358 Class Note for B M B 400 at PSU
358 Class Note for B M B 400 at PSU
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Date Created: 02/06/15
BMB 400 PartThreeAH Chpt ll Transcription Promoters and Terminators B M B 400 Part Three Gene Expression and Protein Synthesis Chapter 11 TRANSCRIPTION PROMOTERS TERMINATORS AND mRNA This second chapter on transcription focusses on the eisiacting elements needed for accurate transcription with a emphasis on promoters The chapter begins with a discussion of techniques used to find the start site for transcription and to identify the segments of DNA bound by protein It then covers promoters elongation termination and mRNA structure The phenomenon of polarity is explored to show the relationships among mRNA structure transcription and translation in E coli A Mapping the 539 ends of mRNA The nucleotide in DNA that encodes the 539 end of mRNA is almost always the start site for transcription Thus methods to map the 539 end of the mRNA are critical first steps in defining the promoter Figure321 Nuclease protection to map 5 end of a gene lquot lgt nontemplate template gtRNA Hybridize RNA with a DNA probe labeled on SEd gt a l S1 nuclease single strand specific 37 l Denaturing gel electrophoresis Size distance from labeled site to first discontinuity between DNA and RNA eg 5 end of gene or beginning of an exon 1 quot51 protection assayquot This assay measures the distance between an end label at a specific known site on DNA and the end of a duplex between RNA and the labeled DNA A fragment of DNA complementary to the RNA that extends beyond the 539 end of the RNA is labeled at a restriction site within the RNAicomplementary region The labeled DNA is hybridized to RNA and then digested with the singleistrand specific nuclease 1 The resulting fragment of protected DNA is run on a denaturing gel to determine its size Note that this fragment runs from the labeled site to the nearest interruption between the DNA and the RNA This could be the beginning of the RNA or it could be an intron or it could be an 51 sensitive site BMB 400 Part ThreeeH Chpt ll Transcription Promoters and Terminators Fig 322 Nuclease protection assay to define the 3 end of a gene l l nont m late a temp atg gtRNA Hvbridize RNA with a DNA probe labeled on Said m S1 nuclease single strand specific l m Denaturing gel electrophoresis l 7 Size distance from labeled site to first discontinuity between DNA and RNA eg 3 end of gene or end of an exon 2 llPrimer extension assayll This assay measures the distance between an end label and the point to which reverse transcriptase can copy the RNA A short fragment of DNA complementary to RNA shorter than the RNA and labeled at the 539 end is hybridized to the RNA It will now serve as a primer for synthesis of the complementary DNA by reverse transcriptase The size of the resulting primer extension product gives the distance from the labeled site to the 539 end of the RNA or to the nearest block to reverse transcriptase Fig 323 Primer extension assay another way to map the 5 ends of genes v lquot l nontem late 5 templatg 3 gtRNA Anneal a primer complementary to RNA labeled on gt M7 1 Reverse transcriptase dNTPs to extend m primerto 5 end of RNA A l Denaturing gel electrophoresis A Size distance from labeled site to the 5 end of the mRNA BMB 400 Part ThreerII Chpt ll Transcription Promoters and Terminators 3 How do you label DNA at the ends a 539 end label T4 polynucleotide kinase and y 32P ATP The reaction is most efficient if the 539 phosphate is removed by alkaline phosphatase prior to the kinase treatment b 339 end label Klenow DNA polymerase plus 023213 dNTP The labeled dNTP is chosen to be complementary to the first position past the primer A restriction fragment with a 539 overhang is ideal for this quotfillrinquot labeling c Digestion with a second restriction endonuclease will frequently work to remove the label at the quototherquot end One can also use electrophoretic gels that separate strands 4 A PCRrbased technique to determine the 539 ends of mRNAs and enes A technique utilizing the high sensitivity of PCR has been developed to determine the 539 ends of mRNAs which can then be mapped onto genomic DNA sequences to find the 