Introductory Microbiology BIOL 228 PDF

Introductory Microbiology BIOL 228 Nusrat J. Urmi Biology Dept. , OC © 2016 Pearson Education, Inc.  Lecture 16 Molecular Information Flow and Protein Processing...

Introductory Microbiology BIOL 228 Nusrat J. Urmi Biology Dept. , OC © 2016 Pearson Education, Inc.  Lecture 16 Molecular Information Flow and Protein Processing Nusrat J. Urmi Biology Dept. , OC © 2016 Pearson Education, Inc. Key Concept 1: DNA and Genetic Information Flow DNA and Genetic Information Flow Functional unit of genetic information is the gene Genes are part of genetic elements: large molecules and/or chromosomes Genome: all genetic elements DNA (genetic blueprint) and RNA (transcription product) – Messenger RNA is translated into protein (amino acid sequence) Informational macromolecules: nucleic acids and proteins Nucleotides: nucleic acid monomers – DNA and RNA are polynucleotides – Three components: pentose sugar (ribose ordeoxyribose), nitrogenous base (Purine: A, G & Pyrimidine: C, T, U), phosphate. Nucleoside: has pentose sugar and nitrogenase base, no phosphate DNA and Genetic Information Flow Figure 6.1 Genetic Information Flow and the Components of the Nucleic Acids DNA and Genetic Information Flow Properties of the DNA Double Helix – Nucleic acid backbone is a polymer of alternating phosphates and the pentose sugar deoxyribose – Phosphodiester bonds connect 3’ carbon of one sugar to 5’ carbon of the adjacent sugar. – DNA is double-stranded and held together by hydrogen bonding between bases – Two strands have complementary base sequences Adenine pairs with thymine Guanine pairs with cytosine – Two strands are antiparallel (5’ to 3’ and 3’ to 5’) forming double helix Contains two grooves, major (where proteins bind) and minor DNA and Genetic Information Flow Arrangement of the DNA Structure DNA Double Helix DNA and Genetic Information Flow Size, Shape, and Supercoiling of DNA – Size is expressed in number of nucleotide base pairs 1000 base pairs = 1 kilobase pair = 1 kbp 1 million base pairs = 1 megabase pair = 1Mbp – E. coli genome = 4.64 Mbp – Linear DNA length is several hundred times longer than cell, so supercoiling compacts DNA to accommodate genome – Topoisomerases insert and remove supercoils – Negative supercoiling: twisted in opposite sense relative to right-handed double helix; found in most cells – DNA gyrase: a topoisomerase enzyme that introduces supercoils into DNA via double-strand breaks – Positive supercoiling: helps prevent DNA melting at high temperatures (e.g., some Archaea) DNA and Genetic Information Flow Figure 6.4 Supercoiled DNA and DNA Gyrase DNA and Genetic Information Flow Genes and the Steps in Biological Information Flow – Central dogma: Genetic information flow can be divided into three stages, DNA to RNA to protein Gene expression transfers DNA information to RNA. Three main RNA classes involved in protein synthesis: – mRNA (messenger RNA): carry information to ribosome – tRNA (transfer RNA): convert mRNA information to amino acid sequence – rRNA (ribosomal RNA): catalytic and structural ribosome components – Three stages of information flow Replication: DNA is duplicated by DNA polymerase Transcription: Information from DNA is transferred to RNA by RNA polymerase Translation: Information in mRNA is used to build polypeptides on ribosome DNA and Genetic Information Flow Figure 6.5 Synthesis of the Three Types of Informational Macromolecules in the Processes of Replication From (DNA D N→A DNA to D N )A, Transcription (DNA →ARNA), From D N to R N A and Translation From (RNA RN→A Protein) to Protein DNA and Genetic Information Flow Genes and the Steps in Biological Information Flow – Many different RNAs can be described from a short DNA region – Eukaryotes Each gene is transcribed individually into a single mRNA Replication and transcription occur in nucleus RNAs must be exported outside nucleus for translation – Prokaryotes Multiple genes may be transcribed in one