6-14 Gene Regulation - BIO 2045 Molecular Biology and...

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Unformatted text preview: BIO 2045 Molecular Biology and Genome Analysis Gene Regulation in Prokaryotes and Eukaryotes To demonstrate an understanding of the structure and expression of nuclear genes in prokaryotic and eukaryotic organisms. Evaluate the techniques that have allowed gene expression to be studied. www.ncbi.nih.gov/entrez/query.fcgi?db=Books What is gene regulation? Genes can either be switched on or switched off : Transcribed/not transcribed Expressed/ not expressed Escherichia coli has a genome size of 4.2 x 106 bp and ~2000 genes. But <5% are actively transcribed at a given time. Bacteria are TOTIPOTENT rRNA -galactosidase Biotin synthetase 1 copy sec-1 1 copy min-1 1 copy10min-1 Transcriptional Regulation Co-ordinate Regulation catabolite repression Modulated Regulation Regulation of initiation of transcription recruitment of RNA polymerase to the promoter Regulation of transcription and translation The contrast between prokaryotes and eukaryotes Transcriptional control in prokaryotes Ribosome Protein mRNA DNA RNA Polymerase Bacterial Cell Bacterial chromosome Transcription and translation occur together Typical mRNA 1/2 life ~ 1- 3 mins Transcription control in Eukaryotes Nuclear mRNA DNA RNA Polymerase Ribosome Protein Cytoplasmic Typical mRNA 1/2 life >30 min Promoter Structure in Prokaryotes -60 -50 -40 -30 -20 -10 +1 5' 3' T80 A95T45 A60A50T 96 -10 Sequence/Pribnow Box T82 T84 G45 A 78C65 A45 -35 Sequence Closer the sequence is to consensus the better gene expression RNA Polymerase -9 +1 Influenced by: Formation of Open Promoter Complex Promoter sequence Conformation of promoter DNA supercoiling DNA-binding proteins RNA-binding proteins Bacterial RNA polymerase Product 2 sub-units 40 kDa sub-unit 155 kDa ' sub-unit 160 kDa sub-unit 32-90 kDa Function Enzyme assembly Promoter recognition ~ activator-binding Catalytic Core Catalytic Core Encoded by rpoA rpoB rpoC rpoD Promoter specificity E. coli holoenzyme = 465 kDa Sigma Factors control DNAbinding by RNA polymerase Gene Factor Use rpoD rpoH rpoE rpoN fliA 70 32 E 54 F -35 seq Separation -10 seq general TTGACA 16-18 bp TATAAT heat shock CCCTTGAA 13-15 bp CCCGATNT heat shock ..........not known................ nitrogen CTGGNA 6 bp TTGCA flagella CTAAA 15 bp GCCGATAA 1. factor controls DNA-binding at promoter for initiation 2. Detaches from core enzyme during transcriptional elongation 3. Development can be controlled by cascades of factors - Bacillus subtilis sporulation 4. Anti-sigma factors exist as form of control/virus infection Regulation of Bacterial Gene Expression Positive regulation activator transcription Negative regulation repressor No transcription X promoter operator No transcription X transcription promoter operator Control of activators and repressors Allosteric site Allosteric effector DNA-binding site X Operator The Lac-Operon A paradigm for gene regulation in bacteria First evidence for repression of gene expression Jacques Monod and Franois Jacob, 1961 Introduced concept of genes being organised in co-ordinately regulated clusters - OPERONS Discovered before molecular biology techniques using classical genetics Provided an explanation for catabolite repression http://vcell.ndsu.nodak.edu/~christjo/vcell/animationSite/lacOperon/ Lac-Operon Lac I Lac O Lac Z Lac Y Lac A mRNA -galactosidase 500 kDa Lac repressor -galactoside -galactoside permease transacetylase 30 kDa O Lac I RNA polymerase Z Y A X No transcription No -galactosidase activity Lactose absent from growth medium O Lac I Z Y A mRNA Lactose added Lactose metabolism How was the lac-operon model formulated? 