Bacterial Genome Replication and Expression.pptx

Bacterial Genome Replication and Expression 1 By the end of this chapter you should be able to: Know and Understand: Bacterial DNA replication Transcription Translation Protein secretion and translocation 2  DNA Replicat...

Bacterial Genome Replication and Expression 1 By the end of this chapter you should be able to: Know and Understand: Bacterial DNA replication Transcription Translation Protein secretion and translocation 2  DNA Replication proteins help ensure accuracy The 2 strands separate, each serving as a template for synthesis of a complementary strand Synthesis is semi- conservative; each daughter cell obtains one old and one new strand 3  Patterns of DNA Synthesis DNA in most Bacteria is circular bidirectional replication from a single origin replication fork is where DNA is unwound (Consist of ATs) replicon – portion of DNA replicated as a unit (entire genome in bacteria) 4 The Replication Machinery - 1 E. coli, consists of at least 30 proteins 5  DNA polymerase catalyzes synthesis of complementary strand of DNA DNA synthesis in 5’ to 3’ direction forming phosphodiester bonds These enzymes require a template – directs synthesis of complementary strand a primer – DNA or RNA strand dNTPs (dATP, dTTP, dCTP, dGTP) – deoxynucleotide triphosphates 6 7 8  E. coli has 5 (I-V) DNA polymerases with polymerase III playing the major role in replication DNA polymerase holoenzyme complex of 10 proteins 3 proteins form core enzyme 3 core enzymes in each polymerase catalyze DNA synthesis proofreading for fidelity 9  Helicases unwind DNA strands Single stranded binding proteins (SSBs) keep strands apart for replication to occur Topoisomerases breaks one strand of DNA to relieve tension from rapid unwinding of double helix and prevents supercoiling 10  DNA gyrase topoisomerase also introduces negative supercoiling to help compact bacterial chromosome Primase synthesizes short complementary strands of RNA (~10 nucleotides) to serve as primers needed by DNA polymerase 11  DNA Synthesis DNA polymerase synthesis is in 5’ to 3’ direction only The lagging strand is synthesized in short fragments called Okazaki fragments A new primer is needed for the synthesis of each 12 13  Events at the Replication Fork in E. coli DnaA proteins bind oriC (origin of replication) causing bending and separation of strands (AT rich) DnaB and other helicases separate strands, SSB attach Primase synthesizes RNA primer Lagging and leading strand is synthesized DNA polymerase I removes RNA primers, fills gaps with DNA 14  Linking the Fragments DNA ligase forms a phosphodiester bond between 3’- hydroxyl of the growing strand and the 5’-phosphate of an Okazaki fragment 15  Proofreading Carried out by DNA polymerase III Removal of mismatched base from 3’ end of growing strand by exonuclease activity of enzyme This activity is not 100% efficient 16 17  Termination of Replication in E. coli stops when Replication Tus=terminus utilization replisome (Tus) reaches substance termination site (ter) on DNA Catenanes form when the two circular daughter chromosomes do not separate Topoisomerases temporarily break the DNA molecules so the stands can separate Dimerized Chromosome; Recombinase 18 Replication of Linear Chromosomes The “end” replication problem shortening of chromosomes after each round of replication solved in eukaryotes by telomerase enzyme solved in bacteria by disguising the ends of the linear chromosome; Telomere resolvase 19 Inverted repeats play a role in: gene regulation DNA replication recombination processes 20  Gene Structure Gene basic unit of genetic information nucleic acid sequence coding for a polypeptide, tRNA or rRNA linear sequence of nucleotides with a fixed start point and end point codons are found in mRNA and code for single amino acids 21  Protein-Coding Genes Template strand of DNA - synthesis directs RNA 1 is read in the 3’ to 5’ direction Complementary DNA strand is coding strand, same nucleotide sequence as mRNA (except in thymine) 22  Sense Antisense strand strand TAC Downstre Upstream am 23 24  Promoter is located at the start of the gene is the recognition/binding site for RNA polymerase functions to orient polymerase Leader sequence is transcribed into mRNA but is not translated into amino acids Shine-Dalgarno sequence important for initiation of translation 25  Protein-Coding Genes - 3 Begins with DNA sequence 3’-TAC-5’ produces codon AUG codes for N-formylmethionine, a modified amino acid used to initiate protein synthesis in bacteria coding region ends with a stop codon immediately followed by the trailer sequence which contains a terminator sequence used to stop transcription 26  tRNA and rRNA Genes DNA sequences that code for tRNA and rRNA are