Bacterial Genome Replication and Expression PDF

Bacterial Genome Replication and Expression Chapter 13 Notice: This material is subject to the U.S. Copyright Law; further reproduction in violatio...

Bacterial Genome Replication and Expression Chapter 13 Notice: This material is subject to the U.S. Copyright Law; further reproduction in violation of the law is prohibited. Steven L. McKnight © McGraw Hill, Central Dogma of Molecular Biology DNA -> RNA -> Protein Incredible diversity of life determined by information within DNA Composed of four nucleotides - Adenine (A) - Thymine (T) - Cytosine (C) - Guanine (G) DNA converted to mRNA via transcription 3 nucleotides encode specific amino acid (codon code) mRNA converted to amino acids to protein via translation - The sequence of AA determines protein structure & function Proteins control every characteristic of the cell Enzymes and structural components of cell © McGraw Hill,  Terminology and Concepts Genome: complete set of genetic information Chromosomes - Bacteria and Archaea generally have one chromosome (haploid – 1N) - Eukaryotes generally have a pair (or more) of each chromosome (ex: diploid - 2N) Plasmids - Generally found in Bacteria and Archaea - Non-essential genes Courtesy: National Human Genome Research Institute The Nucleoid Location of chromosome and associated proteins. Usually 1 closed circular, double- stranded D N A molecule. Supercoiling and nucleoid associated proteins aid in folding and structure through (a) CNRI/SPL/Science Source electrostatic interactions (no covalent bonding). Positively-charged amino acids interact with negatively- charged DNA backbone Structure organized into 1 Mbp macrodomains that are further divided into chromosome interacting domains a) E. coli NAP protein HU creates sharp bend. b) SMC encircles DNA © McGraw Hill,  Terminology and Concepts Genome: complete set of genetic Strain information 1 - Subcategories: - Core genome = set shared among Unique all/most strains Dis i s p. p. D - Dispensable genome = set shared Core among a few strains Unique Disp. Unique - Unique genome = strain specific set of genes Pan-genome The set of all genes present in Strain Strain 2 3 all strains of a group of organisms (species) Pan-genome  Terminology and Concepts Genotype Specific set of genes for a particular trait Phenotype Collection of observable characteristics - influenced both by genotype and the environment Image by OpenStax Microbiology licensed under CC BY Image by Anne Auman (Wikimedia Commons) is licensed under CC BY 2.0. Overview Cells must accomplish two tasks to multiply 1. DNA replication – DNA duplicated to pass on to progeny 2. Gene expression – Synthesis of products encoded by genes - Transcription – DNA  RNA - Translation – RNA  protein © McGraw Hill, DNA Replication DNA replication Begins at origin of replication Specific sequence in genome at which replication begins Bacterial chromosomes © McGraw Hill, and plasmids typically LLC have 1 - oriC Eukaryotes and Archaea can have many Any DNA without origin will not be replicated Image by OpenStax Microbiology licensed under CC BY DNA Replication DNA replication is usually bidirectional Proceeds on both strands for faster processing Replication begins at the origin of replication Proteins recognize and bind to the site Melt double-stranded DNA Creates two replication forks Sites of unwound DNA Ultimately meet at the terminating site (terminus) © McGraw Hill, DNA Replication Replication is semiconservative DNA contains one original (parental), one newly synthesized strand © McGraw Hill, DNA Replication  The start of DNA replication is controlled by DNA methylation, and by the binding of a specific initiator protein to the origin. The oriC origin is a 245-bp sequence that includes a series of repeats. Initiations are triggered by DnaA, a protein that increases in concentration cell during growth and promotes DNA unwinding at oriC. © McGraw Hill,  DNA Replication Replisome—12 proteins involved in replication. Two replisomes move in either direction away from the origin. Helicase—ring encircles DNA, disrupts H-bonds and provides force to move the replisome. © McGraw Hill,  DNA Replication Protein DnaA (replication initiation factor) recognizes oriC sequence causing bending and separation of strands and then recruits DNA helicase and gyrase, which bind to origin Helicase separates DNA – ring encircles DNA, disrupts H-bonds between strands, starting at fork Gyrase unwinds DNA - relieves twist generated by the rapid unwinding of double helix by transiently breaking, then resealing DNA strands. SSB Protein coats ssDNA and prevents the re-annealing of single strands Dna oriC A Cornell, B. 2016. https://ib.bioninja.com.au/higher-level/topic-7-nucleic-acids/71-dna-structure-and-replic/okazaki-fragments.html. [ONLINE] Available at: http://ib.bioninja.com.au. [Accessed 2 February 2023] DNA Replication DNA replication DNA primase initiates DNA synthesis Synthesizes small strand