1. Mutations in structural genes? lacZ loss of -gal activity. No growth on lactose lacY loss of permease activity. No growth on lactose lacA loss of acetylase activity. 2. Mutations in controlling elements lac I : mutants produced induced levels of -gal activity in absence of lactose. Constitutive. lac IS mutants do not produce any -gal activity even in presence of lactose. lacOC mutants produce induced levels of -gal activity all time. Constitutive. 3. Partial diploid construction Constructing Partial Diploids in Bacteria Transformation lacZ+ lacZGenotype Phenotype LacZWild type LacZ+ Conjugation lacZ+ lacZ- Transduction lacZ+ lacZ- COMPLEMENTATION O Lac I RNA polymerase Z Y A X No -galactosidase Structural gene mutations Lactose added Regulatory Gene Mutations - lac I O Lac I Z Y A X RNA polymerase mRNA X Cannot bind to operator DNA Constitutive Lactose metabolism Regulatory Gene Mutations - lac O O Lac I Lac Oc Z Y A X X mRNA Constitutive Lactose metabolism Lac-operon Partial Diploid Phenotypes lacI+ lacZlacI- lacZ+ lacI- lacZlacI+ lacZ+ = Normal inducible expression = Normal inducible expression lacI+ is dominant to lacIlacIS lacI+ = suppressed phenotype: No growth on lactose lacIS is dominant to lacI+ Lac I encodes a diffusible cytoplasmic factor Lac-operon Partial Diploid Phenotypes lacOC lacZ+ lacO+ lacZ+ lacOC lacZ+ lacO+ lacZlacOC lacZlacO+ lacZ+ = constitutive expression = wild type inducible expression = constitutive expression lacO is dominant only when on same piece of DNA as lacZ lacO is a cis-acting factor lacI is a trans-acting factor Reading and associated tasks Core Activities Review lecture material and animations on server Chapter 9. Genomes 2. T.A Brown (p261--) Griffiths et al. Introduction to Genetic Analysis 6th edition. Chapter 11. Further Reading Lewin Genes VII. Chapter 9, 10. For more on cis-trans test and genetic mapping in bacteria read Griffiths Chapter 10-12. BIO 2045 Gene Regulation in Prokaryotes and Eukaryotes Lac-operon partial diploid phenotypes Operon organisation Attenuation Antisense RNA. Lac-operon Partial Diploid Phenotypes lacI+ lacZlacI- lacZ+ lacI- lacZlacI+ lacZ+ = Normal inducible expression = Normal inducible expression lacI+ is dominant to lacIlacIS lacI+ = suppressed phenotype: No growth on lactose lacIS is dominant to lacI+ Lac I encodes a diffusible cytoplasmic factor lacI-/lacI+ partial diploid Lac I O Z Y A Lac I O Z Y A X X Cannot bind to operator DNA Binds normally mRNA lacI-/lacI+ partial diploid Lac I O Z Y A Lac I O Z Y A X X Cannot bind to operator DNA Binds normally Normal Regulated Lactose Metabolism Lac gene repressed in absence of lactose Lac genes induced in presence of lactose Regardless of position of lacZ- mutation O Lac I Z Y A X RNA polymerase X No transcription No -galactosidase activity Lactose added lacIs mutation lacIS/lacI+ partial diploid Lac I O Z Y A Lac I O Z Y A X No transcription lacIS mutation is dominant Faulty repressor bids to both operator sites and cannot be removed by addition of lactose Lac-operon Partial Diploid Phenotypes lacOC lacZ+ lacO+ lacZ+ lacOC lacZ+ lacO+ lacZlacOC lacZlacO+ lacZ+ = constitutive expression = wild type inducible expression = constitutive expression lacO is dominant only when on same piece of DNA as lacZ lacO is a cis-acting factor lacI is a trans-acting factor lacOc/lacO+ partial diploid Lac I O Z X Y A Lac I O Z Y A mRNA Constitutive Lactose metabolism lacOc partial diploid with lacZ- in trans Lac I O Z X X Y A Lac I O Z X Y A mRNA Constitutive Lactose metabolism lacOc/partial diploid with lacZ- in cis Lac I O Z X X Y A Lac I O Z Y A No functional -galactosidase Regulated Lactose metabolism Dominant lacI mutants - lacI-d X Lac I mRNA O Z Y A Lac I mRNA O Z Y A Makes faulty sub-unit Repressor inactive with lacI-d sub-unit Constitutive expression Catabolite Repression Lactose utilization Cell Density OD590 