considered genes genes coding for tRNA may code for more than a single tRNA molecule or type of tRNA genes coding for rRNA are transcribed as single, large precursor spacers between the coding regions of both are removed after transcription, some by the use of special 27 tRNA and rRNA Genes 28  Regulatory genes Regulatory sequences encode regulatory genes control the expression of one or more other genes 5' or 3' to the start/end site of transcription of the gene they regulate. Inverted repeats play a role in: gene regulation DNA replication recombination processes 29  Transcription RNA synthesis under the direction of DNA RNA produced has complementary sequence to the template DNA three types of RNA are produced mRNA carries the message for protein synthesis tRNA carries amino acids during protein synthesis rRNA molecules are components of ribosomes Initiation, Elongation and Termination 30  Transcription in Bacteria Polycistronic mRNA often found in bacteria and archaea Contains directions for >1 polypeptide catalyzed by a single RNA polymerase reaction similar to that catalyzed by DNA polymerase 31  Most bacterial RNA polymerases core enzyme composed of 5 chains and catalyzes RNA synthesis (β` β α α ω) the sigma (σ) factor has no catalytic activity but helps the core enzyme recognize the start of genes holoenzyme = core enzyme + sigma 32 Transcript ion Initiation Only a short segment of DNA is transcribed Promoter site where RNA polymerase binds to initiate transcription is not transcribed has specific sequence before transcription starting point and a 33 34 35 Transcription Elongation After binding, RNA polymerase unwinds the DNA Transcription bubble produced moves with the polymerase as it transcribes mRNA from template strand within the bubble a temporary RNA:DNA hybrid is formed Sigma must dissociate from RNA polymerase 36 ribonucleoside tri- phosphate (rNTP) 37  Transcription Termination Occurs when core RNA polymerase dissociates from template DNA DNA sequences mark the end of gene in the trailer and the terminator Some terminators require the aid of the rho factor for termination 38 Factor-independent Factor- dependent Rho factor =p rut=rho utilization site Helicase activity 39  The Genetic Code - 1 Final step in expression of protein encoding genes mRNA translated into amino acid sequence of polypeptide chain (process = translation) An understanding of the genetic code is necessary before translation is studied Reading frame 40 41  Codon genetic code word, 3 base pairs long specifies an amino acid anticodon on tRNA is complementary Start codon start site for translation always AUG 42  Sense codons the 61 codons that specify amino acids Stop (nonsense) codons the three codons used as translation termination signals: UAA, UGA, UAG do not encode amino acids Code degeneracy/redundancy up to six different codons can code for a single amino acid Wobble effect 43  Differ at the 3rd base 44  Wobble Loose base pairing 3rd position of codon less important than 1st or 2nd Eliminates need for unique tRNA for each codon 45 46  Differ at the 3rd base 47 Exceptions to the Code Several exceptions exist Some protists use a single stop codon; the other two code for amino acids instead Some microbes incorporate two rare amino acids into polypeptides Selenocysteine (SECIS) Pyrrolysine (PYLIS) 48  Translation Synthesis of polypeptide directed by sequence of nucleotides in mRNA direction of synthesis N terminal C-terminal Ribosome = site of translation coupled transcription/translation in Bacteria/Archaea polyribosome – complex of mRNA with several ribosomes 49 50  Transf er RNA Tertiary structure due to base pairing within the tRNA molecule Anticodon is present complementary to the codon binds the codon 3’ end of tRNA binds amino acid Acceptor stem CCA 51  Amino Acid Activation Attachment of amino acid to tRNA Catalyzed by aminoacyl-tRNA synthetases at least 20 each specific for single amino acid and for all the tRNAs to which each may be properly attached (cognate tRNAs) 52  The Ribosome Site of protein synthesis Bacterial ribosome 70S ribosomes = 30S + 50S subunits translational domain on both subunits is responsible for translation 50S exit domain 53  tRNA Binding Sites of Ribosome Aminoacyl (acceptor; A) site binds incoming aminoacyl-tRNA Peptidyl (donor; P) site binds initiator tRNA or tRNA attached to growing polypeptide (peptidyl-tRNA) Exit (E) site briefly binds empty tRNA before it leaves ribosome 54 55  Role of Ribosomal RNA in Translation Contributes to structure of ribosome 16S rRNA ribosomal binding site (RBS) 3` end binds to Shine Dalgarno site (RBS) on mRNA for protein synthesis initiation binds protein needed for initiation of translation and amino acyl-tRNA 23S rRNA ribozyme catalyzes peptide bond