive target for cancer chemotherapy and drug development. Each sister chromatid contains one double-stranded DNA molecule.

The Coding Regions and Intervening Sequences :

The protein coding regions of DNA, the transcripts of which ultimately appear in the cytoplasm as single mRNA molecules, are usually interrupted in the eukaryotic genome by large intervening sequences of nonprotein coding DNA. Accordingly, the primary transcripts of DNA (mRNA precursors, originally termed hnRNA because this species of RNA was quite heterogeneous in size [length] and mostly restricted to the nucleus), contain noncoding intervening sequences of RNA that must be removed in a process which also joins together the appropriate coding segments to form the mature mRNA. Most coding sequences for a single mRNA are interrupted in the genome (and thus in the primary transcript) by at least oneand in some cases as many as 50noncoding intervening sequences (introns). In most cases, the introns are much longer than the continuous coding regions (exons). The processing of the primary transcript, which involves removal of introns and splicing of adjacent exons, is shown below. The function of the intervening sequences, or introns, is not clear. They may serve to separate functional domains (exons) of coding information in a form that permits genetic rearrangement by recombination to occur more rapidly than if all coding regions for a given genetic function were continuous. The relationships among chromosomal DNA, gene clusters on the chromosome, the exon-intron structure of genes, and the final mRNA product are illustrated in the following figure :


The relationship between chromosomal DNA and mRNA.
The human haploid DNA complement of 3 x 109 base pairs (bp) is distributed between 23 chromosomes. Genes are clustered on these chromosomes. An average gene is 2 x 104 bp in length, including the regulatory region (hatched area), which is usually located at the 5' end of the gene. The regulatory region is shown here as being adjacent to the transcription initiation site (arrow). Most eukaryotic genes have alternating exons and introns. In this example, there are nine exons (dark colored areas) and eight introns (light colored areas). The introns are removed from the primary transcript by the processing reaction, and the exons are ligated together in sequence to form the mature mRNA. (nt, nucleotides.)


The DNA in a eukaryotic genome can be divided into different "sequence classes." These are nonrepetitive- sequence DNA and repetitive-sequence DNA. In the haploid genome, nonrepetitive- sequence generally includes the single copy genes that code for proteins. The repetitive DNA includes sequences that vary in copy number from 2 to as many as 107 copies per cell. It is estimated that more than half the DNA in eukaryotic organisms is in unique or nonrepetitive sequences.In human DNA at least 30% of the genome consists of repetitive sequences. These sequences are usually clustered in centromeres and telomeres of the chromosome and are present in about 110 million copies per haploid genome. These sequences are transcriptionally inactive and may play a structural role in the chromosome .
Mitochondrial DNA:
The majority of the peptides in mitochondria (about 54 out of 67) are coded by nuclear genes. The rest are coded by genes found in mitochondrial (mt) DNA. Human mitochondria contain two to ten copies of a small circular double-stranded DNA molecule that makes up approximately 1% of total cellular DNA. This mtDNA codes for mt ribosomal and transfer RNAs and for 13 proteins that play key roles in the respiratory chain. An important feature of human mitochondrial mtDNA is thatbecause all mitochondria are contributed by the ovum during zygote formationit is transmitted by maternal nonmendelian inheritance. Thus, in diseases resulting from mutations of mtDNA, an affected mother would in theory pass the disease to all of her children but only her daughters would transmit the trait. However, in some cases, deletions in mtDNA occur during oogenesis and thus are not inherited from the mother. A number of diseases have now been shown to be due to mutations of mtDNA. These include a variety of myopathies, neurologic disorders, and some cases of diabetes mellitus.
Mutations
An alteration in the sequence of purine and pyrimidine bases in a gene due to a changea removal or an insertionof one or more bases may result in an altered gene product. Such alteration in the genetic material results in a mutation. Genetic information can be exchanged between similar or homologous chromosomes. The exchange or recombination event occurs primarily during meiosis in mammalian cells and requires alignment of homologous metaphase chromosomes, an alignment that almost always occurs with great exactness. A process of crossing over occurs as shown below. This usually results in an equal and reciprocal exchange of genetic information between homologous chromosomes.