Glucose utilization Time Diauxic Growth Curve Catabolite repression in E.coli is controlled by cAMP [High Glucose] [Low cyclic AMP] [Low Glucose] [High cyclic AMP] Mutants isolated: permanently repressed (no -gal activity in absence of glucose) Class I: Phenotype corrected by adding cAMP cyaA mutants: adenylate cyclase gene Class II: Not correctable CAP mutants: catabolite activator protein gene What is CAP? 1. Positive Regulator - An apoactivator protein 2. Binds to pentameric sequence TGTGA ACACT -90 -35 Transcription cAMP 5-6 bp TGTGA ACACT CAP -10 CAP I O Z Y A 1. Glucose present; no lactose cAMP low; full repression CAP 2. Glucose and lactose present cAMP low; some lac mRNA I O Z Y A I CAP/cAMP O Z Y A 3. Glucose absent; lactose present cAMP high; full derepression Positive and negative control of bacterial genes The arabinose operon araC araO araI araB araA araD Promoter (2 operators) Arabinose Structural Genes Activator 1. Presence of arabinose/absence of glucose araC araO araI araB araA araD CAP-cAMP mRNA Arabinose metabolism 1. Absence of arabinose araO araI araC araB araA araD Repression Fine Control of Gene Expression: The attenuator trpR trpO trpE trpD trpC trpB trpA Tryptophan biosynthetic genes Repressed in presence of Trp Trp aporepressor Tryptophan Regulation occurs at two levels (a) Gross control (b) Fine control Charles Yanofsky trpR- mutants No repressor Produce trp-operon mRNA in presence of Tryptpphan = Trp mRNA = 10-fold more Trp mRNA 30 bp AUG X trpE trpD trpR- + TRYPTOPHAN trpR- - TRYPTOPHAN +1 163 bases mRNA trpEC Leader 141-163 terminated 90% of time The Attenuator +1 AUG trpE trpD 14 a.a. leader peptide 1 2 Trp codons 2 3 4 2 can base-pair with 3 3 can base-pair with 2 or 4 4 can base-pair with 3 Transcription termination signal Attenuator 1. Tryptophan in abundance 1 2 3 4 tRNAtrp levels high RNA polymerase terminates 1. No Tryptophan 2 3 4 Transcription of trp genes proceeds Bacterial genes can also be regulated by anti-sense RNA molecules Hyperosmotic stress envZ OmpR ompF micF mRNA mRNA Degraded by endonuclease Reading and associated tasks Review lecture material and animations on WebCT Chapter 9 Genomes 2 Brown TA. Introduction to Genetic analysis Griffiths 8th edition Genes VIII Lewin Summary Bacterial genes are regulated by activator and repressor proteins (transacting factors) These bind to DNA sequences called operators (cis-acting elements) Fine control involves attenuators Anti-sense mRNAs can also regulate bacterial genes BIO 2045 Molecular Biology Gene Regulation in Prokaryotes and Eukaryotes Genetic control of lysogeny in phage Late lysis Establishing lysogeny How the decision is made Maintenance of lysogeny. Genetic control of lysogeny in phage Discovered by Andr Lwoff, Franois Jacob and Jacques Monod in Pasteur Institute Mark Ptashne and Alan Campbell deciphered molecular details. Paradigm for understanding gene control. Genetic Control of Lysogeny Lysis Replicate DNA Make Heads and Tails Assemble phage Lyse Cell Lysogeny Integrate phage DNA into genome - Prevent all of this cIII 10 genes Re co mb ina t io n N cI cro cII DN A Re pl ica tio n 2 genes Q LYSIS Lambda genome ~43 genes 3 genes 12 genes D EA H 10 genes IL TA 1. Very Early Genes mRNA N PL cI PR CRO mRNA N gene and Cro gene transcribed N encodes an anti-terminator protein N cIII Transcription Termination signal N Nut cI CR PL N cIII N Nut cI CR PL N turns on O and P DNA replication genes and Q to right of Cro Recombination genes to left of cIII 2. Late Lytic genesN cIII Re co mb ina t io n Re pl ica Q protein is an anti-terminator binds to Qut sites tion cI cro cIII DN A Q LYSIS Switches on Head and Tail genes IL TA 12 genes D EA H 10 genes 2. Late Lytic Genes PL PR N cI CRO Cro gene transcribed Repressor that prevents early genes being