Bacterial Physiology: Macromolecular Synthesis - DNA Replication and Transcription - Prof., Study notes of Health sciences

Download Bacterial Physiology: Macromolecular Synthesis - DNA Replication and Transcription - Prof. and more Study notes Health sciences in PDF only on Docsity! Bacterial Physiology HSCI 5607 Macromolecular Synthesis Introduction: The synthesis of DNA, RNA, and proteins involve polymerization of their monomeric subunits, deoxynucleotides, nucleotides and amino acids respectively. In all cases the donors of the subunits are the charged species like amino-acyl-tRNA in case of protein synthesis while deoxynucleoside triphosphate or nucleoside triphosphate in case of DNA and RNA synthesis. The polymerization is not a simple condensation but much more complicated process involving several factors including several enzymes and binding proteins. Meselson And Stahl’s Experiment: 15 15N A — I5N-ISN grow in !4N for one generation ISN 14 ISN 4N B — 15N/14N continued growth in }4N ISN 14N 14n 14N, 15N 14N 14 14 — 4/14 c — 15N/4N,  Mechanism of Replication: Usually DNA exists as a covalently closed circle of a right- handed double helix which is tightly folded into supercoiled loops. During the process of replication the DNA strands need to be unwounded forming a replication fork. When two strands are pulled apart, it creates overwound region ahead of the replication fork. This can lead to the formation of positive supercoils which may stop further unwinding. The problem of overwinding of double helix during DNA replication is solved by enzyme called DNA topoisomerase II (DNA gyrase). This enzyme works by making a double-stranded break and passing the unbroken strands through it to release the coiling. The enzyme continuously removes the positive coiling ahead of the replication fork. Another problem is to keep the rapidly replicating strands (1000 nucleotides per second in E.coli) separated and not getting entangled to each other and also they need to be partitioned into two daughter cells. At least 30 different proteins are involved in DNA replication in E.coli. Replication Fork: DNA replication takes place at a replication fork, which is created each time DNA replication is initiated. For the formation of the fork the DNA strands must be unwounded at a particular site termed as origin. In E.coli this site is called oriC locus. As the double strand unwinds it creates ‘Y’ containing single stranded arms. The region where two arms come together are still double stranded. The juncture is called replication fork. There are two replication forks and DNA replication takes place at both the forks proceeding bidirectionally to meet at the opposite end. This also reduces the replication time to half. The Replication of DNA: DNA Polymerases: They are the enzymes which synthesize DNA. There are two important characteristics of these enzymes: 1. These enzymes can only extend existing DNA strand but cannot initiate DNA synthesis. 2. They always extend existing DNA strands with the addition of mononucleotide complementary to the template strand at the 3’- hydroxyl end of the existing DNA strand. Thus the direction of growth of DNA strand is from 5’-Phosphate to 3’-hydroxyl end. E.coli has three kinds of DNA polymerases, DNA polymerase I, DNA polymerase II, and DNA polymerase III. All of them have different function in E.coli cells. DNA polymerase III: It replicates DNA at the replication fork. DNA polymerase II: It does not take part in DNA replication but does function in repair of damaged DNA. DNA polymerase I: It functions both in replication as well as in repair of damaged DNA. Leading and Lagging strand: Two strands of DNA have opposite polarity, that means as one runs from 5’>3’ the other runs from 3’>5’. Due to this the two newly synthesized strands at each fork also have opposite polarity. As the direction of DNA synthesis is always 5’>3’, one strand is synthesized in the direction that the fork is moving while the other in opposite direction. The one which is synthesized in the direction of movement of fork is synthesized continuously and is known as ‘Leading strand’. The one which is synthesized in opposite direction is called ‘Lagging strand’ and is synthesized