539 ends of genes This technique is called rapid ampli cation of cDNA ends and is abbreviated RACE When RACE is used to determine the 539 end of mRNA it is called 5 RACE This method requires that an artificial primer binding site be added to the 539 ends of copies of mRNA or cDNA and knowledge of aspecific sequence within the cDNA which will serve as the second specific primer for amplification during PCR Fig 323b 5 RNA Reverse transcriptase l Copies RNA to end addsS S Os 339 cccco cDNA 5555 l Anneal oligont with G s on 3 end 51 cDNA i Further extension by RT39ase of GGGG oligont template 4 cccco RACEready cDNAquot 4ccccc CDNA gt 4 l Primers Taq polymerase dNTPs 2535 cycles 85858 to amplify 5 end ochNA by PCR Fig 323b Rapid amplification of cDNA ends or 5 RACE BMB 400 Part Three II Chpt 11 Transcription Promoters and Terminators The methods for making cDNA from mRNA are more prone to copy the 3 ends and middle of mRNAs than the 5 ends Thus it is common to have access to this part of the cDNA and that provides the sequence information for the second or internal primer In contrast specialized techniques are often employed to get information about the 5 ends of mRNAs In the technique outlined in Fig 323b the fact that reverse transcriptase tends to add a few C residues to the 3 end of the cDNA is used to design an arti cial template that will anneal to those extra C nucleotides Then reverse transcriptase copies the second template thereby adding the artificial primer binding site This artificial primer binding site is needed because the sequence of the 5 end of the mRNA is not known in this experiment indeed that is what the experimenter is trying to determine Once the artificial primer binding site has been added to the cDNA then the modified cDNA serves as the template for PCR The PCR product is sequenced and compared to an appropriate genomic DNA sequence The first exon or exons of the genes will match the sequence of the PCR product starting right after the first primer B General methods for identifying the site for sequencespeci c binding proteins 1 Does a protein bind to a particular region a Electrophoretic mobility shift assay EMSA or gel retardation assay This assay will test for the ability of a particular sequence to form a complex with a protein Many protein DNA complexes are sufficiently stable that they will remain together during electrophoresis through a nondenaturin g polyacrylamide gel A selected restriction fragment or synthetic duplex oligonucleotide is labeled to make a probe and mixed with a protein or crude mixture of proteins If the DNA fragment binds to the protein the complex will migrate much slower in the gel than does the free probe it moves with roughly the mobility of the bound protein The presence of a slowly moving signal is indicative of a complex between the DNA probe and some proteins By incubating the probe and proteins in the presence of increasing amounts of competitor DNA fragments one can test for specificity and even glean some information about the identity of the binding protein BMB 400 Part Three II Chpt 11 Transcription Promoters and Terminators Figure 324 Diagram of results from an electrophoretic mobility shift assay Competitor E coli Spl OCtl EXUaCt complex A m I Complex B m Free Probe Lane 1234567891011121314 In this example two proteins recognize sequences in the labeled probe forming complexes A and B lane 2 The proteins in complexes A and B recognize speci c DNA sequences in the probe This is shown by the competition assays in lanes 3 8 An excess of unlabeled oligonucleotide with the same sequence as the labeled probe self prevents formation of the complexes with labeled probe whereas nonspecific DNA in the form of E coli DNA does not compete effectively compare lanes 6 7 with lanes 3 5 This experiment also provides some information about the identity of the protein forming complex A It recognizes an Spl binding site as shown by the ability of an oligonucloetide with an Spl binding to compete for complex A but not complex B lanes 9 11 Hence the protein could be Spl or a relative of it The proteins forming complexes A and B do not recognize an Octl binding site lanes 12 14 b Nitrocellulose binding Free duplex DNA will not stick