of RNA that serves as a primer (fragment of RNA) that DNA polymerase III attaches to in order to begin DNA replication DNA polymerase reads template 3’ - > 5’ Dna A Cornell, B. 2016. https://ib.bioninja.com.au/higher-level/topic-7-nucleic-acids/71-dna-structure-and-replic/okazaki-fragments.html. [ONLINE] Available at: http://ib.bioninja.com.au. [Accessed 2 February 2023] Replication fork dynamics Leading strand Leading strand - Synthesized CONSTANTLY as DNA polymerase III moves towards the replication fork - DNA polymerase only synthesizes 5’ to 3’: - On the leading Lagging strand parental strand DNA is 3’ to 5’ and DNA poly can easily add new nucleotides in 5’ to 3’ direction Cornell, B. 2016. https://ib.bioninja.com.au/higher-level/topic-7-nucleic-acids/71-dna-structure-and-replic/okazaki- fragments.html. [ONLINE] Available at: http://ib.bioninja.com.au. [Accessed 2 February 2023] Replication fork Leading strand dynamics Lagging strand - Synthesized INTERMITTENTLY as DNA is unwound (polymerase only synthesizes 5’ to 3’) Lagging strand Cornell, B. 2016. https://ib.bioninja.com.au/higher-level/topic-7-nucleic-acids/71-dna-structure-and-replic/okazaki- fragments.html. [ONLINE] Available at: http://ib.bioninja.com.au. [Accessed 2 February 2023] Replication fork dynamics Lagging strand - Synthesized INTERMITTENTLY as DNA is unwound (polymerase only synthesizes 5’ to 3’) - Produces Okazaki fragments as DNA polymerase moves away from replication fork - DNA Poly I and DNA ligase removes RNA primer and creates covalent bond between nucleotides of adjacent fragments © McGraw Hill, DNA Replication Replication fork dynamics A different type of DNA polymerase (DNA polymerase I) will move along replication fork removing RNA primers and replace them with DNA DNA ligase will join fragments together Dna A Cornell, B. 2016. https://ib.bioninja.com.au/higher-level/topic-7-nucleic-acids/71-dna-structure-and-replic/okazaki-fragments.html. [ONLINE] Available at: http://ib.bioninja.com.au. [Accessed 2 February 2023] Linking the Fragments DNA ligase forms a phosphodiester bond between 3’-OH of growing strand and 5’-phosphate of an Okazaki fragment. © McGraw Hill, Terminating Replication and Segregating Sister Chromosomes  Bidirectional replication results in the two replication forks meeting each other at a termination site halfway around the chromosome. There are as many as ten terminator sequences (ter) on the E. coli chromosome. A protein called Tus (terminus utilization substance) binds to these sequences and ensures that the polymerase complexes do not escape and continue replicating DNA. © McGraw Hill,  Termination of Replication in E. coli Replication stops when replisome reaches termination site on DNA. Catenanes form when topoisomerases break and rejoin DNA strands to ease supercoiling. Recombinase enzymes catalyze an intramolecular crossover that separates the 2 chromosomes. © McGraw Hill, Gene Expression in Bacteria Making gene products involves: Transcription - Synthesizing RNA from DNA Translation - Synthesizing protein from RNA © McGraw Hill,  Transcription Synthesis of RNA from DNA - 3 types of RNA: mRNA, tRNA, and rRNA messenger RNA (mRNA) carries the message from DNA to the ribosome (site of protein synthesis), which is primarily made of ribosomal RNA (rRNA) mRNA transcripts - Monocistronic = one gene for one protein Most mRNA transcripts in eukaryotes - Polycistronic = several genes for multiple proteins Most mRNA transcripts in prokaryotes Genes usually have related functions (ex: all enzymes used in glycolysis) Subunits that build a final complex protein © McGraw Hill, Transcription RNA polymerase synthesizes single-stranded RNA Uses DNA as a template Synthesizes in 5′ to 3′ direction Can initiate without primer Binds to promoter Sequence of DNA upstream of genes Stops at terminator Sequence of DNA that ends transcription © McGraw Hill, Bacterial RNA Polymerases Consist of Five Different Proteins Core enzyme composed of 5 polypeptides (two α subunits, β, β′, and ω); catalyzes RNA synthesis. The sigma factor (σ) has no catalytic activity but helps the core enzyme recognize the start of genes. RNA polymerase holoenzyme = core enzyme + sigma factor. Only the holoenzyme can begin transcription. © McGraw Hill, Transcriptio n RNA polymerase uses the antisense/minus (-)/template DNA strand as the template strand for transcription RNA transcript is identical to the complement, sense/plus (+)/coding DNA strand Except uracil is used in place of thymine © McGraw Hill, Transcription Transcription overview 3 distinct phases - Initiation - Elongation - Termination © McGraw Hill, Transcription Initiation RNA polymerase scans and binds to a promoter - The promoter region contains a specific nucleotide sequence that signals where to begin transcription and which direction to synthesize © McGraw Hill,  Gene Structure Promoter is located at the start of the gene. Recognition/binding site for RNA polymerase. Orients polymerase