mRNA Coupled transcription and translation occur, producing proteins at maximal rate Key Concept 2: Genetic Elements Genetic Elements: Chromosomes and Plasmids Chromosome: main genetic element in prokaryotes Other genetic elements include virus genomes, plasmids, organellar genomes, and transposable elements Plasmids: circular or linear double-stranded DNA that replicate separately from chromosome Transposable elements – segments of DNA inserted into other DNA molecules that can move from one site to another site on the same or a different DNA molecule (e.g., chromosomes, plasmids, viral genomes) – found in prokaryotes and eukaryotes Genetic Elements: Chromosomes and Plasmids Most Bacteria and Archaea have single circular chromosome carrying all/most genes Eukaryotes: two or more linear chromosomes Viruses contain either RNA or DNA genomes (SS/ DS; linear/ circular) Table 6.1 Kinds of Genetic Elements Organism Element Type of nucleic acid Description Virus Virus genome Single- or double- Relatively short, circular or stranded DNA or RNA linear Bacteria, Archaea Chromosome Double-stranded DNA Extremely long, usually circular Eukarya Chromosome Double-stranded DNA Extremely long, linear Mitochondrion or Organellar genome Double-stranded DNA Medium length, usually chloroplast circular All organisms Plasmid super a Double-stranded DNA Relatively short circular or Plasmid a linear, extrachromosomal All organisms Transposable element Double-stranded DNA Always found inserted into another DNA molecule a Plasmids are uncommon in eukaryotes. Chromosomal Gene Arrangements in E. coli – Some features of the Escherichia coli K-12 chromosome About 5 Mbp in size Almost 4300 possible protein-encoding genes make up 88 percent of the genome Compact relative to eukaryotes, which contain extra DNA Many genes encoding enzymes of a single biochemical pathway are clustered into operons, transcribed to form single mRNA and regulated as a unit Many genes for biochemical pathways are not clustered Thus, operons appear to be exceptions instead of the rule Figure 6.7 The Chromosome of Escherichia coli Strain K-12 Plasmids Plasmids – found in many Bacteria and Archaea – mostly nonessential – nearly all double-stranded DNA, mostly circular – typically less than 5 percent of the size of the chromosome – present in different copy number (1 or a few to 100+ copies) – may influence host cell physiology (e.g., survival under certain conditions) – R plasmids ▪ Resistance plasmids; confer resistance to antibiotics or other growth inhibitors ▪ Several antibiotic resistance genes can be encoded on one R plasmid (e.g., R100) Plasmids Major antibiotic resistance genes and other key functions: mer, mercuric ion resistance; sul, sulfonamide resistance; str, streptomycin resistance; cat, chloramphenicol resistance; tet, tetracycline resistance; oriT, origin of conjugative transfer; tra, transfer functions. Figure 6.9 Genetic Map of the Resistance Plasmid R100 Plasmids Plasmids – In several pathogenic bacteria, virulence factors (e.g., ability to attach or produce toxins) are encoded by plasmids – Bacteriocins (proteins that inhibit or kill closely related species or different strains of the same species) can be encoded on plasmids – Rhizobia require plasmid-encoded functions to fix nitrogen – Metabolism (e.g., hydrocarbon degradation) – Important for conjugation (horizontal gene transfer) Key Concept 3: Copying the Genetic Blueprint: DNA Replication Templates, Enzymes, and the Replication Fork DNA replication is semiconservative: each of the two resulting double helices has one new strand and one parental (template) strand Replication Always proceeds from the 5’ end to the 3’ end Figure 6.10 Overview of DNA Replication Templates, Enzymes, and the Replication Fork Replication Enzymes – DNA polymerases catalyze polymerization of deoxynucleotides – Five different DNA polymerases (DNA Pol I-V) in E. Coli DNA Pol III is primary enzyme replicating chromosomal DNA; also