Recombinantion of DNA :
Some bacterial viruses (bacteriophages) are capable of recombining with the DNA of a bacterial host in such a way that the genetic information of the bacteriophage is incorporated in a linear fashion into the genetic information of the host. This integration, which is a form of recombination, occurs by the mechanism shown below. The backbone of the circular bacteriophage genome is broken, as is that of the DNA molecule of the host; the appropriate ends are resealed with the proper polarity. The bacteriophage DNA is figuratively straightened out ("linearized") as it is integrated into the bacterial DNA moleculefrequently a closed circle as well. The site at which the bacteriophage genome integrates or recombines with the bacterial genome is chosen by one of two mechanisms. If the bacteriophage contains a DNA sequence homologous to a sequence in the host DNA molecule, then a recombination event analogous to that occurring between homologous chromosomes can occur. However, some bacteriophages synthesize proteins that bind specific sites on bacterial chromosomes to a nonhomologous site characteristic of the bacteriophage DNA molecule. Integration occurs at the site and is said to be "site-specific."

The integration of a circular genome from a virus (with genes A, B, and C) into the DNA molecule of a host (with genes 1 and 2) and the consequent ordering of the genes.

DNA Synthesis & Replication

Since the primary function of DNA replication is understood to provide the future generation with the genetic information possessed by the parent. Thus, the replication of DNA must be complete and carried out in such a way as to maintain genetic stability within the organism and the species.
In all cells, replication can occur only from a single-stranded DNA (ssDNA) template. Mechanisms must exist to target the site of initiation of replication and to unwind the double-stranded DNA (dsDNA) in that region. The replication complex must then form. After replication is complete in an area, the parent and daughter strands must re-form dsDNA. In eukaryotic cells, an additional step must occur. The dsDNA must precisely re-form the chromatin structure, including nucleosomes, that existed prior to the onset of replication. The major steps are listed in the following table , illustrated in the figure , and discussed later. A number of proteins, most with specific enzymatic action, are involved in this process (see table ).
ProteinFunctionDNA polymerasesDeoxynucleotide polymerizationHelicasesProcessive unwinding of DNATopoisomerasesRelieve torsional strain that results from helicase-induced unwindingDNA primaseInitiates synthesis of RNA primersSingle-strand binding proteinsPrevent premature reannealing of dsDNADNA ligaseSeals the single strand nick between the nascent chain and Okazaki fragments on lagging strandSteps Involved in DNA Replication in Eukaryotes.

1. Identification of the origins of replication

2. Unwinding (denaturation) of dsDNA to provide an ssDNA template


3. Formation of the replication fork

4. Initiation of DNA synthesis and elongation

5. Formation of replication bubbles with ligation of the newly synthesized DNA segments

6. Reconstitution of chromatin structure





This figure describes DNA replication in an E coli cell, but the general steps are similar in eukaryotes. A specific interaction of a protein (the O protein) to the origin of replication (ori) results in local unwinding of DNA at an adjacent A+T-rich region. The DNA in this area is maintained in the single-strand conformation (ssDNA) by single-strand-binding proteins (SSBs). This allows a variety of proteins, including helicase, primase, and DNA polymerase, to bind and to initiate DNA synthesis. The replication fork proceeds as DNA synthesis occurs continuously (long arrow) on the leading strand and discontinuously (short arrows) on the lagging strand. The nascent DNA is always synthesized in the 5' to 3' direction, as DNA polymerases can add a nucleotide only to the 3' end of a DNA strand.