to a nitrocellulose membrane but a protein DNA complex will bind 2 To What sequence in the probe DNA is the protein binding The presence of a protein will either protect a segment of DNA from attack by a nuclease or other degradative reagent or in some cases will enhance cleavage e g to an adjacent sequence that is distorted from normal B form An end labeled DNA fragment in complex with protein is treated with a nuclease or other cleaving reagent and the protected fragments are resolved on a denaturing polyacrylamide gel and their sizes measured a Exonuclease protection assay The protein will block the progress of an exonuclease so the protected fragment extends from the labeled site to the edge of the protein furtherest from the labeled site BMB 400 Part Three II Chpt 11 Transcription Promoters and Terminators One can use a combination of a 339 to 5 exonuclease EonII and a 5 to 3 exonuclease O exonuclease to map both edges b DNase footprint analysis DNase I will cut at many but not all phosphodiester bonds in the free DNA The protein DNA complex is treated lightly with DNase I so that on average each DNA molecule is cleaved once The presence of a bound protein will block access of the DNase and the bound region will be visible as a region of the gel that has no bands ie that was not cleaved by the reagent Any reagent that will cleave DNA in a non sequence specific manner can be used in this assay Some chemical probes such as copper ortho phenanthroline are very useful Figure 325 presents a schematic diagram of the in vitro DNase I footprint analysis in the top two panels and then an example of the results of binding a purified transcriptional regulator to its cognate site on DNA B1VIB 400 Part Three II Chpt 11 Transcription Promoters and Terminators Figure 325 DNase footprint analysis DNA only 0 cut with DNase I remove protein and J denature DNA DNA only i i DNA plus protein 39 oi 0 J i DNA plus protein separate labeled products on gel Increasing Protein Concentration Iquot quot quot u I 39 I Site 1 Site 2 BMB 400 Part Three II Chpt ll Transcription Promoters and Terminators 3 What are the contacts between the protein and the binding site in DNA a Methylation interference reactions When a purine that makes contact with the protein is methylated by dimethyl sulphate DMS the DNA will no longer bind to the protein Thus DNA is gently methylated about one hit per molecule mixed with the protein and then the bound complexes are separated from the unbound probe The unbound probe Will be modified at all sites when the Whole population of molecules is examined but the bound DNA Will not be modified at any critical contact points The methylated DNA is then isolated cleaved with piperidine at high temperature just like a Maxam and Gilbert sequencing reaction and resolved on a denaturing gel The critical contact points will be identified by the clear areas on the gel the ones that correpond to fragments that when methylated at that site will no longer bind to the protein DMS reacts mainly with G s at N 7 which is in the major groove of the DNA so these are the contacts most sensitive to this reagent b Other reagents are specific for the minor groove or for the phosphodiester backbone endlabeled DNA methylate about once 0le per molecule CiHS 0le CH3 CH3 CH3 gt O I gt CH 3 CH3 l Remove Separate CH3 CH3 protein by Slze l Blndl denature on gel CH3 proteln CH3 DNA CIHS 0 39 to cleave at CH3 methylated methyl 0 c39H3 0 CH3 DNA CH3 groups 3le I l Cleave at run on gel methyl groups Bound DNA 1 39D 39D Z gt Figure 316 Methylation interference assay BMB 400 Part Three II Chpt 11 Transcription Promoters and Terminators 4 DNA sequenceaf nity chromatography to purify DNA binding proteins The specific binding sites often 6 to 8 bp can serve as an affinity ligand for chromatography Multimers of the binding site are made by ligating together duplex oligonucleotides that contain the specific site After a few crude initial steps eg isolating all DNA binding proteins on DNA sepharose the extract is applied to the affinity column Most of the proteins do not bind and subsequently the specifically bound proteins are eluted C Promoters and the Initiation of Transcription General Properties 1 A promoter is the