in fragments of about 1000 nucleotides. These fragments are known as ‘Okazaki fragments’, named after the investigator who first detected them. 1 2 Synthesis of Leading Strand: DNA polymerases cannot initiate DNA synthesis but can only extend existing polynucleotide chain, therefore there is a need for primers. Primers are short stretch of ribonucleotides (5-60) synthesized by RNA polymerases. DNA polymerase III extends the RNA primers at 3’ end continuously as the replication fork moves along the DNA strand to complete the replication. Synthesis of Lagging strand: In the opposing strand the growing 3’ end is in the opposite direction of the movement of fork. Thus the polymerase disengages itself after synthesizing stretch of 1000 nucleotides and returns back to the new primer which is synthesized at the replication fork. Ligation Reaction: 9 I It R—O—1—OH + ATP —> R—-O-T'~ AMP + PP; OH OH I R-O-P+ AMP + HO—R, ——> R-O-P—O-R, + AMP OH OH Fig. 10.13 DNA ligase reaction. The enzyme catalyzes the adenylylation of the 5’-phosphate. Depending upon the ligase, the AMP can be derived either from ATP, in which case pyrophosphate is displaced, or NAD*, in which case nicotinamide monophosphate (NMN) is displaced. E. coli DNA ligase uses NAD* as the AMP donor. Then the 3’-hydroxy! of the ribose attacks the AMP derivative and displaces the AMP. The result is a phosphodiester bond.  Termination: At the end of the replication the replication forks meet at the region called ‘Termination region’. In E.coli this region has specific sequences called ‘Ter’ sequences. The unique property of this region is that it allows the replication fork to pass through only in one direction. After two forks meet here the daughter DNA molecules are separated. E.coli has more than one such ‘Ter’ sequences. In E.coli there are six Ter sites divided into two groups. Each one prevents the rotation of one replication fork. Multiple Ter sites perhaps provide back ups. There is another protein in E.coli called ‘Tus’ protein which binds to Ter site and imposes one way travel. Chromosome Partitioning: Chromosomal partitioning involves separation and segregation of the newly synthesized daughter DNA molecules. If there are no covalent linkages between the daughter molecules then they are separated by the activity of enzyme ‘Topoisomerase IV’. In case of covalent linkage due to unequal recombination events, site specific recombination is required to separate the daughter molecules. The recombinase proteins involved in site specific recombination are XerC and XerD. Specific location where this takes place in E.coli is called ‘dif’ region located in ‘Ter’ region. Chromosomal segregation refers to the movement of separated chromosomes to opposite poles prior to the cell division. Segregation involves several different proteins like, ‘Par’ and ‘Muk’ proteins. Par proteins get attached to DNA and align it while in E.coli the Muk proteins seem to be involved in moving the DNA to the opposite poles. Mismatch Repair: Proof reading is also not error proof, as a result few wrong bases always get inserted in newly replicated DNA. These are removed by ‘Methyl-directed mismatch(MMR)’ system. This system removes incorrect base from the newly synthesized strand and not from the template strand. This happens since the template strand is protected by methylation. In E.coli the enzyme which methylates is called ‘Deoxyadenosine methylase’ (DAM). It methylates all adenines at N6 in sequence 5’-GATC-3’. Several proteins are involved in mismatch repair. These include products of mutH, mutL, and mutS. The repair process is described in the next slide. Mismatch Repair: 5 A 3 t G CH; MutH MutS MutL 3 Ts 3 4 G CH3 B helicase IT exonuclease I SSB 5 T_e& 3 + G CH; BNA polewe a DNA ligase 5 t_£ c 3 AG CH,  Inhibition of DNA Replication: Many antibiotics and drugs are known to either directly inhibit DNA replication or inhibit the synthesis of its precursors. The antibiotics include, novobiocin, mitomycin C, and nalidixic acid. Novobiocin and nalidixic acid inhibit the activity of DNA gyrase while mitomycin C crosslinks the guanine bases in either the same strand or the opposing strands of DNA. Crosslinking opposing strands prevents strand separation during replication while in the same strand, it distorts the helix. Chemically produced drugs include, acridine dyes, 5-fluoro- deoxyuridine-5’monophosphate, aminopterin, and methotrexate. 