The generation of "replication bubbles" during the process of DNA synthesis. The bidirectional replication and the proposed positions of unwinding proteins at the replication forks are shown.
The Origin of Replication At the origin of replication (ori), there is an association of sequence-specific dsDNA-binding proteins with a series of direct repeated DNA sequences(A-T ).
Unwinding of DNA The interaction of proteins with ori defines the start site of replication and provides a short region of ssDNA essential for initiation of synthesis of the nascent DNA strand. This process requires the formation of a number of protein-protein and protein-DNA interactions. A critical step is provided by a DNA helicase that allows for processive unwinding of DNA. In cooperation with SSB, this leads to DNA unwinding and active replication.
Formation of the Replication Fork A replication fork consists of four components that form in the following sequence: (1) the DNA helicase unwinds a short segment of the parental duplex DNA; (2) a primase initiates synthesis of an RNA molecule that is essential for priming DNA synthesis; (3) the DNA polymerase initiates nascent, daughter strand synthesis; and (4) SSBs bind to ssDNA and prevent premature reannealing of ssDNA to dsDNA. The polymerase III holoenzyme binds to template DNA as part of a multiprotein complex that consists of several polymerase accessory factors. DNA polymerases only synthesize DNA in the 5' to 3' direction, and only one of the several different types of polymerases is involved at the replication fork. Because the DNA strands are antiparallel , the polymerase functions asymmetrically. On the leading (forward) strand, the DNA is synthesized continuously. On the lagging (retrograde) strand, the DNA is synthesized in short (15 kb; see Figure ) fragments, the so-called Okazaki fragments. Several Okazaki fragments (up to 250) must be sequentially synthesized for each replication fork. To ensure that this happens, the helicase acts on the lagging strand to unwind dsDNA in a 5' to 3' direction. The helicase associates with the primase to afford the latter proper access to the template. This allows the RNA primer to be made and, in turn, the polymerase to begin replicating the DNA. This is an important reaction sequence since DNA polymerases cannot initiate DNA synthesis de novo. The mobile complex between helicase and primase has been called a primosome. As the synthesis of an Okazaki fragment is completed and the polymerase is released, a new primer has been synthesized. The same polymerase molecule remains associated with the replication fork and proceeds to synthesize the next Okazaki fragment.
The DNA Polymerase Complex A number of different DNA polymerase molecules engage in DNA replication. These share three important properties: (1) chain elongation, (2) processivity, and (3) proofreading. Chain elongation accounts for the rate (in nucleotides per second) at which polymerization occurs. Processivity is an expression of the number of nucleotides added to the nascent chain before the polymerase disengages from the template. The proofreading function identifies copying errors and corrects them. polymerase III (pol III) functions at the replication fork. Of all polymerases, it catalyzes the highest rate of chain elongation and is the most processive. It is capable of polymerizing 0.5 Mb of DNA during one cycle on the leading strand.Polymerase II (pol II) is mostly involved in proofreading and DNA repair. Polymerase I (pol I) completes chain synthesis between Okazaki fragments on the lagging strand. Eukaryotic cells have counterparts for each of these enzymes plus some additional ones.