DNA Pol I plays lesser role Others repair damage – DNA polymerases can only add nucleotides to pre-existing 3’-OH and require a primer: short stretch of RNA Primer made from RNA by primase Primer eventually removed and replaced with DNA Figure 6.11 The RNA Primer Table 6.2 Major Enzymes That Participate in DNA Replication in Bacteria Enzyme Encoding genes Function DNA gyrase gyrAB Replaces supercoils ahead of Replisome Origin-binding protein dnaA Binds origin of replication to open double helix Helicase loader dnaC Loads helicase at origin Helicase dnaB Unwinds double helix at replication fork Single-strand binding protein ssb Prevents single strands from annealing Primase dnaG Primes new strands of DNA Blank DNA polymerase three III Main polymerizing enzyme Sliding clamp dnaN Holds Pol three III on DNA Clamp loader holA–E Loads Pol III three onto sliding clamp Dimerization subunit (Tau) dnaX Holds together the two core enzymes for the leading and lagging strands Polymerase subunit dnaE Strand elongation Proofreading subunit dnaQ Proofreading DNA polymerase I one polA Excises RNA primer and fills in gaps DNA ligase ligA, ligB Seals nicks in DNA Tus protein tus Binds terminus and blocks progress of the replication fork Topoisomerase IV four parCE Unlinking of interlocked circles Templates, Enzymes, and the Replication Fork Initiation of DNA Synthesis – Double helix must be unwound to expose template strands at replication fork – DNA helicase unwinds double helix – DNA synthesis begins at the origin of replication (oriC) in prokaryotes, where DnaA protein binds and opens double helix – Helicase (DnaB) and loader protein (DnaC) bind – Primase and DNA polymerase enzymes loaded and DNA replication begins – Replication fork moves along DNA Figure 6.12 DNA Helicase Unwinding a Double Helix Templates, Enzymes, and the Replication Fork Leading and Lagging Strands and the Replication Process – DNA replication at replication fork (Figure 6.13) occurs continuously on the leading strand 5 to 3 (always free 3′ − OH) discontinuously on lagging strand—no 3′-OH; primase synthesizes multiple RNA primers – Primase replaced by DNA Pol III, DNA synthesis continues until it reaches previously synthesized DNA (Figure 6.14) – DNA polymerase I removes the RNA primer and replaces it with DNA DNA ligase seals nicks in the DNA Templates, Enzymes, and the Replication Fork Figure 6.13 Events at the DNA Replication Fork on the Nucleoid Templates, Enzymes, and the Replication Fork Figure 6.14 Sealing Two Fragments on the Lagging Strand Proofreading DNA synthesis is bidirectional in prokaryotes because of circular chromosome – two replication forks moving in opposite directions (j Figure 6.15 Replication of Circular DNA: The Theta Structure Proofreading The Replisome – large replication complex of multiple proteins ▪ Primosome: helicase and primase subcomplex within replisome – When replication forks collide at terminus of replication (opposite origin), replisome is finished – DNA is partitioned; facilitated by FtsZ Figure 6.16 The Replisome Copyright © 2021, 2018, 2015 Pearson Education, Inc. All Rights Reserved Proofreading Fidelity of DNA Replication: Proofreading – DNA replication is extremely accurate ▪ Errors introduce mutations: changes in sequence ▪ Mutation rates in cells are 10−8 to 10−11 errors per base pair inserted ▪ Proofreading (Figure 6.17) helps to ensure high fidelity. – During replication, DNA Pol I and Pol III can detect base pair mismatch through double helix distortion; remove with 3 → 5 exonuclease and insert again – Exonuclease proofreading occurs in prokaryotes, eukaryotes, and viral DNA replication systems Copyright © 2021, 2018, 2015 Pearson Education, Inc. All Rights Reserved Proofreading Figure 6.17 Proofreading by the 3 → 5 3 prime to 5 prime Exonuclease Activity of DNA Polymerase III three Copyright © 2021, 2018, 2015 Pearson Education, Inc. All Rights Reserved Key Concept 4: RNA Synthesis: Transcription Transcription in