5’-fluorodeoxyuridine-5’ monophosphate inhibits thymidylate synthase converting dUMP to dUTP while aminopterin and methotrexate are dihydrofolate analogs and inhibit dihydrofolate reductase involved in nucleotide biosynthesis. Steps involved in initiation: 1. Formation of the closed complex: The RNA polymerase holoenzyme (core plus sigma factor) binds to the promoter region. DNA at this stage still exists as a double helix thus known as closed complex. 2. Formation of open complex: The RNA polymerase unwinds the promoter region including the start site without any helicase or ATP requirement. 3. Binding of initiating ribonucleotides: Transcription is usually initiated by the binding of either ATP or GTP, whose ribose provides 3’ hydroxyl group that attacks the  phosphate of the incoming nucleotide displacing pyrophosphate. All the newly synthesized RNA therefore contain terminal 5’-phosphate at 5’ end. 3 5 35. =10 3 5  Elongation: After about 12 nucleotides are added, the transcription is said to be in elongation stage as the sigma factor dissociates and the core enzyme continues to elongate the chain. About 18 bases long ‘transcription bubble’ which is the unwounded region of DNA where RNA polymerase is bound moves along the DNA/RNA hybrid during elongation. The rate at which RNA is synthesized which is 40-50 ribonucleotides per second matches the speed of protein synthesis (about 16 amino acids per second). The ribosomal RNA in the fast-growing cells is synthesized at the rate of 90 nucleotides per second. 2. Rho Dependent Termination: This type of termination requires protein factor and do not rely on hairpin loop structures. In E.coli there are three such factors found: Rho, Tau and NusA. Rho is the best studied and is an RNA-dependent ATPase and an ATP-dependent RNA/DNA helicase. Rho factor binds to ‘rut’(rho utilization sites) in RNA behind the transcribing RNA polymerase in the absence of translating ribosome. Rho moves along the RNA molecule at the expense of ATP hydrolysis and when RNA polymerase stops moving at terminator site, its helicase activity unwinds RNA/DNA duplex disengaging the RNA polymerase to terminate transcription. Frequency of Initiation: Different promoters vary in the frequency of initiation of transcription (Promoter Strength). Strong promoter cause initiation every few seconds while the weaker ones start every few minutes. Various transcription factors also influence the frequency of transcription either positively or negatively. Role of Topoisomerase: During transcription same as in case of DNA replication, unwinding creates positive coiling to the downstream of unwinding. If this is not released then it can stop the unwinding of DNA and thereby the transcription. Topoisomerases change positive supercoiling to negative supercoils and facilitate DNA unwinding. The Sigma subunit: The bacterial RNA polymerase is made up of five subunits (2’). The sigma factor is responsible for the recognition of promoter and not needed for chain elongation. Different sigma factors recognize different -10 and -35 sequences. Bacteria usually contain more than one types of sigma factor. The core polymerase can exchange one sigma factor for another changing the specificity of gene transcription. E.coli has at least six such different sigma factors which recognize different consensus sequences. For example the main sigma factor in E.coli is 70 which has a molecular weight of 70 kD and recognizes the consensus sequence of most of the promoters. Then there are s/ 38 and 32 which recognize genes that are expressed during starvation and heat-shock respectively. In Bacillus there is a specific sigma factor which stimulate genes during sporulation. RNA Processing: Ribosomal RNA(rRNA) : Ribosomal RNAs are synthesized as a long 30S preribosomal transcript along with one or more tRNAs which is processed to produce 16S, 23S, 5S rRNA and tRNA. Transfer RNA(tRNA) : Transfer RNAs are extensively processed after their transcription. This includes removal of segments from both the ends 