Initiation & Elongation of DNA Synthesis

The initiation of DNA synthesis requires priming by a short length of RNA, about 10–200 nucleotides long. This priming process involves the nucleophilic attack by the 3'-hydroxyl group of the RNA primer on the α phosphate of the first entering deoxynucleoside triphosphate with the splitting off of pyrophosphate. The 3'-hydroxyl group of the recently attached deoxyribonucleoside monophosphate is then free to carry out a nucleophilic attack on the next entering deoxyribonucleoside triphosphate (N + 1 in the figure), again at its α phosphate moiety, with the splitting off of pyrophosphate. Of course, selection of the proper deoxyribonucleotide whose terminal 3'-hydroxyl group is to be attacked is dependent upon proper base pairing with the other strand of the DNA molecule according to the rules proposed originally by Watson and Crick . When an adenine deoxyribonucleoside monophosphoryl moiety is in the template position, a thymidine triphosphate will enter and its α phosphate will be attacked by the 3'-hydroxyl group of the deoxyribonucleoside monophosphoryl most recently added to the polymer. By this stepwise process, the template dictates which deoxyribonucleoside triphosphate is complementary and by hydrogen bonding holds it in place while the 3'-hydroxyl group of the growing strand attacks and incorporates the new nucleotide into the polymer. These segments of DNA attached to an RNA initiator component are the Okazaki fragments. In mammals, after many Okazaki fragments are generated, the replication complex begins to remove the RNA primers, to fill in the gaps left by their removal with the proper base-paired deoxynucleotide, and then to seal the fragments of newly synthesized DNA by enzymes referred to as DNA ligases.
As has already been noted, DNA molecules are double-stranded and the two strands are antiparallel, ie, running in opposite directions. The replication of DNA in prokaryotes and eukaryotes occurs on both strands simultaneously. However, an enzyme capable of polymerizing DNA in the 3' to 5' direction does not exist in any organism, so that both of the newly replicated DNA strands cannot grow in the same direction simultaneously. Nevertheless, the same enzyme does replicate both strands at the same time. The single enzyme replicates one strand ("leading strand") in a continuous manner in the 5' to 3' direction, with the same overall forward direction. It replicates the other strand ("lagging strand") discontinuously while polymerizing the nucleotides in short piecess of 150250 nucleotides, again in the 5' to 3' direction, but at the same time it faces toward the back end of the preceding RNA primer rather than toward the unreplicated portion. This process is called semidiscontinuous DNA synthesis. In the mammalian nuclear genome, most of the RNA primers are eventually removed as part of the replication process, whereas after replication of the mitochondrial genome the small piece of RNA remains as an integral part of the closed circular DNA structure.








.
Formation of Replication Bubbles Replication proceeds from a single ori in the circular bacterial chromosome, composed of roughly 6 x 106 bp of DNA. This process is completed in about 30 minutes, a replication rate of 3 x 105 bp/min. The entire mammalian genome replicates in approximately 9 hours, the average period required for formation of a tetraploid genome from a diploid genome in a replicating cell. If a mammalian genome (3 x 109 bp) replicated at the same rate as bacteria (ie, 3 x 105 bp/min) from but a single ori, replication would take over 150 hours! Metazoan organisms get around this problem using two strategies. First, replication is bidirectional. Second, replication proceeds from multiple origins in each chromosome (a total of as many as 100 in humans). Thus, replication occurs in both directions along all of the chromosomes, and both strands are replicated simultaneously. This replication process generates "replication bubbles".
Reconstitution of Chromatin Structure There is evidence that nuclear organization and chromatin structure are involved in determining the regulation and initiation of DNA synthesis. As noted above, the rate of polymerization in eukaryotic cells, which have chromatin and nucleosomes, is tenfold slower than that in prokaryotic cells, which have naked DNA. It is also clear that chromatin structure must be re-formed after replication. Newly replicated DNA is rapidly assembled into nucleosomes, and the preexisting and newly assembled histone octamers are randomly distributed to each arm of the replication fork.
Damaged DNA The maintenance of the integrity of the information in DNA molecules is of utmost importance to the survival of a particular organism as well as to survival of the species. Thus, it can be concluded that surviving species have evolved mechanisms for repairing DNA damage occurring as a result of either replication errors or environmental insults. Replication errors, even with a very efficient repair system, lead to the accumulation of mutations. Fortunately, most of these will probably occur in DNA that does not encode proteins or will not affect the function of encoded proteins and so are of no consequence. In addition, spontaneous and chemically induced damage to DNA must be repaired. Damage to DNA by environmental, physical, and chemical agents may be classified into four types (see table below). Abnormal regions of DNA, either from copying errors or DNA damage, are replaced by four mechanisms: (1) mismatch repair, (2) base excision-repair, (3) nucleotide excision-repair, and (4) double-strand break repair . The defective region in one strand can be returned to its original form by relying on the complementary information stored in the unaffected strand.
Mismatch Repair Mismatch repair corrects errors made when DNA is copied. For example, a C could be inserted opposite an A. Specific proteins scan the newly synthesized DNA, using adenine methylation . The template strand is methylated, and the newly synthesized strand is not. This difference allows the repair enzymes to identify the strand that contains the error nucleotide which requires replacement. If a mismatch is found an endonuclease cuts the strand bearing the mutation . An exonuclease then digests this strand through the mutation, thus removing the faulty DNA. This defect is then filled in by normal cellular enzymes according to base pairing rules. Other cellular enzymes, including ligase, polymerase, and SSBs, remove and replace the strand. Faulty mismatch repair has been linked to hereditary nonpolyposis colon cancer (HNPCC), one of the most common inherited cancers.