expressed Prevents expression of cI - Lambda Repressor Establishing lysogeny PL N Int cII Integrase cI PR CRO cII cII PRE PI cII cII is a transcriptional activator Promoter left Promoter for repressor maintenance Promoter right PL PRM OR3 PR OR2 OR1 CRO N cI X N gene off - Q gene off - No lysis X Cro off Lysogeny established Phage Infection Rich medium (high) cIII cII Bacterial Proteases Active Starving (low) Inactive Lysis Lysogeny Maintenance of Lysogeny cI OR3 OR2 OR1 PRM PR Cro 236 Operator sites - 17 bp Repressor protein Resistance to superinfection C 132 92 N 1 C C N N C C N N C C N N C C N N OR 2 Minor groove Major groove OR 1 Lambda repressor shows co-operativity in binding OR1 binding facilitates binding at OR2 OR1 > OR2> OR3 Functions of Lambda Repressor Positive control Binding at OR1 and OR2 Activates cI Negative control Binding at OR1 prevents Cro transcription Binding at OL prevents N transcription C C N N OR1 X C C N N OR3 C C N N OR2 PRM PR X Why are E.coli lysogens immune to superinfection? Phage C N C N C N C N C C C C C CN N N N N N Prophage Lysogen UV Light induces lyss of lysogens of E. coli. UV exposure can cause lysis to occur Normal response to UV light in E. coli involves the SOS response and recA protein This is co-opted by phage lambda How Does UV Light induce Lysis? 1. UV Light damages DNA 2. Induces SOS response recA ssDNA lexA recA umuC uvrA uvrB sfiB lexA repressor How Does UV Light induce Lysis? 1. UV Light damages DNA 2. Induces SOS response recA ssDNA lexA recA umuC uvrA uvrB sfiB lexA repressor ON!!! C C N N cI OR3 C C N N OR2 C C N N OR1 Cro PRM C C N N PR Lysogen is exposed to UV Light recA produced C C N N cI OR3 C C N N OR2 C C N N OR1 Cro PRM C N C N PR recA cleaves Lambda repressor Transcription decreases rapidly X C C OR2 N N OR1 Cro cI OR3 PRM C N C N PR recA cleaves Lambda repressor RNA Polymerase X cI OR3 OR2 OR1 Cro PRM C N C N PR 66 aa Cro produced Cro repressor produced Genetic switch is flipped X cI OR3 OR2 OR1 Cro PRM Cro repressor has opposite affinity OR3 > OR2> OR1 PR 66 aa Cro produced A Genetic Switch Once Cro is transcribed it will repress cI repressor expression N gene will be derepressed N will activate gene expression to excise prophage Q will activate head and tail genes Lysis will occur Mark Ptashne The Key Experiments in understanding repressor/operator function 1. The repressor (a) Clear Mutants plaques normally turbid due to background frequency of lysogeny Occasional clear plaques Mutants unable to carry out lysogeny cI -----------------cII -----------------cIII -----------------Encodes repressor Activates repressor and integrase Protect cII from proteases Virulent mutants vir mutants are able to superinfect lysogens - very rare class of mutant caused by two mutations vir Phage Mutations at OL and OR C C N N C C N N CC CC NN C CN N NN XX Immune to repressor Prophage LYSIS Lysogen How do we know that free repressor is in the cytoplasm and prophage is in the bacterial genome? C C N N C C N N C C N N C C C N N C N N DNA transferred Not cytoplasm X C C N N X X Ex-conjugants lyse Because cI concn is too low Zygotic induction Lysogen Non-lysogen 3. How do we know that repressor binds DNA? A. Purification of repressor Infect E.coli cells with 3H-labelled cI- 14C-labelled Mix and rupture cells. Isolate proteins fractionate by size Repressor H 14C A subtractive screen 3 Experiment to show binding of repressor to DNA DNA Repressor Radio-labelled Phage B DNA 1 Centrifuge 2 DNA in pellet Protein in supernatant 2 1 bottom top 4. How do we know the structure of repressor? A. Purified repressor treated with PAPAIN (protease) to produce two globular proteins B. Each analyse by NMR C. Dimerization shown by gel filtration of protein at high concentration 5. How do we know that repressor binds