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The nucleotides • BIOMEDICAL IMPORTANCE 1-Serving as precursors of nucleic acids purine and pyrimidine nucleotides 2- When linked to vitamins or vitamin derivatives, nucleotides form a portion of many coenzymes (NAD). 3- nucleoside tri- and diphosphates such as ATP and ADP are the principal players in the energy transductions. 4- The cyclic nucleotides cAMP and cGMP serve as the second messengers in hormonally regulated events. 5- Use of synthetic purine and pyrimidine analogs that contain halogens, thiols, or additional nitrogen atoms in the chemotherapy of cancer and AIDS, and as suppressors of the immune response during organ transplantation. Purines & Pyrimidines :are nitrogen-containing heterocycles, structures that contain, in addition to carbon, other atoms such as nitrogen. The smaller pyrimidine molecule has the longer name and the larger purine molecule the shorter name, and that their six-atom rings are numbered in opposite directions (Figure1). chemistry of purines ,pyrimidines , nucleosides & nucleotides • Both DNA and RNA contain the same purine bases: adenine (A) and guanine (G).and contain the pyrimidine cytosine (C), but they differ in their second pyrimidine base: DNA contains thymine (T), whereas RNA contains uracil (U). • T and U differ by only one methyl group, which is present on T but absent on U Nucleosides Are N -Glycosides Nucleosides are derivatives of purines and pyrimidines that have a sugar linked to a ring nitrogen of a purine or pyrimidine. The sugar in ribonucleosides is D-ribose, and in deoxyribonucleosides is 2-deoxy-D-ribose. Both sugars are linked to the heterocycle by a -N-glycosidic bond, almost always to the N-1 of a pyrimidine or to N-9 of a purine. The ribonucleosides of A, G, C, and U are named adenosine, Guanosine, cytidine, and uridine, respectively. The deoxyribonucleoside of A, G, C, and T have the added prefix, "deoxy-", for example deoxyadenosine . Nucleotides Are Phosphorylated Nucleosides Mononucleotides are nucleosides with a phosphoryl group esterified to a hydroxyl group of the sugar. The 5'-nucleotides are nucleosides with a phosphoryl group on the 5'-hydroxyl group of the sugar (nucleoside 5'phosphate or a 5'-nucleotide). If one phosphate group is attached to the 5'-carbon of the pentose →nucleoside monophosphate (NMP:AMP or CMP). a second or third phosphate is added to the nucleoside, a nucleoside diphosphate (eg. ADP) or triphosphate (eg.ATP) results. The second and third phosphates are each connected to the nucleotide by a "high-energy" bond. [The phosphate groups are responsible for the negative charges associated with nucleotides, and cause DNA and RNA to be referred to as "nucleic acids."] Heterocyclic N -Glycosides Exist as Syn and Anti Conformers • In nucleosides or nucleotides there is no freedom of rotation about the -Nglycosidic bond. Both therefore exist as non interconvertible syn or anti conformers . • syn and anti conformers can only be interconverted by cleavage and reformation of the glycosidic bond. Both syn and anti conformers occur in nature, but the anti conformers predominate. • Modification of Polynucleotides Small quantities of additional purines and pyrimidines occur in DNA and RNAs. Examples include 5-methylcytosine of bacterial and human DNA. 5-hydroxymethylcytosine of bacterial and viral nucleic acids DNA & RNA ARE POLYNUCLEOTIDES The 5'-phosphoryl group of a mononucleotide can esterify a hydroxyl group, forming a phosphodiester. Most commonly, this hydroxyl group is the 3'-OH of the pentose of a second nucleotide. This forms a dinucleotide in which the pentose moieties are linked by a 3',5'phosphodiester bond to form the "backbone" of RNA and DNA. Nucleic acids • Nucleic acids are required for the storage and expression of genetic information. There are two types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid.(RNA) • DNA, the storehouse of genetic information, is present not only in chromosomes in the nucleus of eukaryotic organisms, but also in mitochondria and the chloroplasts of plants. Prokaryotic cells, which lack nuclei, have a single chromosome, but may also contain DNA in the form of plasmids. • The genetic information found in DNA is copied and transmitted to daughter cells through DNA replication. • Transcription (RNA synthesis) is the first stage in the expression of genetic information. • The code contained in the nucleotide sequence of messenger RNA molecules is translated (protein synthesis), thus completing gene expression. • This flow of information from DNA to RNA to protein is termed the "central dogma STRUCTURE OF DNA • DNA is contains many deoxyribonucleotides covalently linked by bonds. With the exception of a few viruses that contain singlestranded DNA, DNA exists as a double-stranded molecule, in which the two strands wind around each other, forming a double helix. • eukaryotic cells, DNA is found associated with various types of proteins (known collectively as nucleoprotein) present in the