Table 358. Types of Damage to DNA.

I. Single-base alteration

A. Depurination

B. Deamination of cytosine to uracil

C. Deamination of adenine to hypoxanthine

D. Alkylation of base


E. Insertion or deletion of nucleotide

F. Base-analog incorporation

II. Two-base alteration

A. UV lightinduced thymine-thymine (pyrimidine) dimer

B. Bifunctional alkylating agent cross-linkage

III. Chain breaks

A. Ionizing radiation

B. Radioactive disintegration of backbone element

C. Oxidative free radical formation

IV. Cross-linkage

A. Between bases in same or opposite strands


B. Between DNA and protein molecules (eg, histones)

Base Excision-Repair The depurination of DNA, which happens spontaneously owing to the thermal lability of the purine N-glycosidic bond, occurs at a rate of 500010,000/cell/d at 37 C. Specific enzymes recognize a depurinated site and replace the appropriate purine directly, without interruption of the phosphodiester backbone.Cytosine, adenine, and guanine bases in DNA spontaneously form uracil, hypoxanthine, or xanthine, respectively. Specific N-glycosylases can recognize these abnormal bases and remove the base itself from the DNA.
Nucleotide Excision-Repair This mechanism is used to replace regions of damaged DNA up to 30 bases in length. Common causes of such DNA damage include ultraviolet (UV) light, and smoking, which causes formation of benzo[a]pyrene-guanine adducts. Ionizing radiation, cancer chemotherapeutic agents, and a variety of chemicals found in the environment cause base modification, strand breaks, cross-linkage between bases on opposite strands or between DNA and protein, and numerous other defects. These are repaired by a process called nucleotide excision-repair . Xeroderma pigmentosum (XP) is an autosomal recessive genetic disease. The clinical syndrome includes marked sensitivity to sunlight (ultraviolet) with subsequent formation of multiple skin cancers and premature death. The inherited defect seems to involve the repair of damaged DNA, particularly thymine dimers. Cells cultured from patients with xeroderma pigmentosum exhibit low activity for the nucleotide excision-repair process.
Double-Strand Break Repair The repair of double-strand breaks is part of the physiologic process of immunoglobulin gene rearrangement. It is also an important mechanism for repairing damaged DNA, such as occurs as a result of ionizing radiation or oxidative free radical generation. Some chemotherapeutic agents destroy cells by causing ds breaks or preventing their repair.

