362 Review Sheet for B M B 400 at PSU
362 Review Sheet for B M B 400 at PSU
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Date Created: 02/06/15
B M B 400 PART FOUR IV Chapter 18 Regulation after initiation of transcrition B M B 400 Part Four Gene Regulation Section IV Chapter 18 REGULATION AFTER INITIATION OF TRANSCRIPTION Although regulation of the initiation of transcription appears to be a dominant factor in control of expression of many genes the importance of regulation after initiation is becoming better appreciated in an increasing number and variety of systems The classic systems in which these issues have been explored are antitermination in bacteriophage 9t and in attenuation of transcription in bacterial biosynthetic operons in particular the trp operon in E coli Although some of the mechanistic details may be peculiar to bacteria especially the need for coupled transcription and translation in the trp attenuation system the phenomenon of regulation after initiation is seen in a wide variety of organisms ranging from bacteria to humans Some of this work was discussed in the sections on elongation of transcription in Chapter II of Part Three Both systems discussed in this chapter control the frequency of termination of transcription Antitermination in bacteriophage t can prevent RNA polymerase from stopping at pdependent terminators thus leading to transcription of downstream genes Attenuation in the trp operon also controls the frequency at which RNA polymerase stops at an early terminator in the operon hence regulating the transcription of downstream genes In contrast to the system in t attenuation in trp regulates termination at a pindependent terminator A Antitermination in bacteriophage 7 1 Just to quickly review one of the points in Chapter III antitermination occurs at two different times in the 7 life cycle The N protein allows read through transcription in the shift from immediate early to early transcription and the Q protein allows read through transcription of the late genes Fig 441 Antitermination by N protein leads to early Lyric cascade Cro turns off cl Q protein gene expression action leads to late gene expression t 9am attm ii d GILAI cl crol cllOP Q AJ Pm In PL PPM PF lmPRE In Pnilas 4 as OR IRS l I l as RNA a i i h b N protein Cro l l Clll CH O protein Lytic functions Replication proteins Recombma on Pl melr IS Replication proteins Viral head amp tail proteins B M B 400 PART FOUR IV Chapter 18 Regulation after initiation of transcrition 2 Recall from Part Three of the text that p dependent terminators do not have a well conserved sequence or secondary structure Also the protein p tracks along protein free regions of the RNA until it hits a paused transcriptional complex at a p dependent site at which point its RNA helicase actiVity can cause termination and dissociation of the polymerase and transcript from the template DNA Fig 442 Action of p protein at terminators of transcription p hexamer binds lo protein free RNA and moves along it pdependenl site 1 i RNA polymevmelrznscnbes along the Iemplale and pmoves along the RNA Termination of transcription in E coli Li MW RNA polyrnemse pauses althe Rhodependent srte l rrdeperdmltwninalorsne and p catches up 539 AUCGCUACCUCAUAUCCGCACCUCCUCMACGCUACCUCGACCAGMAGGCGUCUE Termination occurs at one WMMMWA Slructureln RNA um causes pausing of these 3 nucleotides i punwinds theRNA DNA hybrid and transcription terminates Little sequence specificity rich in C poor in G Requires action of rho p in vitro and in vivo Many most genes in E coihave rhodependent WMWMM terminators 3 Sites on the DNA needed for antitermination in bacteriophage t a nut sites N utilization sites for pN qut site for pQ b The nut sites are Within the transcription unit not at the promoter and not at the terminator 1 nutL is in the 5 untranslated region of the N gene and nutR is in the 3 untranslated region of the cm gene 2 In both cases the nut site precedes the terminator at which pN will act B M B 400 PART FOUR IV 2 Chapter 18 Regulation after initiation of transcrition Figure 443 nut sites are located Within transcription units delayed early txn N gt present immediate gt l I early txn cro CH O P Q nutR PR I tRl tRZ 5 I I 339 I tL1 nutL PL CHI N CI immediate early txn delayed early txn N present 3 Both nutL and nutR are 17 bp sequences with a dyad symmetry 539 AGCCCTGAARAAGGGCA TCGGGACTTYTTCCCGT 539 0 Model The protein pN recognizes the nut site and binds to RNA