Bacteria Transcription: RNA synthesis off DNA template – Catalyzed by RNA polymerase – Chain growth is 5′ to 3′ just like DNA replication – no priming needed RNA Polymerases and the Promoter Sequence: – RNA polymerase has five different subunits forming RN polymerase holoenzyme complex Sigma not as tightly bound, easily dissociates to yield RNA polymerase core enzyme Core enzyme synthesizes RNA Sigma recognizes initiation sites on DNA called promoters to begin transcription – Transcription occurs in opposite directions on DNA strands – Transcription terminators mark end of transcription Transcription in Bacteria Steps in RNA synthesis: – The initiation site (promoter) and termination site are specific nucleotide sequences on the DNA – A sigma factor recognizes promoter and initiation sites along the strand of DNA – Sigma binds to the promoter region and then RNA core polymerase binds to begin transcription. – RNA polymerase moves down the DNA chain, temporarily opening the double helix (creating a transcription bubble) and transcribing one of the DNA strands. – Termination site is reached and the chain growth stops. Polymerase and RNA is then released. Figure 6.18 Transcription Transcription in Bacteria Sigma factors (s) recognize two highly conserved regions of promoter: – Pribnow box: located 10 bases before the start of transcription (−10 region) – −35 region: located ~35 bases upstream of transcription Transcription in Bacteria Units of Transcription and Polycistronic mRNA – Transcriptional units: DNA segments transcribed into 1 RNA molecule bounded by initiation and termination sites can result from 1 or 2+ (cotranscribed) genes – Most genes encode proteins, but some encode untranslated RNA s (e.g., rRNA, tRNA) e.g., three types of rRNA: 16S, 23S, and 5S + one tRNA Figure 6.21 A Ribosomal rRNA Transcription Unit From Bacteria and Its Subsequent Processing Transcription in Bacteria Units of Transcription and Polycistronic mRNA – Operons are transcribed into a single mRNA called a polycistronic mRNA (Figure 6.22) containing multiple open reading frames that encode amino acids Figure 6.22 Operon and Polycistronic mRNA Structure Transcription in Bacteria Termination of Transcription – Termination governed by specific DNA sequences – Example: GC-rich sequence containing an inverted repeat and a central nonrepeating segment followed by several adenines RNA forms stem-loop by intra-strand base pairing, RNA polymerase pauses, DNA and RNA dissociate – Rho-dependent termination: Rho protein recognizes specific DNA sequences (Rho-dependent termination site) and releases RNA polymerase from DNA Figure 6.23 Inverted Repeats and Transcription Termination Transcription in Archea Many archaeal transcription and translation details are more similar to eukaryotes than bacteria ✓ Archaea contain one RNA polymerase: resembles eukaryotic polymerase II ✓ Most important promoter recognition sequence is 6–8 base pair “TATA” box, recognized by TATA-binding protein (TBP) transcription factor ✓ B recognition element (BRE), upstream of TATA box, recognized by archaeal transcription factor B (TFB) ✓ Binding of TBP to TATA and TFB to BRE enables RNA polymerase binding and transcription initiation Figure 6.24 Promoter Architecture and Transcription in Archaea – Less known about termination in Archaea Some archaeal genes have inverted repeats followed by AT-rich sequence similar to bacterial terminators Transcription in Archaea RNA Processing in Eukaryotes and intervening Sequences in Archaea – Eukaryotic genes have coding and noncoding regions exons: coding sequences introns: intervening noncoding sequences – found in tRNA and rRNA genes of Archaea RNA processing of primary transcript (original transcribed RNA) required to form mature RNAs for translation – splicing: removing introns and joining exons Eukaryotes: splicing occurs in nucleus via the spliceosome (RNA + protein) Archaea: introns rare in protein encoding-genes but need to be removed