nucleus, whereas in prokaryotes, the protein-DNA complex is present in the nucleoid. • Phosphodiester bonds join the 5'-hydroxyl group of the deoxypentose of one nucleotide to the 3'-hydroxyl group of the deoxypentose of an adjacent nucleotide through a phosphate group • The resulting long, unbranched chain has polarity, with both a 5'end (free phosphate) and a 3'-end (free hydroxyl) that are not attached to other nucleotides. The bases located along the resulting deoxyribose-phosphate backbone are always written in sequence from the 5'-end of the chain to the 3'-end (5'-TACG-3'). Phosphodiester linkages between nucleotides (in DNA or RNA) can be cleaved hydrolytically by chemicals, or hydrolyzed by a family of nucleases: deoxyribonucleases for DNA and ribonucleases for RNA. [endonucleases: cleave the nucleotide chain at positions in the interior exo nucleases: cleave the chain only by removing individual nucleotides from one of the two ends ] Double helix • the two chains are coiled around a common Axis called the axis of symmetry. The chains are paired in an antiparallel manner, the 5'-end of one strand is paired with the3'-end of the other strand • the hydrophilic deoxyribose- Phosphate backbone of each chain is on the outside of the molecule, whereas the hydro phobic bases are stacked inside. The overall structure resembles a twisted ladder. The spatial relationship between the two strands creates a major and a minor groove . • Certain anticancer drugs, such as dactinomycin (actinomycin D), exert their cytotoxic effect by intercalating into the narrow groove of the DNA double helix, thus interfering with RNA and DNA synthesis.1] Base pairing • Base pairing: The bases of one strand of DNA are paired with the bases of the second strand, so that an adenine is always paired with a thymine and a cytosine is always paired with a guanine. Therefore, one polynucleotide chain of the DNA double helix is always the complement of the other. • The specific base pairing in DNA leads to Chargaff's Rules: In any sample of doublestranded DNA, the amount of adenine equals the amount of thymine, the amount of guanine equals the amount of cytosine, and the total amount of purines equals the total amount of pyrimidines. • The base pairs are held together by hydrogen bonds : two between A and T and three between G and C. These hydrogen bonds, plus the hydrophobic interactions between the stacked bases, stabilize the structure of the double helix. Circular DNA molecules Each chromosome in the nucleus of a eukaryote contains one long linear molecule of double-stranded DNA, which is bound to a complex mixture of proteins to form chromatin. Eukaryotes have also closed circular DNA molecules in their mitochondria, as do plant chloroplasts. A prokaryotic organism contains a single, double-stranded, supercoiled, circular chromosome. In addition, most species of bacteria also contain small, circular, extra chromosomal DNA molecules called plasmids. Quadruple DNA G-quadruplexes (also known as G-tetrads or G4DNA) are nucleic acid sequences that are rich in guanine and are capable of forming a four-stranded structure. Four guanine bases can associate through hydrogen bonding to form a square planar structure called a guanine tetrad, and two or more guanine tetrads can stack on top of each other to form a G-quadruplex. The quadruplex structure is further stabilized by the presence of a cation, especially potassium, which sits in a central channel between each pair of tetrads . • The formation of these quadruplexes in telomeres [consists of many repeats of the sequenced(TTAGGG)]. has been shown to decrease the activity of the enzyme telomerase which is responsible for maintaining length of telomeres and is involved in around 85% of all cancers. This is an active target of drug discovery • DNA Exists in Relaxed & Supercoiled Forms In some organisms such as bacteria, 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. 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. • Negative supercoils the molecule is twisted in the direction opposite from the clockwise turns of the right-handed double helix found in B-DNA. • Enzymes that catalyze topologic changes of DNA are called topoisomerases. Topoisomerases can relax or insert supercoils, using ATP as an energy source. Homologs of this enzymes are important Sense and antisense • A DNA sequence is called "sense" if its sequence is the same as that of a messenger RNA copy that is translated into protein. • The sequence on the opposite strand is called the "antisense" sequence. • Both sense and antisense sequences can exist on different parts of the same strand of DNA . Separation of the two DNA strands in the double helix • The two strands of the double helix separate when hydrogen bonds between the paired bases are disrupted. • Disruption can occur if the pH is altered (nucleotide bases ionize) or if the solution is heated. [Phosphodiester bonds are not broken by such treatment.] • When DNA is heated, the temperature at which one half of the