polymerase as it transcribes through the site The complex of pN with the RNA polymerases is highly processive and overrides the efforts of p at the terminator 4 E coli host proteins needed for action of pN a These were isolated as host functions that when mutated prevented action of pN b NusA encoded by nusA for N utilization substance complementation group A is the best characterized 1 Can form part of the transcription complex 2 Has been proposed to bind to the core RNA polymerase after 0 dissociates B M B 400 PART FOUR 7 1V Chapter 18 Regulation a er initiation of transcrition 3 Can also bind pN 4 Model NusA binds the core polymerzse after a dissociaes As this complex trarscribes through and site pN binds also The complex uzf f 39rNusArpN prevents pedependem lerminalion a KR mg and KL Pigure444 pdepencbm site N plus N N NUSA RNA polymerase with N and NusA US transcribes alongthe template and p moves alongthe FlNA factors block rho RNA polymerase does NOT pause at the pdependem terminator site and action pnever catches up Transcription continues pas1 the ierminmor e Several ahermu genes have been identified NusG is the bacterial homolog of a family of conserved proteins involved in elongation It is homologous to the large subunit ofDSIF which is an elongaion faoor in mammals DSIF is the DRBsensitivity inducing fador Current studies implicate it in both negaive and positive effeds on elongaion It has twosubunits one of 160 hDatha is homologous to the yeast transcriptional regulaory protein Spts and one of 14 hDatha is homologous to the yeast spt4 protein Another m4 gene encodes a ribosomal protein Much more needs to be learned about both termination and antiterminaion The ma phenotype of mutaions in a gene encoding a ribosomal proteinsuggests tha translaion is also ooupledtothis process B M B 400 PART FOUR 7 IV Chapter 18 Regulation after initiation of transcrition B Components of the E cali tn operon l The trp operon encodes the enzymes required for biosynthesis of ptophan More specifically its five genes trpEDCBA encode five subunits of proteins that in total catalyze five enzymatic steps convening chorismic acid to ptophan However there is not a 11 correspondence between a cistron and an enzyme For example trpB and trpA encode respectively the 5 and or subunits of tryptophan synthase which catalyzes the replacement of glycerolr3r phosphate from indolee3rglycerolephosphate with serine to form tryptophan with glyceraldehydee3ephosphate as the other product Figure 445 Organization of the E coli trp operon po 1705 FWD Irpc W75 pl t t l l l l l leader attenuator chgr39sm39c gt gt gt gt gt tryptophan aCI COOH CW azic w mH ij N 0H 000H 2 A leader sequence separates the promoter and operator from the first structural gene of the operon trpE 3 An attenuator of transcription follows the leader As we will see in more detail below the efficiency of quotprematurequot termination at this attenuator is determined by the extent of translation of the leader which in turn is determined by the availability ofTrpetRNALrP This is an important part of the regulation of the operon 4 Two terminators of transcription follow the structural genes one dependent on p and one independent of p B M B 400 PART FOUR 7 IV Chapter 18 Regulation after initiation of transcrition C Modes ofregulatjon turn operon off in presence of Trp l Repressorioperator requires a protein binding to a specific site in the presence of Trp to decrease the efficiency of initiation of transcription 2 Attenuation the elongation and termination of transcription by RNA polymerase is linked to the progress oftranslation by a ribosome In the presence ofTrp the translation by the ribosome causes transcription of the subsequent genes in the operon to terminate D Repressor aporepressor and corepressor Trp l The apoirepressor is encoded by trpR at a distant locus The apoirepressor is a homoetetramer It has a high affinity for the operator only when it is bound by the amino acid Tm which serves as a coirepressor Thus the actiVe repressor is a tetramer of formerl a170 repressor in complex with Tm The actiVe repressor binds to the operator to preVent initiation of transcription 2 The operator overlaps the promoter including the 710 region of the promoter It has a dyad axis ofsymmetry Figure 446 The trp operon is regulated in part by an aporepressor po 1705 FWD 17 pC W75 pl t t l l l l l p 0 1705 i OperonON p 0 1705 trp 2 39 4 39 I Flt339rel0resslt3r Repressor Operon OFF with