Protein Biosynthesis

The genetic information within the nucleotide sequence of DNA is transcribed in the nucleus into the specific nucleotide sequence of an RNA molecule. The sequence of nucleotides in the RNA transcript is complementary to the nucleotide sequence of the template strand of its gene in accordance with the base-pairing rules. Several different classes of RNA combine to direct the synthesis of proteins. The primary transcript is much larger than the mature mRNA. The large mRNA precursors contain coding regions (exons) that will form the mature mRNA and long intervening sequences (introns) that separate the exons. The mRNA is processed within the nucleus, and the introns, which often make up much more of this RNA than the exons, are removed. Exons are spliced together to form mature mRNA, which is transported to the cytoplasm, where it is translated into protein. The nucleotide sequence of an mRNA molecule consists of a series of codons that specify the amino acid sequence of the encoded protein. The amino acid codon Twenty different amino acids are required for the synthesis of the cellular complement of proteins; thus, there must be at least 20 distinct codons that make up the genetic code. Since there are only four different nucleotides in mRNA, each codon must consist of more than a single purine or pyrimidine nucleotide. Codons consisting of two nucleotides each could provide for only 16 (42) specific codons, i.e : the A G C U nucleotides would form only 16 different codons whereas codons of three nucleotides could provide 64 (43) specific codons. ie, it is a triplet code. Three of the 64 possible codons do not code for specific amino acids; these have been termed nonsense codons. These nonsense codons are utilized in the cell as termination signals; they specify where the polymerization of amino acids into a protein molecule is to stop. The remaining 61 codons code for 20 amino acids. Some amino acids are encoded by several codons; for example, six different codons specify serine. Other amino acids, such as methionine and tryptophan, have a single codon. The recognition of specific codons in the mRNA by the tRNA adapter molecules is dependent upon their anticodon region and specific base-pairing rules. Each tRNA molecule contains a specific sequence, complementary to a codon, which is termed its anticodon. For a given codon in the mRNA, only a single species of tRNA molecule possesses the proper anticodon. Since each tRNA molecule can be charged with only one specific amino acid, each codon therefore specifies only one amino acid. With few exceptions, given a specific codon, only a specific amino acid will be incorporated. The main function of the tRNA molecules requires the charging of each specific tRNA with its specific amino acid. At least 20 specific enzymes are required for the proper attachment of the 20 amino acids to specific tRNA molecules. The process of recognition and attachment (charging) proceeds in two steps by one enzyme for each of the 20 amino acids. These enzymes are termed aminoacyl-tRNA synthetases.

The (TψC) arm is involved in binding of the aminoacyl-tRNA to the ribosomal surface at the site of protein synthesis. The D arm is one of the sites important for the proper recognition of a given tRNA species by its proper aminoacyl-tRNA synthetase. The acceptor arm, located at the 3'-hydroxyl adenosyl terminal, is the site of attachment of the specific amino acid.