Leader sequence is transcribed into mRNA but is not translated into amino acids. Shine-Dalgarno sequence important for translation initiation. Coding region Begins with D N A sequence 3’-T A C-5’. Produces codon A U G. Ends with a stop codon, immediately followed by the trailer sequence which prepares R Transcription Transcription Promoter orientation dictates direction of transcription and which strand is used as template Found -10 to -35 bp upstream of where transcription starts Once RNA polymerase has moved past, another RNA polymerase can bind Allows rapid and repeated transcription of a single gene © McGraw Hill, Transcription Initiation RNA polymerase needs help attaching - Sigma factor recognizes the promoter sequence - Protein loosely attached to a subunit of RNA polymerase - Enables specific binding of RNA polymerase to the gene promoter - Detaches from RNA polymerase after binding to the promoter is complete RNA polymerase then denatures (disrupts H-bonds) a short stretch of DNA exposing nucleotides of DNA to form transcription bubble © McGraw Hill, Sigma Promoter Sigma factor helps position the core enzyme at the promoter. Bacterial promoter features: 35 basepairs upstream (TT GACA) Sigma factor recognizes this sequence directing holoenzyme to “settle” here. 10 basepairs upstream (TAT AAT) Where DNA strands start to separate. © McGraw Hill, Transcription Initiation RNA polymerase needs help to recognize promoter - Sigma factors - Different sigma factors recognize different promoter sequences in the genome - The number and type of sigma factors vary between bacterial species - By controlling the availability of sigma factors, the cell controls what genes are expressed © McGraw Hill, Transcription Initiation RNA polymerase needs help - Sigma factor - E. coli have seven - RpoD (σ70 ) - the "housekeeping" sigma factor transcribes most genes in growing cells - RpoH (σ32) - the “heat shock” sigma factor, turned on when bacteria are exposed to heat. Expresses heat shock proteins (ex: DNA-repair enzymes), which enable the cell to survive higher temperatures. © McGraw Hill, © McGraw Hill, Rodriguez Ayala et al. 2020 Frontiers in Microbiology CC Transcription Initiation RNA polymerase then denatures (disrupts H- bonds) a short stretch of DNA exposing nucleotides of DNA to form transcription bubble © McGraw Hill,  Transcription Initiation - Sigma factors are unique to prokaryotes - Homologous to - Archaea: transcription factor B - Eukaryote: Transcription factor II B By Kelvin13 - Own work, CC BY 3.0, https://commons.wikimedia.org/w/index.php? Transcription Elongation RNA polymerase continues to move along the template, synthesizing RNA at ~ 45 bases/sec. Nucleotides are added only to the 3’ end using (–)/antisense strand as a template ‣ Uracil (replaces thymine) with Adenine ‣ Cytosine with Guanine Fueled by hydrolyzing high-energy phosphate bond from incoming nucleotide Boris Wikimedia Commons Public Domain Transcription Termination  RNA polymerase continues until it reaches a terminator  All bacterial genes use one of two known transcription termination signals: 1. Rho factor-dependent Relies on a protein called Rho and a strong pause site at the 3′ end of the gene 2. Rho-independent (intrinsic termination) - GC-rich terminator sequence results in a hairpin loop in the RNA transcript - Causes newly synthesized mRNA to be released and polymerase to disassociate The Termination of Transcription 1. Rho-dependent Rho factor binds to rut site on mRNA and scans until it reaches RNA polymerase, which is stalled at a pause site (specific sequence on DNA template strand) Rho’s helicase activity causes everything to disassociate © McGraw Hill, The Termination of Transcription 2. Rho-independent GC-rich terminator sequence results in a hairpin loop in the RNA transcript Hairpin loop along with NusA protein causes newly synthesized mRNA to be released and RNA polymerase to disassociate © McGraw Hill, Translation Translation overview Converting the information from mRNA (strand of nucleic acids) to synthesize protein (chain of amino acids) This process requires three major structures 1. Messenger RNA (mRNA) – carries the information 2. Ribosomes – ribosomal RNA (rRNA) serves as a scaffold for ribosome 3. Transfer RNA (tRNA) – converts the information from mRNA to protein by carrying amino acids to the ribosome  Translation Translation overview Messenger RNA (mRNA) carries the information in a code - mRNA must be decoded into amino acids by ribosomes - Codon A genetic code word, 3 base pairs long Specifies an amino acid Courtesy: National Human Genome Research Institute The Genetic Code—Example of Gene to mR NA to Protein Final step in expression of protein encoding genes. mRNA translated into amino acid sequence of polypeptide chain. Access the text alternative for slide images. © McGraw Hill,  Translation Genetic code: three nucleotides long = codon 64 different codons (# of unique NT# of bp in codon => 43) Universal – used