DNA sequence required for correct initiation of transcription 2 Phenotype of promoter mutants a cis acting A cis acting regulatory element functions as a segment of DNA to affect the expression of genes on the same chromosome that it is located on Cis acting elements do not encode a diffusible product The promoter is a cis acting regulatory element Compare the phenotypes of mutations in the gene encoding S galactosidase lacZ versus mutations in its promoter 17 Consider a heterozygote that is p lacZ p lacZ The phenotype is Lac lacZ complements 6102 in trans In this case lacZ is dominant to lacZ Consider a heterozygote that is p lacZ p lacZ The phenotype is Lac p does not complement p in trans p operates in cis to prevent expression of lacZ on this chromosome The mutant promoter is dominant over the wild type when the mutant promoter is in gis to the Wt lacZ Consider a heterozygote that isp lacZ p lacZ The phenotype is Lac lacZ now complements lacZ in trans because it is driven by a functional promoter in gs p b Dominance in cis the promoter allele that is in 9 to the wild type structural gene lacZ is dominant over the other promoter allele BMB 400 C D Part Three II Chpt 11 Transcription Promoters and Terminators Promoter mutations affect the amount of product from the gene but do Lot affect the structure of the gene product Bacterial promoters 1 Bacterial promoters occur just 5 to and overlap the start site for 2 holoenzyme transcriptionusually Bacterial promoters are the binding site for E coli RNA polymerase The promoter covers about 70 bp from about 50 to about 20 Consensus sequences in the E coli promoter a 35 and 10 sequences 35 16 19bp 10 1 TTGACA TATAAT CAT Recognition by Allows binary complex to convert b with 0 RNA polymerase holoenzyme from closed to open The sequences are conserved in all E coli genes transcribed by holoenzyme 70 4 Promoter mutants a Tend to fall into or close to one of these hexanucleotides b Affect the level of gene expression not the structure of the gene product c Down promoter mutations decrease the level of transcription Tend to make the promoter sequence less like the consensus d Up promoter mutations increase the level of transcription Tend to make the promoter sequence more like the consensus e Down promoter mutations in the 35 sequence decrease the rate of formation of the closed complex indicating this is the sequence needed for intial recognition by the polymerase holoenzyme f Down promoter mutations in the 10 sequence decrease the rate of conversion from the closed to the open complex again supporting the proposed role for this AT rich hexanucleotide g The critical contact points between RNA polymerase and the promoter tend to be in or immediately upstream from the consensus 35 and 10 boxes See Fig BMB 400 Part ThreeilI Chpt ll Transcription Promoters and Terminators 327 Thus the biochemical and genetic data all support the importance of these conserved sequences Figure 327 Correlation of conserved sequences location of promoter mutants and regions of contact with polymerase at bacterial promoters 10 48 bp 1 mT GTA v AT Unwound in open complex Contacts with RNA polymerase The sigma subunit of RNA polymerase contacts both the 35 and the 10 boxes 5 Alternate 0 factors can control the expression of sets of genes a Alternative 0 factors make complexes with the core polymerase to direct the new holoenzyme to a particular set of promoters that differ in sequence from the general E coli promoter sequence Thus the polymerase can be directed to trancribe a new set of genes This is one way to control gene expression b Examples include 0 factors for heatishock response 032 transcription of genes involved in chemotaxis and flagellar formation 028 and nitrogen starvation 054 The 0 factors are named by their size in kDa c Three of the E colio factors have regions of sequence similarity 070 032 and 028 whereas 054 is a distinctly different molecule that works rather differently Factor Gene Use 735 Separation 710 O U rpoD General TTGACA 16719 bp TATAAT O32 rpoH Heat shock CCCTTGAA 13715 bp CCCGATNT O28 iA Flagella CTAAA 15 bp GCCGATAA O54 rpoN Nitrogen CTGGNA 6 bp TTGCA starvation BMB 400 Part Threerll Chpt 11 Transcription Promoters and Terminators