to operator sites? O R2 O R1 O R3 Promoter cloned into Plasmid vector Nitrocellulose filter-binding assay DNA with: OR1 OR1 and OR2 OR1 and OR2 and OR3 Used in DNA binding assays with excess Repressor protein 6. How do we know the nature of the operator? A. Cloned and sequenced. - Shown to be 17-bp palindromic sequences - Dyad symmetry - Similar to lac operator sequences B. repressor and lac repressor are related - both are HELIX-TURN-HELIX proteins Mark Ptashne - A Genetic Switch Summary Lambda lysogeny genetic control represents precise genetic switch Vast majority of gene expression control mechanisms work using same principles Investigated using multi-disciplinary approach molecular genetics, biochemistry A paradigm for investigating gene regulation Genetic information flow in Eukaryotic Cells Transcription - Initiation - Elongation DNA hnRNA Nucleosomes Intron Splicing Nuclear mRNA Nuclear Export Post-translational Regulation Translation Cytoplasmic Comparative analysis of transcription in eukaryotes and prokaryotes Differs spatially (nuclear/cytoplasmic) Organisationally Complexity/Diversity of RNA polymerases Promoter structure Basal transcription/Activated transcription Transcription factors Enhancers Silencers RNA Polymerases in Eukaryotes RNA polymerase I - Transcribes rRNA (Major activity) RNA polymerase II - mRNA (protein-encoding genes) RNA Polymerase III - tRNA and snRNAs Eukaryotic RNA Polymerase II has 10 sub-units 500 kDa in size - largest sub-unit is 200 kDa and related to ' - Contains C-terminal domain (CTD) that becomes highly phosphorylated during initiation on serine, threonine residues -Inhibited by -amanatin Promoter elements in mRNAencoding genes GC -90 GGGCGG CAAT -75 TATA -25 TATAAT Site of initiation of transcription Increases promoter strength Recruits RNA pol II Initiator Py2 CA Py5 -3 +5 Basal Transcription Step 1. TBP-binding TAFS TFIID TBP TATA TBP TATA-Box binding protein - Saddle-shaped - Binds to minor groove TBP-associated factors (TAFs) DNA Step 2. Building the pre-initiation complex TFIIJ TFIIH TFIIF P P TBP TATA TFIIB TFIIA TFIIE P P Transcription elongation occurs Promoter clearance Transcription factors are analogous to Sigma factors Activated Transcription in Eukaryotes PIC Enhancer elements TATA 500 bp - 3 kb Promoter 200-400 bp Enhancers bind Activator proteins (UAS) Silencers bind Repressor proteins Activator proteins Transcription activated PIC TATA Activation at a distance involves looping and/or co-activators Co-activator PIC GAL4 System of Yeast: A paradigm for Eukaryotic Gene Regulation GAL80 GAL4 GAL1 UASG Promoter Add Galactose GAL80 GAL4 UASG PIC GAL1 Promoter Switched on RNA Processing 1. Capping - 5' cap structure always added - 5' end of nascent hnRNA is always a purine A/G - Addition of a new G by GUANYLYL TRANSFERASE - Condensation reaction between GTP and 5' triphosphate group causes 5'5' bond. - Terminal G in opposite orientation to rest of nucleotides Methylated at position 7 Called Cap 0 site Cap seals end of transcript from exonuclease activity increasing longevity of molecule CH3 RNA Processing 2. Polyadenylation - A polyadenylic acid tail is added at 3' end of molecule - Up to 200 A residues - PolyA tail added by Poly(A) polymerase - Poly(A)-binding protein - Consensus sequence AAUAAA - Poly(A)-tail confers stability - Poly(A)-tail is useful for isolating mRNA using oligo-dT Summary Eukaryotic gene regulation involves compartmentalisation of transcription and translation Vast majority of gene expression control mechanisms work using same principles as prokaryotes A multi-protein pre-initiation complex is built at TATA box Activation/repression can occur at a distance away from promoter Reading and associated tasks Brown, T.A. Genomes 2. Chapter 3 Transcriptomes and proteomes