3’ and 5’. The enzyme involved here is RNase P and interestingly it is a ribonucleoprotein, and the enzyme activity is due to its RNA content which is also called ‘Ribozyme’. An additional processing occurs at 3’ end in many bacteria and in all eukaryotes to add CCA-3’ sequence. Apart from these processing, several bases at specific site in different tRNAs are chemically modified after transcription. Inhibitors of Transcription: These include antibiotics like Actinomycin D, Rifamycin (or derivative Rifampicin), and Streptolydigin while the chemical inhibitors include acridine and Amanitin. Actinomycin D is inserted and intercalated between the bases deforming the DNA resulting in the inhibition of the movement of RNA polymerase along DNA inhibiting transcription. Acridine also inhibits the same way while Rifamycin or Rifampicin bind to the  subunit of the RNA polymerase and inhibit initiation of transcription. It does not recognize eukaryotic RNA polymerase. Streptolydigin also inhibit transcription in the same way as Rifamycin while Amanitin inhibit eukaryotic RNA polymerase II but not bacterial RNA polymerase. Protein Synthesis: Protein synthesis is also known as ‘Translation’ since the codons on mRNA are translated to amino acids. The process of translation can be divided into three stages: 1. Initiation; 2. Elongation; and 3. Termination. 1.Initiation: During initiation the first tRNA which is formyl methionine- tRNA binds at the 5’ end of mRNA at the P(peptidyl) site. Amino acid alone can not recognize and bind to mRNA directly, it is the tRNA molecule carrying specific amino acid specified by codon on mRNA places the amino acids in the growing polypeptide chain. Thus the sequence of amino acid in the polypeptide chain is specified by the sequence of nucleotides in mRNA. Transfer RNA (tRNA): The transfer RNAs are small RNA molecules which are folded in a manner that forms three hairpin loops resembling cloverleaf. All tRNAs have modified nucleotide bases in the loops. The amino acid it carries is esterified at 3’-OH group of the ribose moiety of the terminal adenylic acid at the 3’end. All tRNAs have terminal CCA sequence at 3’ end. Out of three loops one loop which recognizes and binds the codon on mRNA is known as ‘Anticodon loop/arm’. Anticodon arm has the nucleotide triplet which hybridyzes to the codon. Prior to the initiation of the protein synthesis, each amino acid is covalently bound to specific tRNAs. For 20 different amino acids there are 20 different enzymes called “aminoacyl-tRNA synthetases” that catalyze the reaction synthesizing aminoacyl- tRNA. These enzymes recognize both the amino acids as well as certain structural features of tRNA. The process of formation of aminoacyl- tRNA is known as charging of tRNA molecules. Charging of tRNA: The charging of tRNA is a two step process, both catalyzed by 20 different aminoacyl-tRNA synthetase. Each enzyme recognizes specific amino acid and tRNA molecule. In the first reaction there is a formation of aminoacyl-AMP where the carboxyl oxygen of amino acid attacks the -phosphate of ATP and displaces PP to form aa-AMP. In the second step the oxygen atom of the 3’-OH group of the terminal ribose of tRNA attacks aminoacyl-AMP and displaces AMP to form aminoacyl-tRNA. The linkage between the carboxyl group of amino acid and the hydroxyl group of ribose is through ester bond. Formylmethionine-tRNAf :The first amino acid is always formylmethionine (fMet) in bacteria. This is made by first attachment of methionine to tRNAfMet and then the methionine is formylated by an enzyme transformylase ( tRNA methionyl transformylase).This enzyme transfers formyl group from 10- Formyl-THF to Met-tRNAfMet to form fMet-tRNAfMet. Transfer RNA Charging ATP pecaactibaee —adenine oO. oO a H 9 NH,—C—C—' I 4 AMP an~, we t oe a t—RNA—OH OH 2 “NH; o=C FOF. aden I H-C-R 0. oO il} aa~tRNA a “NI HO adenine tRNA—O—| 3 transesterification. ————— #Q_O> f 5 ‘eat H-C-R 4S ‘NH; 9 W tRNA —O—P—O- I 0.  