trp bound B MB 400 PART FOUR 7 IV Chapter 18 Regulation after initiation of transcrition E Attenuation l The attenuator is aconditional transcriptional terminator used to regulate expression of biosynthetic operons in bacteria a The attenuator is upstream of the structural genes trpEDCBA b It is a prindependent termination site Its ability to terminate transcription is dependent on its ability to form the stem of duplex RNA that is characteristic of prindependent termination sites Fig 447 The trp operon is also regulated by attenuation Eideranen zrpE zrp zrpc 1505 zrpA tt lt l l l llP 1 27 54 70 90 114 126 140 RNA I AUG UGGUGG UGA txn trp trp attenuator pause J gt Conditional terminator Leader peptide of transcription 14 amino acids Terminates in high trp 2 are trp Y Allows readthrough in low trp 2 The fraction of transcripts that read through the attenuator is determined by the TrptRNAtrP a The concentration of charged tRNAs is a measure of the amount of Trp available for protein synthesis If most tRNAC P is charged there is an abundance ofTrp and the cell does not need to make more b Low 39I rpetRNAt P allows readethrough transcription through the attenuator so that trpEDCBA is expressed c High TrpetRNAt P causes termination of transcription at the attenuator BMB400 3 PART FOUR IV 2 Chapter 18 Regulation after initiation of transcrition The TrptRNAtI39P determines the progress of ribosomes as they translate a short leader peptide a b The leader peptide is a short 14 amino acid polypeptide encoded by trpL Two codons for Trp are in the leader and the progress of ribosomes past these Trp codons will be determined by the availability of Trp tRNAtI39P When the concentration of tryptophanyl tRNA is high translation of the tip leader will be completed but when it is low translation will stall at the tryptophan codons The extent of progress of the ribosomes determines the secondary structures formed in the leader RNA a When the Trp tRNAtrP is high the ribosomes translate past the Trp codons to complete the synthesis leader of the peptide This allows the nascent RNA to form the structure for p independent terminator Thus transcription terrninates before the RNA polymerase reaches trpEDCBA When the Trp tRNAtrP is low the ribosomes stall at the Trp codons which prevents formation of the secondary structures in the RNA necessary for termination at the attenuator Thus read through transcription continues through trpEDCBA and the operon is expressed so that more Trp is made Table 441 The basic components of regulation at the attenuator of the E coli tip operon are tabulated below trp tRNA translation of trpL secondary structures Attenuator Operon formed in RNA High complete 3 4 stern terminate transcription OFF Low stalls at Trp codons 2 3 stern allow read through ON transcription B M B 400 PART FOUR 7 IV Chapter 18 Regulation after initiation of transcrition 5 Alternative basepaired structures in leader RNA a Four regions of the leader RNA can be inVolVed in secondary structure formation in particular baseepaired stems These are referred to simply as regions 1 23 and 4 b Potentially 1 can pair with 2 2 can pair with 3 and 3 can pair with 4 Fig 448 Alternative basepaired structures in leader RNA 1 27 54 70 90 114 126 140 I I I I I I gt AUG UGGUGG UGA txn attenuator tl Ptl P pause 1 2 3 4 gt Termination of transcription No termination c A stem formed by pairing between 3 and 4 makes a GC rich stem followed by U39s which is sufficient for peindependent termination of transcription When the TrpetRNALrP is high the 374 baseepaired structure forms and transcription terminates at the attenuator This turns the operon OFF d The formation ofa baseepaired stem between regions 2 and 3 precludes formation of the 374 terminator and transcription will continue into the structural genes trpEDCBA This turns the operon ON BMB400 Fig 449 e PART FOUR 7 IV Chapter 18 Regulation after initiation of transcrition Progress of ribosome determines secondary structure of trp leader RNA attenuator th ml termination of transcription trp trp ribosome UGGUG UGGUGG 1 4 Low trp No termination 2 3 The choice between a 273 stem or a34 stem is dictated by the progress of the ribosome 1 If the ribosome can translate past the Trp codons when the Trpe tRNAtrP is high then it will reach a natural translation termination codon When the ribosome is in that position region 2 of the leader RNA is covered by the ribosome so the 273 stem cannot form but the 37 4 stem