E Promoters for enkaryo c RNA polymerases Promoters contain binding sites for nuclear proteins but which of these binding sites have afunction in gene exmsion l This requires a genetic approach for an answer 1 Use of quotsurrogate gene tsquot to de ne the promoter a In virru mutgenesis deletions or point mutations 1 Mutations of the binding sites for activator proteins lead to a decreme in the level of transcription of thegene Loss of function 2 Addition of a DNA fragment containing these binding sites will activate some heterologous promoters Gain of function 3 Sequences ofthe binding sites arefrequently well conserved in promoters for homologous genes from re1ated species 4 A potential regulatory region is initially examined by constructing progressive deletions from the 5 end with respect to the direction of lnnscription and alsofrom the 3 end Subsequendy one can make c1usters ofpoint mutations eg by linkerscanning mutagenesis or individual point mutaions RNA pdymerose ii promoter Upstream bmdmg was TATA box HBB encodes betaglobln initiator TFHD binds I Conserved in many Class II genes I D Conserved in mammalian HBB genes EE ES I Directed mutation loss olrans crlption Mutations cause betathalassemia bin ng of transcription E E DE SE E Mutation of gene encoding transcription factor that binds here prevents HBB expression Figure 313 Evidence for an RNA polymerase IIpromoter BMB 400 Part Three II Chpt 11 Transcription Promoters and Terminators b Test in an expression assay 1 The mutagenized promoter is linked to a reporter gene so that RNA or protein from that gene can be measured quantitatively a Gene itself measure RNA production by 81 protection primer extension or other assay that is specific for a particular RNA 17 Heterologous reporter gene encodes an enzyme whose activity is easy to measure quantitatively Note that these measures of expression require both transcription and translation in contrast to measurement of RNA directly Eg the genes encoding 1 S galactosidase colorimetric assay monitor the cleavage of o nitrophenyl S galactoside 2 chloramphenicol Cm acetyl transferase CAT measure the acetylation of Cm ususally use 14C Cm this is the enzyme that confers resistance to Cm in bacteria 3 luciferase monitor the emission of photons resulting from the ATP dependent oxidation of luciferin this is the enzyme that catalyzes light production in firefly tails 2 The promoter reporter DNA constructs are introduced into an assay system that will allow the reporter to be expressed a Whole cells microinjection into Xenopus oocytes transfection of cell lines introduce the DNA via electroporation or by getting the cells to take up a precipitate of DNA and Ca phosphate by pinocytosis 17 Whole animals 2 transgenic animals Introduce the DNA into the germ line of an animal in mammals by microinjecting into a fertilized egg and placing that into a pseudopregnant female This technology allows one to examine the effects of the mutation throughout the development of the animal 0 Cell free systems Extracts of nuclei or purified systems ie with all the necessary components purified BMB 400 Part ThreelT Chpt 11 Transcription Promoters and Terminators 2 Promoter for RNA P01 11 a The minimal moter is needed for basal activiu and accurate initiation 1 Needed for assembly of the initiation complex at the correct site 2 DNA sequences a TATA box 1 Initially identified as awell conserved sequence motif about 25 bp 5 to the cap site The cap site is the usual start site for uanscnption 2 The uanscnption factor TFDD binds to the TATA box 5 Mutations at the TATA box generates heterogeneous 5 ends of the mRNAs r indicative of aloss of start site speci city b Initiator 1 Sequences at the start site for tianscription have consensus YANWYY Y c ort WTorA 2 Mode of action is still und investigation Recent data indicate that TFJID also binds to the initiator binds to one of the TAFs see below 3 TATA plus initiator is the simplest minimal promoter RNA pdyrnerase ll promoter Upstream blrldlrlg sites TATA borner 39 TFllD binds Regulate efficiency Minimal of utilization of promoter minimal promoter TATA lnr gure 329 Two general parts of promoters for RNA polymerase 11 b The amount of exmsion is guided Via ughearn