. The Genetic Code (Codon Assignments in Mammalian Messenger RNA).1

First Nucleotide
Second Nucleotide
Third Nucleotide

U
C
A
G


U
Phe
Ser
Tyr
Cys
U

Phe
Ser
Tyr
Cys
C

Leu
Ser
Term
Term2
A

Leu
Ser
Term
Trp
G


C
Leu
Pro
His
Arg
U

Leu
Pro
His
Arg
C

Leu
Pro
Gln
Arg
A

Leu
Pro
Gln
Arg
G


A
Ile
Thr
Asn
Ser
U

Ile
Thr
Asn
Ser
C

Ile2

Thr
Lys
Arg2
A

Met
Thr
Lys
Arg2
G


G
Val
Ala
Asp
Gly
U

Val
Ala
Asp
Gly
C

Val
Ala
Glu
Gly
A

Val
Ala
Glu
Gly
G




Mutations

Some mutations occur by base substitution : Single-base changes may occur . A given pyrimidine may change to the other pyrimidine or a given purine may change to the other purine. or changes from a purine to either of the two pyrimidines or the change of a pyrimidine into either of the two purines. If the nucleotide sequence of the gene containing the mutation is transcribed into an RNA molecule, then the RNA molecule will possess a complementary base change at this corresponding point. Single-base changes in the mRNA molecules may have one of several effects when translated into protein: (1) There may be no detectable effect ; such mutations are often referred to as silent mutations. This would be more likely if the changed base in the mRNA molecule were to be at the third nucleotide of a codon. Because the translation of a codon is least sensitive to a change at the third position. (2) A missense effect will occur when a different amino acid is incorporated at the corresponding site in the protein molecule. This mistaken amino acidor missense, depending upon its location in the specific proteinmight be acceptable, partially acceptable, or unacceptable to the function of that protein molecule. If an acceptable missense effect occurs, the resulting protein molecule may not be distinguishable from the normal one. A partially acceptable missense will result in a protein molecule with partial but abnormal function. If an unacceptable missense effect occurs, then the protein molecule will not be capable of functioning in its assigned role. (3) A nonsense codon may appear that would then result in the premature termination of amino acid incorporation into a peptide chain and the production of only a fragment of the intended protein molecule. The probability is high that a prematurely terminated protein molecule or peptide fragment will not function in its assigned role. Examples : The lack of effect of a single-base change is demonstrable in the gene system that encodes hemoglobin in humans . The sequencing of a large number of hemoglobin mRNAs and genes from many individuals has shown that the codon for valine at position 67 of the β chain of hemoglobin is not identical in all persons who possess a normally functional β chain of hemoglobin. Hemoglobin Milwaukee has at position 67 a glutamic acid; hemoglobin Bristol contains aspartic acid at position 67. Hemoglobin Sydney contains an alanine at position 67.
Acceptable Missense Mutations An example of an acceptable missense mutation in the structural gene for the β -chain of hemoglobin could be detected by the presence of an electrophoretically altered hemoglobin in the red cells of an apparently healthy individual. Hemoglobin Hikari has been found in at least two families of Japanese people. This hemoglobin has asparagine substituted for lysine at the 61 position in the β chain. .The replacement of the specific lysine with asparagine apparently does not alter the normal function of the β chain in these individuals. A partially acceptable missense mutation is best exemplified by hemoglobin S, which is found in sickle cell anemia. Here glutamic acid, the normal amino acid in position 6 of the β chain, has been replaced by valine..This missense mutation affects normal function and results in sickle cell anemia when the mutant gene is present in the homozygous state. The glutamate-to-valine change may be considered to be partially acceptable because hemoglobin S does bind and release oxygen, although abnormal.
Unacceptable Missense Mutations An unacceptable missense mutation in a hemoglobin gene generates a nonfunctioning hemoglobin molecule. For example, the hemoglobin M mutations generate molecules that allow the Fe2+ of the heme moiety to be oxidized to Fe3+, producing methemoglobin. Methemoglobin cannot transport oxygen .
Protein Synthesis (part 2)
Protein synthesis can be described in three phases: Initiation, Elongation, & Termination Initiation of protein synthesis requires that an mRNA molecule be selected for translation by a ribosome (see figure). Once the mRNA binds to the ribosome, the latter finds the correct reading frame on the mRNA, and translation begins. This process involves tRNA, rRNA, mRNA, and at least ten eukaryotic initiation factors (eIFs), some of which have multiple (three to eight) subunits. Also involved are GTP, ATP, and amino acids. Initiation can be divided into four steps:
(1) dissociation of the ribosome into its 40S and 60S subunits; (2) binding of a ternary complex consisting of met-tRNAi, GTP, and eIF-2 to the 40S ribosome to form a preinitiation complex; (3) binding of mRNA to the 40S preinitiation complex to form a 43S initiation complex; (4) combination of the 43S initiation complex with the 60S ribosomal subunit to form the 80S initiation complex.
The first step in this process involves the binding of GTP by eIF-2. This binary complex then binds to met-tRNAi, a tRNA specifically involved in binding to the initiation codon AUG. (There are two tRNAs for methionine. One specifies methionine for the initiator codon, the other for internal methionines. Each has a unique nucleotide sequence.) This ternary complex binds to the 40S ribosomal subunit to form the 43S preinitiation complex, which is stabilized by association with eIF-3 and eIF-1A.
Elongation Elongation is a cyclic process on the ribosome in which one amino acid at a time is added to the nascent peptide chain (see figure). The peptide sequence is determined by the order of the codons in the mRNA. Elongation involves several steps catalyzed by proteins called elongation factors (EFs). These steps are: (1) binding of aminoacyl-tRNA to the A site, (2) peptide bond formation, and (3) translocation.
Binding In the complete 80S ribosome formed during the process of initiation, the A site (aminoacyl or acceptor site) is free. The binding of the proper aminoacyl-tRNA in the A site requires proper codon recognition. Elongation factor EF1A forms a ternary complex with GTP and the entering aminoacyl-tRNA (see figure ). This complex then allows the aminoacyl-tRNA to enter the A site with the release of EF1A ,GDP and phosphate. GTP hydrolysis is catalyzed by an active site on the ribosome. As shown in the Figure , EF1A-GDP then recycles to EF1A-GTP with the aid of other soluble protein factors and GTP.
Peptide Bond Formation
The α-amino group of the new aminoacyl-tRNA in the A site carries out a nucleophilic attack on the esterified carboxyl group of the peptidyl-tRNA occupying the P site (peptidyl or polypeptide site). At initiation, this site is occupied by aminoacyl-tRNA meti. This reaction is catalyzed by a peptidyltransferase, a component of the 28S RNA of the 60S ribosomal subunit. Because the amino acid on the aminoacyl-tRNA is already "activated," no further energy source is required for this reaction. The reaction results in attachment of the growing peptide chain to the tRNA in the A site.