can This generates the secondary structure needed for termination of transcription at the attenuator 2 In contrast ifthe ribosome stalls at the Trp codons in the leader because the TrpetRNAtrP is low then region 2 of the leader RNA is not covered by the ribosome It can then base pair with region 3 This preVents formation of the 374tem1inator and RNA polymerase can continue elongation through tTpEDCBA BMB400 PART FOUR IV 2 Chapter 18 Regulation after initiation of transcrition E Mutational analysis selected examples 1 Translation of an is needed for regulation by attenuation Mutation of the AUG for initiation of translation of the leader RNA prevents transcription past the attenuator In the absence of translation both the 1 2 and 3 4 stems can form The latter 3 4 stem is the terminator Charged tRNAtrP is required for regulation Mutation of the genes for tRNAtrP or Trp tRNAtrP synthetase leads to constitutive expression of trpEDCBA In these mutants translation will stall at Trp codons regardless of the intracellular Trp and no terminator will form at the attenuator Speci c secondary structures in the trp leader RNA are needed for regulation E g mutations that decrease the number of base pairs between the 3 and 4 regions will decrease the amount of transcriptional termination ie increase expression of the operon Compensatory mutations that increase the number of base pairs between 3 and 4 will suppress the original mutations F Attenuation requires coupled transcription and translation 1 Requires no regulatory proteins charging of cognate tRNA is the regulatory signal Need a transcriptional pause site at 90 to allow the ribosomes to catch up with the RNA polymerase and thereby affect the secondary structures in the nascent RNA G Attenuation is a common mechanism for regulating biosynthetic operons Many operons that encode the enzymes catalyzing biosynthesis of amino acids are regulated by attenuation In each case the leader polypeptide is rich in the amino acid that is the product of the pathway E g his phe leu thr ilv B M B 400 PART FOUR IV 2 Chapter 18 Regulation after initiation of transcrition Additional readings Friedman DI and Count DL 1995 Transcriptional antitermination The lambda paradigm updated Molecular Microbiology 18 191 200 Henkin T 2000 Transcriptional termination in bacteria Current Opinions in Microbiology 3 149 153 Gusarov I and Nudler E 2001 Control of intrinsic transcriptional termination by N and NusA The basic mechanism Cell 107 437 449 BMB400 181 182 183 184 185 PART FOUR IV 2 Chapter 18 Regulation after initiation of transcrition Questions on Chapter 18 Regulation after initiation of transcription Which of the following statements concerning the action of N protein are true 1 N action requires sequences on the A DNA called nutL and nutR 2 N activity requires a host function encoded by nusA 3 N protein acts to promote rho dependent termination 4 N protein can relieve the polarity of certain amber mutations Antitermination at tL1 of A by N protein allows read through transcription through int which encodes the integrase enzyme However large amounts of the Int protein are not produced lytic infection because these transcripts continue past the p dependent terminator tint This allows the formation of a secondary structure in the RNA that serves as a signal for RNases to degrade the transcripts from the 3 end Why are large amounts of Int made during lysogeny Sketch the RNA secondary structures in the trp leader attenuator region being translated by a ribosome under conditions of low and high concentrations of tryptophan What determines the progress of the ribosome and how does this affect trp expression Which of the following events occur when E coli is starved for the amino acid tryptophan 1 No tryptophanyl tRNA is made 2 The ribosome translates the leader peptide completely to the UGA stop codon 3 A GC rich stem loop structure forms in the nascent RNA regions 3 and 4 at the attenuator site 4 A step loop structure forms in the nascent RNA regions 2 and 3 that precludes formation of the GC rich stem loop at the attenuator site 5 Transcription reads through the attenuator into trp EDCBA FOB Transcription attenuation In the leader region of the trp mRNA what would be the effect of a Increasing the distance number of bases between the leader peptide gene and sequence 2 b Increasing the distance between sequences 2 and 3 c Removing sequence 4
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