by all living things (with exception of a few minor changes) Redundancy: code is degenerate - Up to six different codons can code for a single amino acid - Wobble position © McGraw Hill, Wobble Loose base pairing 3rd position of codon less important than 1st or 2nd. Eliminates need for unique tRNA for each codon. Decreases effect of some mutations. © McGraw Hill,  Translation Correct reading frame is critical Incorrect will yield different, nonfunctional Start codon protein Start site for translation Always AUG - encodes for the amino acid methionine Aligns ribosome on correct reading frame (3 possible) Sense codons The 61 codons that specify amino acids (~20 total) Stop (nonsense) codons Three codons used to terminate translation UAA, UAG, UGA These do not encode amino acids Recognized by release factor Kellyg19 Wikimedia Commons CC BY-SA 4.0 Translation Ribosomes serve as translation “machines” Made from protein and ribosomal RNA (rRNA) link amino acids together to make polypeptide Recognizes and binds to Ribosome-binding site Begin translation at start codon, moves along mRNA in 5′ to 3′ Aligns and catalyzes the formation of a peptide bond between amino acids © McGraw Hill, The Ribosome Site of protein synthesis. A (acceptor) site Receives tRNA carrying amino acid. P (peptidyl) site Holds tRNA attached to growing polypeptide. E (exit) site Empty tRNA leaves ribosome. © McGraw Hill, Role of Ribosomal RNA in Translation Contributes to ribosome structure. 16S rRNA Needed for initiation of translation. Binds to Shine-Dalgarno sequence (ribosome binding site). Binds to 3’CCA end of aminoacyl-tRNA. 23S rRNA Ribozyme that catalyzes peptide bond formation. © McGraw Hill, The Three Stages of Protein Synthesis  Polypeptide synthesis occurs in three stages: 1. Initiation: brings the two ribosomal subunits together with mRNA, placing the first amino acid in position at the start codon 2. Elongation: sequentially adds amino acids as directed by mRNA transcript via tRNA 3. Termination: stop codon and release factor release the completed protein and recycles ribosomal subunits . © McGraw Hill, Antibiotics That Affect Translation  Streptomycin: bactericidal narrow- spectrum (G- bacteria) drug that inhibits 70S ribosome formation by binding to rRNA. Side- effects – can bind to host ribosomes  Tetracycline: bacteriostatic, broad- spectrum drug, inhibits aminoacyl-tRNA binding to the A site © McGraw Hill, Differences Between Eukaryotes and Prokaryotes Eukaryotic transcription, translation differs transcription occurs in the nucleus mRNA is transported to the cytoplasm where translation occurs Therefore, transcription and translation cannot happen simultaneously mRNA synthesized in precursor form: pre- mRNA Must be processed during and after transcription to form mature mRNA Image by OpenStax Microbiology licensed under CC BY  Differences Between Eukaryotes and Prokaryotes Eukaryotic transcription, translation differs Pre-mRNA must be processed during and after transcription to form mature mRNA 5′ end capped with methylated guanine derivative Binds specific proteins that stabilize & enhance translation 3′ end modified via polyadenylation mRNA is cleaved at specific sequence and Adds ~200 adenine derivatives Poly A tail stabilizes mRNA, enhances translation © McGraw Hill, Differences Between Eukaryotes and Prokaryotes Eukaryotic transcription, translation differs Splicing removes introns (Non-coding sequences) Exons are expressed regions mRNA typically monocistronic (one gene) Ribosomes are 80S 40S and 60S subunits An important difference for targeting with antibiotics © McGraw Hill, Differences Between Eukaryotes and Prokaryotes Translation in prokaryotes begins before transcription is complete  Before RNA polymerase has even finished making an mRNA molecule, ribosomes will bind to the 5′ end of the mRNA and begin translating protein. This is called coupled transcription and translation © McGraw Hill, Polysomes  Once a ribosome begins translating mRNA and moves off the ribosome-binding site, another ribosome can immediately jump onto that site. The result is an RNA molecule with many ribosomes moving along its length at the same time. This multiribosome structure is known as a poly(ribo)some.  Ribosomes in a polysome are closely packed and arranged helically along the mRNA.  Polysomes help protect the message from degrading RNases and enable the speedy production of protein from just a single mRNA molecule. © McGraw Hill, Colliding Polymerases—Head On 2-4 replisomes at the replication fork. 1,000+ RNA polymerases on the chromosome. Replisome must be able to navigate around RNA polymerases competing for the same template. Head-on collisions—stall replisome Dissociation, fidelity compromised or increased supercoiling. © McGraw Hill, Colliding Polymerases—Rear End Co-directional collisions Rear-end collisions Helicases help move through RNA polymerase barriers. Mfd protein evicts paused/stalled RNA polymerases. © McGraw Hill,