helical structure is lost is defined as the melting temperature. • The loss of helical structure in DNA, called denaturation. • Because there are three hydrogen bonds between G and C but only two between A and T, DNA that contains high concentrations of A and T denatures at a lower temperature than G- and C-rich DNA Under appropriate conditions, • complementary DNA strands can reform the double helix by the process called renaturation (or reannealing). Chromatin is the chromosomal material in the nuclei of eukaryotic • Chromatin consists of very long doublestranded 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 repair, and the proteins involved in RNA synthesis, processing, and transport to the cytoplasm. • Electron microscopic studies of chromatin have demonstrated dense spherical particles called nucleosomes, which are approximately 10 nm in diameter and connected by DNA filaments . • Nucleosomes are composed of DNA wound around a collection of histone molecules chromosomes possess a 2-fold symmetry, with the identical duplicated sister chromatids connected at a centromere. The centromere is an adenine-thymine (A–T)-rich region containing repeated DNA DNA IS ORGANIZED INTO CHROMOSOMES • the kinetochore, provides the anchor for the mitotic spindle. •The ends of each chromosome contain structures called telomeres. Telomeres consist of short TG-rich repeats. • Human telomeres have a variable number of repeats of the sequence 5'-TTAGGG-3. Telomerase, a multisubunit RNA-containing complex , 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. DNA SYNTHESIS & REPLICATION In all cells, replication can occur only from a single-stranded DNA (ssDNA) template. Steps 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; synthesis of RNA primer 4. Initiation of DNA synthesis and elongation 5. Formation of replication bubbles with ligation of the newly synthesized DNA segments A-origin of replication . • prokaryotic organisms, begins at a single, unique nucleotide site . • In eukaryotes, begins at multiple sites along the DNA helix .These sites include a short sequence composed almost exclusively of AT base pairs. • B- Separation (Unwinding )of the two complementary DNA strands In order for the two strands of DNA to be replicated, they must first separate (or "melt"), at least in a small region, because the polymerases use only single-stranded DNA as a template. Proteins required for DNA strand separation : a. dnaA protein: bind to specific nucleotide sequences at the origin of replication. This causes the strands separate, forming single-stranded DNA. b. Single-stranded DNA-binding (SSB) proteins 1. keep the two strands of DNA separated, 2. protect the DNA from nucleases that cleave single-stranded DNA. c. DNA helicases: These enzymes bind to single-stranded DNA near the replication fork, and then move into the neighboring double-stranded region, forcing the strands apart in effect, unwinding the double helix. Formation of the Replication Fork A replication fork consists of: (1) 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. (4) SSBs bind to ssDNA and prevent premature reannealing of ssDNA to dsDNA. The DNA polymerases responsible for copying the DNA templates are only able to "read" the parental nucleotide sequences in the direction 3'-»5, and they synthesize the new DNA strands in the 5'->3' .the two newly synthesized stretches of nucleotide chains must grow in opposite directions—one in the 5'->3' direction toward the replication fork and one in the 5'>3' direction away from the replication fork . 1. Leading strand: The strand that is being copied in the direction of the advancing replication fork and is synthesized almost continuously. 2. Lagging strand: The strand that is being copied in the direction away from the replication fork is synthesized discontinuously, with small fragments of DNA copied near the replication fork. These short stretches of discontinuous DNA, termed Okazaki fragments, are eventually joined to become a single ,continuous strand. The DNA Polymerase Complex A number of different DNA polymerase molecules engage in DNA replication. These share three important properties: 1-chain elongation accounts for the rate (in nucleotides per second; ntd/s) at which polymerization occurs. 2- processivity is an expression of the number of nucleotides added to the nascent chain before the polymerase disengages from the template. 