formation Peptidyl transferase-transpeptidation 56 57  Initiation of Protein Synthesis Involves ribosome subunits and numerous additional molecules N-formylmethionine-tRNA – bacterial initiator tRNA archaea and eukaryotes use methionine- tRNA 58  In bacteria initiation begins when Shine Dalgarno sequence of mRNA is aligned with 16S rRNA initiator codon binds 16S rRNA in 30S subunit 3 initiation factors (IFs) in bacteria required for formation of the initiation complex GTP catalyzes 59 60  Elongation of the Polypeptide Chain Elongation cycle sequential addition of amino acids to growing polypeptide consists of three phases aminoacyl-tRNA binding transpeptidation reaction translocation involves several elongation factors (EFs) 61 62  Peptidyl transferase 63 64 65  Transpeptidati on Reaction Catalyzed by peptidyl transferase of 23S rRNA Amino group of the A site amino acid reacts with the carboxyl group of the C- terminal amino acid on the P site tRNA Peptide chain transferred from P site to A site 66  Final Phase in Elongation Three simultaneous events peptidyl-tRNA moves from A site to P site ribosome moves down one codon empty tRNA leaves P site Requires GTP hydrolysis 67  Termination of Protein Synthesis Takes place at any one of three codons Peptidyl nonsense (stop) codons – transferas e UAA, UAG, and UGA Release factors (RFs) aid in recognition of stop codons 3 RFs function in prokaryotes only 1 RF active in eukaryotes GTP hydrolysis required 68 69  Protein Maturation and Secretion Protein function depends on 3-D shape Occurs as post or co-translational event requires folding association with other proteins delivered to proper subcellular or extracellular site 70  Protein Folding and Molecular Chaperones Molecular chaperones proteins that aid the folding of nascent polypeptides protect cells from thermal damage e.g., heat-shock proteins aid in transport of proteins across membranes 71 72  Protein Splicing Removal of part of polypeptide before folding Inteins – removed portion Exteins – portions that remain in protein 73  Protein Translocation and Secretion Numerous in Bacteria protein secretion pathways have been identified some reside in all 3 domains some unique to Bacteria and Archaea some unique to Gram-negative bacteria 74  Translocation movement of proteins from cytoplasm to plasma membrane or periplasmic space include transport proteins, ETC proteins, proteins involved in chemotaxis and cell wall synthesis, enzymes Sec and Tat systems Secretion movement of proteins from the cytoplasm to external environment hydrolytic enzymes for nutrient break down Type ?? secretion systems (Type I, II, III etc.; T1SS, T2SS T3SS) 75  Common Translocation and Secretion Systems Sec-dependent pathway the major pathway for all bacteria for transporting proteins across the plasma membrane Gram-negative bacteria may use Sec system also must cross the outer membrane using Types I, II, III, IV, V systems All pathways require energy 76  Also called general secretion Sec- pathway Dependent highly conserved in all Pathway domains secY, secE, and secG Translocates proteins from form a channel in the cytoplasm across or into membrane plasma membrane secA translocates pre- Secreted proteins synthesized protein through the as pre-proteins having amino- terminal signal peptide plasma membrane signal peptide delays When pre-protein protein folding emerges from plasma chaperone proteins (SecB) membrane a signal keep pre-proteins unfolded peptidase removes77 Sec-Dependent Pathway 78  Tat System Tat=twin arginine translocase Protein translocation system in Bacteria and some archaea Moves across plasma membrane Tat pathway translocated folded proteins with “twin” arginine residues in their signal sequence Works with Type II secretion system 79  Other Protein Secretion Type I secretion Pathways systems related to ABC transport systems Gram-positive/ Gram-negative bacteria, and Archaea Secretion of toxins, proteases, other proteins 80  Other Protein Secretion Pathways Type IV secretion system secrete proteins secrete DNA from donor to recipient bacterium during conjugation found in both Gram-positive and Gram- negative 81  Protein Secretion in Gram-Negative Bacteria Six proteins secretion systems identified Types I and IV also in Gram-positives Types II, III, and V are unique to Gram- negatives most secrete virulence factors 82 83  Type II pathways transport proteins across outer membrane that were first translocated across plasma membrane by Sec-dependent pathway Types III is sec independent forms injectisomes transports virulence factors and other proteins Type V are sec-dependent autotransporters – transport themselves out Type VI are similar to bacteriophage genome injection systems 84 Follow something that will teach you…. 85