elements 1 Proteins bind to specific sequences usually 5 to the TATA box to regulate the efficiency of utilization of the promoter 2 These are frequently activators but proteins that exert negative control are also being characterized BMB 400 Part Three II Chpt 11 Transcription Promoters and Terminators 3 Examples of activator proteins Spl binds GGGGCGGGG GC box Octn binds ATTTGCAT octamer motif Octl is a general factor ubiquitous Oct2 is specific for lymphoid cells CPI CTF 2 NFL CEBP bind to CCAAT CCAAT box pronounced quotca quot box These are different families of proteins CPI and CTF are found in many cell types CEBP is found in liver and adipose tissue 4 These upstream control elements may be inducible e g by hormones may be cell type specific or they may be present and active in virtually all cell types ie ubiquitous and constitutive Figure 3210 Comparisons of promoters for eukaryotic RNA polymerases RNA polymerase II promoter TATA box Upstream binding sites Initiator Gene gt I TFIID binds RNA ol merase l romoter Upstream control element Core promoter 180 107 45 20 Gene gt I UBF1 binds UBF1 binds SL1 binds cooperatively with UBF1 RNA polymerase III promoter 58 RNA gene Gene Core promoter 55 80 IIIB IO N IIC 4 Binding byTFIIIn BMB 400 Part Threerll Chpt 11 Transcription Prornorers and Tenninawrs Promoter for RNA Pol I The core promoter covers the 5m sine of cranscripu39on from about 40 in about 30 The promoter also contains an upsueam conuol element lowed about 70 bp further 539 extending from 7170 to 7110 The faewr U39BFl binds in a GC rien sequence in boih the upstream control element and in the core prornorer A mullisubunit complex called SL1 binds in the U39BFerNA complex again at boih the upstream and core elements one of the subunlis of SL1 is 1131 RNA polymemse Iihen binds to ihis complex of DNAUBF1SL1 to ini abe cranscripu39on at ihe correct nucleou39de and the eloxgabe in make prerrRNA Fig 3211 Binding ofproteins for promoter for RNA polymearase 1 Up ream control element 1 80 Core prommer 7 45 20 UBF1 nds UBF1 binds SL1 binds coopermively with UBF1 BMB 400 Fig 3211 Part Threerll Chpt 11 Tmnscription Promoters and Terminators Promoter for RNA Pol III This promoter bas internal controlsequences Deletion ofS39 anking DNA still permits ef cient tmnscription ofmost genes transcribed by RNA PolIEL Even the intial part ofthe gene is expendable as is the 3 end Sequences internal to the gene eg 55 to 80 in SS rRNA genes are required for ef cient initiation in contrmt to the familiarsituation in bacteria where most of the promoter sequences are 539 to the gene As discussed above TFJIIIA binds totbe internal control region of genes that encode 55 RNA type 1 internal promoter TFIC binds to internal control regions of genes forSS RNA alongside TFDIA and for tRNAs type 2 internal promoters The binding ofTFJIIIC directs TFJIIB to bind to sequences 40 to 11 that overlap the start site fortranscription One subunit ofTFDIB is TBP even though no TATA box is required for transcription TFDIA and MC can now be removed without affecting the ability ofRNA polymerase Ito initiate transcript39on Thus TFJIIIA and TFIC are assembly factors andTFJIIIB is the initiation factor RNA polymerase n1 binds to the complex ofTFDJBDNA to accurately and ef ciently initiated transcription Binding of proteins for promoter for RNA polyiu muse n1 SS FINA gene Gene Core promoter BMB 400 Part Three II Chpt 11 Transcription Promoters and Terminators F Enhancers 1 Enhancers are DNA sequences that cause an increase in the level of expression of a gene with an intact promoter They may act to increase the efficiency of utilization of a promoter or they may increase the probability that a promoter is in a transcriptionally competent chromatin conformation This will be explored further in Part Four 2 They are operationally defined by their ability to act in either orientation and at a variety of positions and distances from a gene ie act independently of orientation and position This contrasts with promoters that act usually in only one orientation and usually are at or close to the 5 end of the gene 3 They consist of binding sites for specific activator proteins Always have multiple binding sites often for several