Chapter 9. DNAse I footprinting to investigate DNA-binding proteins Gel retardation assays DMA modification protection assay RNA Processing 1. Capping - 5' cap structure always added - 5' end of nascent hnRNA is always a purine A/G - Addition of a new G by GUANYLYL TRANSFERASE - Condensation reaction between GTP and 5' triphosphate group causes 5'5' bond. - Terminal G in opposite orientation to rest of nucleotides Methylated at position 7 Called Cap 0 site Cap seals end of transcript from exonuclease activity increasing longevity of molecule CH3 RNA Processing 2. Polyadenylation - The 3'-ends of mRNAs are generated by cleavage followed by poladenylation - Cleavage of 3' end by endonuclease (CFI/CFII) - Polyadenylic acid tail is added at 3' end of molecule - Up to 200 A residues added by Poly(A) polymerase - Consensus sequence AAUAAA recognised by specificity component (CPSF) and a GU-rich tract by stimulatory factor (CstF) - Inhibition of polyadenylation by cordycepin prevents mRNA production. 5' AAAAAAAAAAAAA 3' G G Selection of Poly(A)+ RNA G G 5' AAAAAAAAAAAAA TTTTTTTTTTTTTT 3' Oligo-dT- Sepharose or magnetic beads Allows fractionation of RNA to obtain Poly(A)+ fraction (mRNA) Used to make cDNA libraries Poly(A) tail can be up to 200 nucleotides in length Intron Splicing Eukaryotic genes are interrupted ATG intron Exon1 Exon2 intron Exon3 Splicing mRNA Translation Protein product intron Exon4 TGA TAA TAG Intron Splicing 5' GU UACUAAC Lariat Site AG 3' I N T R O N Introns follow GT-/ -AG rule These are the 5' and 3' splice site flanking introns There is also a lariat sequence in the middle of an intron This is conserved in some organisms e.g. yeast Intron Splicing 5' Cut 5' 3' 5' 2' UACUAAC UG UG GU UACUAAC AG 3' 3' AG Cut 5' Exon1 Exon2 3' Components of nuclear intron splicing Splicing involves transesterification reactions - Free OH attacks a phosphodiester bond The Spliceosome Complex Composed of small nuclear RNAs (snRNAs) and proteins Form snRNPs (snurps) Spliceosome is 50-60S ribonulceoprotein particle snRNPs include U1, U2, U4, U5 and U6 U1 binds to 5' splice site U2 binds to lariat site U5/U4/U6 trimer binds to lariat during 5'-2' bond formation U2, U4, U5 and U6 involved in exon splicing and intron removal Evolution of Intron Splicing Some mitochondrial genes have self-splicing introns Group II introns Form lariat without need for protein complex Self-splicing introns are an example of catalytic RNA and may pre-date nuclear introns Introns early/introns late theories Alternative Splicing Was originally thought that one gene always gave rise to one mRNA after splicing Alternative splicing is common, however, allowing single hnRNA to be processed into distinct mRNAs with different products Alternative Splicing 1 2 3 1 2 3 1 Three distinct transcripts lead To three distinct proteins 1 2 3 Example of alternative splicing Human slo gene encodes a K+ pump in plasma membrane Gene possesses 35 exons, of which 8 are involved in alternative splicing Leads to more than 500 distinct mRNA products Involved in inner ear, cochlear hair cells Different hair cells respond to sound frequencines between 20 and 20,000 Hz. Auditory range partly dictated by Slo protein form. Consequences of alternative splicing The proteome may be much more complex than indicated by genome sequence As many as 35% of human genes may show alternative splicing This may provide reason that we possess `only' 35,000 genes. Drosophila has fewer genes (13K) than Caenorhabditis elegans (19K) Chapter 10 Brown, TA Genomes 2. Summary Transcripts must be processed before mRNA leaves nucleus for translation Capping Polyadenylation Intron splicing Alternative intron splicing may have been a key innovation in genome evolution ...
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