from tRNA- and rRNA-encoding transcripts by special ribonuclease Transcription in Archaea RNA Processing in Eukaryotes and intervening Sequences in Archaea: two unique eukaryotic mRNA processing steps occur in nucleus before splicing- – capping: addition of methylated guanine to 5’ end of mRNA in reverse orienation, needed to initiate translation – polyadenylation: trimming 3’ end; addition of 100–200 adenylate residues (poly(A) tail) to stabilize mRNA; must be removed before mRNA can be degraded Figure 6.26 Processing of the Primary Transcript Into Mature mRNA in Eukaryotes Key Concept 5: Protein Synthesis: Translation Translation in Bacteria Unique features in bacterial translation – 60 different tRNA’s for 22 amino acids (prokaryotic specific and rarely used) Start and Stop Codons and Reading Frames: – Start codon: Translation begins with AUG, encodes N-formylmethionine in Bacteria and methionine in Archaea and Eukarya – Reading frame: Triplet code requires translation to begin at the correct nucleotide. – Shine–Dalgarno sequence, or the ribosome-binding site (RBS), ensures proper reading frame in Bacteria – Stop (nonsense) codons: terminate translation (UAA, UAG, and UGA) Sometimes unusual amino acids selenocysteine and pyrrolysine can be encoded by stop/nonsense codons – Open reading frame (ORF): AUG followed by a number of codons and a stop codon Translation in Bacteria Unique features in bacterial translation – Bacterial ribosomes: rRNA subunits: – 70s=50s large subunit (5s + 23s + 31 proteins) + 30s small subunit (16s + 21 proteins) – Small subunit initiates translation, large subunit (23s) handles peptidyl transferase reaction Figure 6.35 Ribosome Structure Translation in Bacteria Ribosomes and the Initiation of Translation – Initiation begins with free 30S ribosomal subunit – Initiation complex (30S subunit, mRNA, formylmethionine tRNA, initiation factors (I F1, I F2, I F3) forms – 50S ribosomal subunit added = 70S ribosome – Ribosome binding site: 3–9 nucleotides toward 5’ end of mRNA, complementary to sequences on 3’ end of 16S rRNA – Base pairing holds ribosome-mRNA complex in frame Figure 6.36 The Ribosome and Initiation of Protein Synthesis Translation in Bacteria 1. Initiation: – two ribosomal subunits + formylmethionine tRNA + initiation factors assemble with mRNA – begins at an AUG start codon 2. Elongation: – amino acids brought to the ribosome and added to the growing polypeptide – occurs in the A (acceptor) and P (peptide) sites of ribosome – mRNA threads through ribosome – tRNAs interact at A (acceptor) and P (peptide) sites on 50S – A site: incoming charged tRNA first attaches; loading assisted by elongation factor EF-Tu – P site: growing polypeptide chain is attached to prior tRNA – Growing polypeptide moves to tRNA at the A site as new peptide bond is formed – After elongation, tRNA holding peptide transferred to P site – Amino acid-free tRNA pushed to E (exit site) and released from ribosome Figure 6.37 Elongation Cycle of Translation Translation in Bacteria Elongation, Translocation, and Termination – Polysomes: a complex formed by multiple ribosomes simultaneously translating a single mRNA – Termination occurs at stop codon Release factors (RFs): recognize stop codon and cleave polypeptide from tRNA Ribosomal subunits dissociate Subunits free to form new initiation complex and repeat process Figure 6.38 Polysomes Translation in Bacteria Freeing trapped Ribosomes and Trans-Translation – ribosomes trapped if no stop codon at end of mRNA – Release factor cannot bind, ribosome cannot be released = “trapped” – Trans-translation: produces small RNA (tmRNA) that frees stalled ribosomes Mimics both tRNA (carries alanine) and mRNA (contains stretch that can be translated) Binds next to defective mRNA, allowing protein synthesis to proceed. Also encodes degradation signal Figure 6.39 Freeing of a Stalled Ribosome by tmRNA Protein folding in Bacteria Polypeptides go through primary →quaternary folding to