different activator proteins 4 Particular sets of genes can be regulated by their need for defined sets of activator proteins at their enhancers G Elongation of transcription 1 RNA polymerase must be released from the initiation complex to transcribe the rest of the gene Elongation must be highly processive ie once the polymerase begins elongation it must transcribe that template all the way to the end of the gene 2 The factors required for initiation are not needed and may inhibit elongation and they dissociate O in bacteria The conformation of the polymerase changes upon dissociation of O to that it enters a processive mode for elongation For eukaryotic transcription by RNA polymerase II TFlID and TFHA are thought to stay behind after the transcription complex clears the promoter The release of the transcription complex from the promoter appears to be dependent on the phophorylation of the CTD of RNA polymerase 11 One of the protein kinases implicated in this process is TFHH but others such as P TEFb have also been implicated BMB 400 Part Threeell Chpt ll Transcription Promoters and Terminators Fig 3213 Model for role of phosphorylation of RNA polymerase in shift from initiating to elongating enzyme Eukaryotic RNA polymerase II Kin2 ATP F39oi iia lt7 mosmatae OTD or large subunit of Pol ii F39oi iio CTD or large Subunit of Pol ii CTD has repeat of YSPTSPT 2550 Model Phosphorylation of Pol Ilato make Pol No is needed to release the polymerase from the initiation complex and allow it to start elongation Fig 3214 Supportive evidence lmmunofluoresence shows Pol Ila is on heat shock genes when quiescent stalled polymerases but Pol Ho is present once the genes are actively transcribed elongating polymerases 39 9311 IIIA l ililCi E li QE 3151 ll39Ml 5min HS BMB 400 Part Three II Chpt 11 Transcription Promoters and Terminators 3 There is some indication that factors that increase the processivity of the transcription complex bind to the elongating polymerase Examples include the following NusA in bacteria GreA and GreB in bacteria TFIIS in eukaryotes possibly many others 4 GreA and GreB in E coli and TFIIS in eukaryotes induce hydrolytic cleavage of the transcript Within the RNA polymerase followed by release of the 339 terminal RNA fragment This process has been implicated in overcoming pausing of the polymerase Fig 3215 Cleavage of RNA to help overcome pausing Elongau39un facmr depen em realigmmm of the 3 RN A and n Backward farmlandm 1 Gm ormindumd til9ng nea39l igrg the 339 Hui with the mnhrtb he s noel on anticn can he ume WT 5 4 Regulation of elongation is an under studied area at present In fact many transcription complexes pause about 20 nt into the gene and stay there primed for transcription until they are released for elongation in response to some stimulus The classic example are the heat shock genes in Drosophila but this may be a fairly general phenomenon 5 The regulation of transcription is primarily at initiation in most cases but that regulation can be exerted at the frequency of assembling an initiation complex or BMB 400 Part Three II Chpt 11 Transcription Promoters and Terminators by the frequency of release into the elongation mode or any step prior to elongation The elongation rate averages about 50 nt per sec This is not a constant rate and many pause sites are seen Also some templates may be transcribed at different rates Variation in elongation rate will not affect the output of gene product e g transcript It will affect the lag time between initiation and the first appearance of a product Of course a sufficiently long pause ie when no elongation occurs can reduce the amount of RNA synthesized from a gene As an illustration of the importance of elongation in regulation consider the Tat and tar system in the human immunodeficiency virus HIV This case study also illustrates the complexity of the system Elongation of transcription in HIV requires the virally encoded protein Tat that binds to an RNA structure centered at about 60 called the tar Elongation requires the CTD of RNA polymerase II and now it is clear that Tat leads to phosphorylation of the CTD One step probably promoter clearance uses the kinase activity in the CDK7 subunit of TFIIH or a trimeric