3. Shine-Dalagarno Sequences: This sequences are about 5 to 10 bases long and are located at the 6 to 10 nucleotide upstream of the start codon. These sequences base pair to the complementary sequences on the 3’ region of the 16S rRNA in the 30S ribosome. This helps to place the start codon in the right place at the P site. 2. Elongation: The chain elongation begins when an incoming aminoacyl- tRNA binds to the A (aminoacyl) site. The incoming aminoacyl- tRNA is correctly positioned by the bound elongation factor Ef-Tu- GTP complex. Once the AA-tRNA is positioned to the A site, GTP is hydrolyzed and EF-Tu-GDP is released from the ribosome. The released EF-Tu-GDP then reacts with EF-Ts, which displaces GDP for GTP to regenerate EF-Tu-GTP. Formation of Peptide Bond: After positioning of the AA-tRNA to the A site, in the next step the free -amino group of amino acid bound to the tRNA on the A site attacks ester linkage and displaces the tRNAf from the P site. This reaction is catalyzed by enzyme ‘Peptidyl transferase’ which is a part of 50S subunit and interestingly it is not a protein but 23S rRNA. At the end of the reaction there is a formation of first peptide on the A site. The energy required for the formation of the peptide bond is obtained from the the breakage of ester linkage between the carboxyl and hydroxyl group of the tRNAf. Thus there is no requirement of ATP as energy donor but the energy conserved in the form of ester linkage is initially derived from ATP while charging the tRNAs. Translocation: Once the peptide bond is formed, the uncharged tRNA from the P site is removed. The peptidyl-tRNA moves from A site to the P site and mRNA moves one more codon in 3’ direction to be occupied by newly formed A site. This process is called translocation and it requires elongation factor EF-G (translocase). During translocation GTP is hydrolyzed to GDP and Pi. At the end of translocation the empty A site is ready to accept another aminoacyl-tRNA molecule to continue the elongation. Termination: The polypeptide chain is elongated till the ribosome reaches termination codon which may be UAA, UGA, or UAG. These codons are not recognized by any tRNA and thus no aminoacyl tRNA enters A site when it is occupied by any one of the termination codons. There are termination factors which bind to termination codons and cause release of the polypeptide chain as well as ribosomal complex. There are three such factors RF-1 recognizes UAA and UAG, RF-2 recognizes UAA and UGA while RF-3 facilitates the activity of RF1 and RF2. The release factors are believed to stimulate peptidyl transferase to hydrolyzed the ester linkage between the polypeptide and the tRNA at the P site to release the protein which is followed by the release of ribosomal subunits and mRNA. IF-3 at this stage prevents the reassociation of the 30S and 50S subunits before the initiation complex is formed. Table 10.1 Antibiotics that inhibit protein synthesis Antibiotic Site of action Bacteria Archaea Eukaryotes Erythromycin Inhibits translocation cs = - Streptomycin? Inhibits initiation and causes mistakes + - - in reading mRNA at low concentrations Tetracycline? Inhibits binding of aminoacyl-tRNA + + + (blocks A site) + ~ Chloramphenicol Inhibits peptidyl transferase + - = Puromycin¢ Resembles charged tRNA and causes causes premature chain termination + + + Cycloheximide Inhibits peptidy! transferase - - + Diphtheria toxin Inhibits elongation factor 2 (eEF2), which is analogous to bacterial EF-G, by ADP-ribosylation - + + 4 Streptomycin binds to the $12 protein in the 30S ribosomal subunit and inhibits the binding of f{Met-tRNAMet to the P site. Neomycin and kanamycin act in a manner similar to streptomycin. 6 Tetracyclines inhibit bacterial protein synthesis by binding to the 30S ribosomal subunit and preventing attachment of aminoacylated tRNA. They also inhibit eukaryotic ribosomes but not at the concentrations used to treat bacterial infections. This is apparently because eukaryotic membranes are not very permeable to tetracycline. ¢ Puromycin resembles the aminoacyl portion of aminoacylated tRNA. It has a free amino group that attacks the ester linkage in the growing peptide in the P site, with the result that a peptidyl puromycin forms at the A site. However, the peptidyl puromycin cannot be transferred to the P site (i.e., translocation does not occur), and therefore chain elongation is terminated as the peptidy! puromycin dissociates from the ribosome.