form 3D protein – domains structurally independent regions of polypeptide separated from each other by less structured portions of polypeptide – in eukaryotes domains fold independently right after being synthesized molecular chaperones not as important – in bacteria polypeptide does not fold until after synthesis of entire polypeptide molecular chaperones play important role Protein folding in Bacteria In bacteria, some polypeptides require assistance from molecular chaperones or chaperonins for folding to occur They only assist in the folding, are not incorporated into protein. Can also aid in refolding partially denatured proteins (heat shock proteins) – In Escherichia coli, DnaK, DnaJ, GroEL, GroES are key. DnaK and DnaJ are ATP-dependent enzymes that slow polypeptide folding GroEL and GroES fold partially folded proteins Figure 6.40 The Activity of Chaperone Proteins Protein secretion Some proteins must be transported outside cytoplasmic membrane into periplasm or inserted into cytoplasmic/outer membrane Some proteins (e.g., toxins, exoenzymes) must be secreted into the environment Signal sequences: found on proteins requiring transport from cell – 15–20 residues long – Found at the beginning of the protein molecule – Signal the cell’s secretory system (Sec system); Prevent protein from completely folding and prepares it for transfer SecA: bind protein that are exported to periplasm SRP (Signal recognition Particle): binds proteins destined for membrane insertion Protein secretion Figure 6.42 Export of Proteins via the Sec A Secretory System Protein secretion Secretion of Folded Proteins: The Tat System – Proteins that fold in the cytoplasm are exported by a transport system distinct from Sec, called the Tat protein export system TatBC proteins bind folded protein and transfer to membrane spanning TatA which uses PMF for expulsion Iron–sulfur proteins Redox proteins Figure 6.43 Secretion Systems in Gram-Negative Bacteria Protein Synthesis in Archaea Archaea share 67 of 78 proteins of the ribosome in common with Eukarya Eukarya and Archaea have twice as many translation factors as Bacteria Translational machinery is more similar between Eukarya and Archaea Transcription and Translation in Eukarya Eukaryotes have multiple RNA polymerases: – RNA polymerase I: transcribes genes for two large rRNAs (23S & 16S) – RNA polymerase II: transcribes protein-encoding genes – RNA polymerase III: transcribes genes for tRNA, 5S rRNA, and other small RNA molecules Each class of RNA polymerase recognizes a distinct promoter Transcription and Translation in Eukarya Protein synthesis is more complex in eukaryotes than in Bacteria ↓ cuteraynease DNA bigger so – Eukaryotic ribosomes are larger than bacterial ribosomes – More initiation factors in Eukarya – Eukaryotic mRNA is monocistronic – Eukaryotic mRNA has no ribosome-binding site Eukaryotic mRNA is recognized by its cap Translation comparison Shared Features of Bacteria, Archaea, and Eukaryotes Bacteria and Archaea share several fundamental properties that are absent from eukaryotes – Typically single-celled, most divide by binary fission – Neither possesses a nucleus or membrane-bound organelles – Archaea and Bacteria have coupled transcription and translation – Both possess a single, circular chromosome – Both use Shine–Dalgarno sequences to indicate translation start  Il Bacteria Eukarya 1 RNA. polymerase I multiple 2. Promoter - 35 region Zsigma factor TATA DOX - TBP recognized by - 10 region/Pribnow box monocistronic polycistronic 3. Transcriptions I transcript Unit 1 RFI ORF I. ORFI T protein I transcript. It Unit = I protein 4. Operon present absent present 5. RNA absent Y I processing splicing S'cap +. 3 all I I I 22 a a 20 a Eukarya.. a Bacteria 1 Start methionine. Formyl methionine codon 3 codons 3 stop codons 2 Stop stop t Uga. Vaa UAG , codon selenocysteine + pyroleucine , MRNA- SD sequence/. 3 "captPODat ribosome interaction RBS n 70S Type 4 ⑳ G of 80s. ribosome ↳ 305