Translocation
The now deacylated tRNA is attached by its anticodon to the P site at one end and by the open CCA tail to an exit (E) site on the large ribosomal subunit . At this point, elongation factor 2 (EF2) binds to and displaces the peptidyl tRNA from the A site to the P site. In turn, the deacylated tRNA is on the E site, from which it leaves the ribosome. The EF2-GTP complex is hydrolyzed to EF2-GDP, effectively moving the mRNA forward by one codon and leaving the A site open for occupancy by another ternary complex of amino acid tRNA-EF1A-GTP and another cycle of elongation.
Termination Occurs When a Stop Codon Is Recognized
The Polysomes Many ribosomes can translate the same mRNA molecule simultaneously. Because of their relatively large size, the ribosome particles cannot attach to an mRNA any closer than 35 nucleotides apart. Multiple ribosomes on the same mRNA molecule form a polyribosome,
Factors affecting protein synthesis : The machinery of protein synthesis can respond to environmental threats. Ferritin, an iron-binding protein, prevents ionized iron (Fe2+) from reaching toxic levels within cells. Elemental iron stimulates ferritin synthesis by causing the release of a cytoplasmic protein that binds to a specific region in the 5' nontranslated region of ferritin mRNA. Disruption of this protein-mRNA interaction activates ferritin mRNA and results in its translation. This mechanism provides for rapid control of the synthesis of a protein that sequesters Fe2+, a potentially toxic molecule.
Many Viruses Co-Opt the Host Cell Protein Synthesis Machinery
The protein synthesis machinery can also be modified in deleterious ways. Viruses replicate by using host cell processes, including those involved in protein synthesis. (eg, encephalomyocarditis virus
Posttranslational Processing
In animal cells, many proteins are synthesized from the mRNA template as a precursor molecule, which then must be modified to achieve the active protein. The prototype is insulin, which is a low-molecular-weight protein having two polypeptide chains with interchain and intrachain disulfide bridges. The molecule is synthesized as a single chain precursor, or prohormone, which folds to allow the disulfide bridges to form. A specific protease then clips out the segment that connects the two chains which form the functional insulin molecule.


Inhibition of protein synthesis :
Many effective antibiotics interact specifically with the proteins and RNAs of prokaryotic ribosomes and thus inhibit protein synthesis. This results in growth arrest or death of the bacterium. The most useful members of this class of antibiotics (eg, tetracyclines, lincomycin, erythromycin, and chloramphenicol) do not interact with components of eukaryotic ribosomal particles and thus are not toxic to eukaryotes. Tetracycline prevents the binding of aminoacyl-tRNAs to the A site. Chloramphenicol and the macrolide class of antibiotics work by binding to 23S rRNA, Other antibiotics inhibit protein synthesis on all ribosomes (puromycin) or only on those of eukaryotic cells (cycloheximide). Puromycin has a structural similar to tyrosinyl-tRNA..










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