3- proofreading function identifies copying errors and corrects them. Polymerase I (pol I) and II (pol II) are mostly involved in proofreading and DNA repair. Eukaryotic cells have a large number of additional DNA polymerases In mammalian cells, the polymerase is capable of polymerizing at a rate that is somewhat slower than the rate of polymerization of deoxynucleotides by the bacterial DNA polymerase complex. This reduced rate may result from interference by nucleosomes STRUCTURE OF RNA : There are three major types of RNA that participate in the process of protein synthesis: ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA) A. Ribosomal RNA (rRNAs) are found in association with several proteins serve as the sites for protein synthesis . There are three distinct size species of rRNA (23S, 16S, and 5S) in prokaryotic cells • In the eukaryotic cytosol, there are four rRNA size species (28S, 18S, 5.8S, and 5S). Transfer RNAs (tRNAs), the smallest of the three major species of RNA molecules (4S), There is at least one specific type of tRNA molecule for each of the twenty amino acids commonly found in proteins. Each tRNA serves as an "adaptor" molecule that carries its specific amino acid together. Messenger RNA: mRNA carries genetic information from the nuclear DNA to the cytosol, where it is used as the template for protein synthesis. Special structural characteristics of eukaryotic mRNA include a long sequence of adenine nucleotides (a "poly-A tail") on the 3'-end of the RNA chain, plus a'cap" on the 5'-end . RNA IS SYNTHESIZED FROM A DNA TEMPLATE BY AN RNA POLYMERASE The process of RNA synthesis is called transcription, and its substrates are ribonucleoside triphosphates. The enzyme that synthesizes RNA is RNA polymerase The processes of DNA and RNA synthesis are similar in that they involve: (1)general steps of initiation, elongation, and termination with 5' to 3' (2) large, multicomponent initiation complexes. (3)adherence to Watson–Crick base-pairing rules. DNA and RNA synthesis do differ in several important ways, including: (1) ribonucleotides are used in RNA synthesis rather than deoxyribonucleotides. (2) U replaces T as the complementary base for A in RNA. (3) only portions of the genome are vigorously transcribed or copied into RNA, whereas the entire genome must be copied during DNA replication (4) there is no highly active, efficient proofreading function during RNA transcription. The strand that is transcribed or copied into an RNA molecule is referred to as the template strand of the DNA. the non-template strand, is frequently referred to as the coding strand of that gene.The information in the template strand is read out in the 3' to 5' direction. Steps in RNA synthesis: The process of transcription of a typical gene can be divided into three phases: initiation, elongation, and termination. 1. Initiation of transcription: involves the binding of the RNA polymerase to a region on the DNA that determines the specificity of transcription of that particular gene. That DNA sequence is known as the promoter region . many different promoters are recognized by prokaryotic RNA polymerase ,include: a.Pribnow box: This is a stretch of six nucleotides (5'-TATAAT-3') b. -35 sequence: A second consensus nucleotide sequence (5-TTGACA3,), [A mutation in either the Pribnow box or the -3 5 sequence can affect the transcription of the gene controlled by the mutant promoter.] Elongation: the promoter region has been recognized by the holoenzyme. RNA polymerase begins to synthesize a transcript of the DNA sequence (usually beginning with a purine). Unlike DNA polymerase, RNA polymerase does not require a primer and has no known endonuclease or exonuclease activity. It therefore, has no ability to repair mistakes in the RNA. RNA polymerase uses ribonucleoside triphosphates, and releases pyrophosphate each time a nucleotide is added to the growing chain. Termination: The process of elongation of the RNA chain continues until a termination signal is reached. termination requires an additional protein : P-Rho-independent termination , requires that the newly synthesized RNA have two important structural features. 1. the RNA transcript must be able to form a stable hairpin turn . 2. palindrome .[A palindrome is a region of double-stranded DNA in which each of the two strands have the same nucleotide sequence when read in the same (for example, 5'->3' ) beyond the hairpin turn. Some antibiotics prevent bacterial cell growth by inhibiting RNA synthesis. For example, rifampin inhibits the initiation of transcription by binding to the β-subunit of prokaryotic RNA polymerase, thus interfering with the formation of the first phosphodiester bond .Rifampin is useful in the treatment of tuberculosis. • D actinomycin :It binds to the DNA template and interferes with the movement of RNA polymerase along the DNA. TRANSCRIPTION OF EUKARYOTIC GENES Promoters of eukaryotic genes: 1. TATA or Hogness box . 2. CAAT box . 3. GC box (GGGCGG). Nuclear RNA polymerases of eukaryotic cells: There are three distinct classes of RNA polymerase in the nucleus of eukaryotic cells: 1. RNA polymerase I : synthesizes the precursor of the large ribosomal RNAs in the nucleolus. 2. RNA polymerase II: This enzyme synthesizes the precursors of messenger RNAs also synthesizes certain small nuclear RNAs (snRNA Inhibitors of RNA polymerase II: This enzyme is inhibited by αamanitin— potent toxin produced by the poisonous mushroom. Forms a tight complex with the polymerase, thereby inhibiting mRNA synthesis and, ultimately, protein synthesis. 3. RNA polymerase III : produces the small RNAs, including tRNAs, the small 5S ribosomal RNA, and some snRNAs. POSTTRANSCRIPTIONAL MODIFICATION OF RNA Protein Synthesis • The pathway of protein synthesis is called translation because the "language" of the nucleotide sequence on the mRNA is translated into the language of an amino acid sequence. • The process of translation requires a genetic code, through which the information contained in the nucleic acid sequence is expressed to produce a specific sequence of amino acids. • Codons are usually presented in the messenger RNA language of adenine (A), guanine (G), cytosine (C), and uracil (U). Their nucleotide sequences are always written from the 5'-end to the 3'-end. The four nucleotide bases are used to produce the three-base codons. For example, the codon 5-AUG-3' codes for methionine. Sixty-one of the 64 codons code for the twenty common amino acids. Termination ("stop" or "nonsense") codons: Three of the codons, UAG, UGA, and UAA, do not code for amino acids, but rather are termination codons. When one of these codons appears in an mRNA sequence, it signals that synthesis of the peptide chain is completed. Characteristics of the genetic code include the following: 1. Specificity: The genetic codes specific that is, a specific codon always codes for the same amino acid. 2. Universality: the specificity of the genetic code has been conserved from very early stages of evolution. 3. Redundancy: Although each codon corresponds to a single amino acid, a given amino acid may have more than one triplet coding for it. For example, arginine is specified by six different codons . 4. Nonoverlapping and commaless, that is, the code is read from a fixed starting point as a continuous sequence of bases, taken three at a time. For example ABCDEFGHIJKL , is read as ABC/DEF/GHI/JKL without any "punctuation" between the codons. mutation :Changing a single nucleotide base on the mRNA chain (a "point mutation") can lead to any one of three results 1. Silent mutation: The codon containing the changed base may code for the same amino acid. For example, if the serine codon UCA is given a different third base—U—to become UCU, it still codes for serine. Therefore, this is termed a "silent" mutation. 2. Missense mutation: The codon containing the changed base may code for a different amino acid. For example, if the serine codon UCA is given a different first base—C—to become CCA, it will code for a different amino acid, in this case, proline. This substitution of an incorrect amino acid is called a "missense" mutation. 3. Nonsense mutation: The codon containing the changed base may become a termination codon. For example, if the serine codon UCA is given a different second become UAA, thenew codon causes termination of translation at that point. The creation of a termination codon at an inappropriate place is calleda "nonsense" mutation. COMPONENTS REQUIRED FOR TRANSLATION A large number of components are required for the synthesis of a polypeptide chain. These include 1) all the amino acids that are found in the finished product. 2) the mRNA to be translated. 3) Transfer RNA: A specific type of tRNA is required per amino acid. 4) functional ribosome . 5) energy sources. 6) enzymes, as well as protein factors needed for initiation, elongation, and termination of the polypeptide chain. STEPS in PROTEIN SYNTHESIS The pathway of protein synthesis translates the three-letter alphabet of nucleotide sequences on mRNA into the twenty-letter alphabet of amino acids that constitute proteins. The mRNA is translated from its 5'-end to its 3'-end, producing a protein synthesized from its amino-terminal end to its carboxyl-terminal end. The process of translation is divided into three separate steps: initiation, elongation, and termination. A. Initiation The codon AUG at the beginning of the message is recognized by a special initiator tRNA that enters the ribosomal P, site. [ Only the initiator tRNA goes to the P site- other charged tRNAs enter at the A site. B. Elongation of the polypeptide chain involves the addition of amino acids to the carboxyl end of the growing chain. During elongation, the ribosome moves from the 5‘- end to the 3‘-end of the mRNA that is being translated. After the peptide bond has been formed, the ribosome advances three nucleotides toward the 3'-end of the mRNA. This causes movement of the uncharged tRNA into the ribosomal E site (before being released) and movement of the peptidyl-tRNAinto the P site C. Termination occurs when one of the three termination codons moves into the A site. These codons are recognized by release factors: RF-1, which recognizes the termination codons UAA and UAG. RF-2, which recognizes UGA and UAA. RF-3,which binds GTP and stimulates the activity of RF-1 and RF-2. These factors cause the newly synthesized protein to be released from the ribosomal complex, and cause the dissociation of the ribosoma from ricin (from castor beans) is a very potent toxin that exerts its effects'"by removing an adenine from 28S ribosomal RNA, thus inhibiting eukaryotic ribosome.