complex of CDK7 cyclin H and MATl referred to as CAK Thiswas shown by the ability of a pseudosubstrate inhibitor of CDK7 to block Tat dependent elongation Further phosphorylation of the CTD of RNA polymerase II is catalyzed by the positive transcription elongation factor b called P TEFb which contains a kinase subunit known as PITALRE or CDK9 P TEFb is needed for Tat stimulated elongation of transcripts from the HIV promoter a combination of promoter and enhancer called a long terminal repeat or LTR A stylized example of these data is shown below The inhibitor of elongation DRB blocks the P TEFb kinase Indeed a random screen of gt100000 compounds for the ability to block Tat stimulated HIV transcription found several new compounds All of these blocked elongation and many structurally diverse compounds also inhibit the P TEFb kinase Thus Tat dependent activation works through both TFIIH perhaps at promoter clearance and P TEFb for full elongation BMB 400 Part Three II Chpt 11 Transcription Promoters and Terminators Fig 3216 PTEFb is needed for elongation in HIV Hela nuclear complete depleted of PTEFb extract LTR template encodes TAR TAR deleted encodes TAR 700 nts m Transcripts from HIV LTR 70ms I II II n n I I II II I I II II II I Lane 1 2 3 4 5 6 7 8 9 10 11 12 Figure legend When a DNA template containing the LTR and encoding the TAR is used for in vitro transcription in a HeLa cell nuclear extract which is competent for transcription by RNA polymerase II and associated general transcription factors plus all 4 ribonucleoside triphosphates a short RNA of about 70 nucleotides is produced lane 1 in the gure below Addition of increasing amounts of Tat indicated by the triangle labeled Tat causes transcription to continue to the end of the template to produce a quotrun offquot transcript of about 700 nucleotides lanes 2 4 darker shading indicates greater abundance The results of removing the segment of DNA encoding the TAR from the template is shown in lanes 5 8 A cellular protein kinase complex called P TEFb has been found associated with Tat It can be removed from the HeLa cell nuclear extract and the effects of this treatment are shown in lanes 9 12 For a review of this work see the article by K A Jones 1997 quotTaking a new TAK on Tat transactivation quot Genes amp Development 11 2593 2599 BMB 400 Part Three II Chpt 11 Transcription Promoters and Terminators H Termination of transcription in E coli 1 Terminator sequences in E coli cause pausing by RNA polymerase Figure 3217 A pindependent site for transcription termination I AG UU U A G G A A UG GO GO CGA C UAU UA GOG ACG AU AU AU GO GC rich re ion in stem CG 9 CG 8 Run of Us 339tostemIoo AU I P 539 GCAU UUUU 339 A pdependent site for termination of transcription These sites tend to be rich in C and poor in G preceding the sites of termination 39 539 AUCGCUACCUCAUAUCOGCACCUCCUCAAACGCUACCUCGACCAGAAAGGCG UCUCU U i Termination occurs at one of these 3 nucleotides a p independent sites Note p rho 1 Identified in Vitro 2 GC rich hairpin followed by about 6 Us 3 Hairpin is thought to be a site at which RNA polymerase pauses and the weak rU dA base pairs in the RNA DNA heteroduplex allow melting of the duplex and termination 4 Some of the best examples of p independent terminators are integral parts of the mechanism of regulation Examples include the attenuators in the tip operon and other amino acid biosynthetic operons The p independent terminators may be a specialized adaptation for regulation BMB 400 Part Three II Chpt 11 Transcription Promoters and Terminators b p dependent sites 1 2 C rich G poor stretch Requires the action of the protein 9 both in Vitro and in Vivo 3 The p dependent terminators are used at the 3 ends of many eubacterial genes N 9 factor a Hexamer each subunit 46 kDa b RNA dependent ATPase c Gene for p is essential for E coli 3 Model for action of 9 factor a p binds to protein free RNA and moves along it b When it reaches a paused polymerase it causes the polymerase to dissociate and unwinds the RNA DNA duplex thereby terminating transcription This last step utilizes the energy of ATP hydrolysis The protein 9 serves as the ATPase BMB 400 Part Three H Chpt 11 Transcription Promoters and Terminators Figure 3218 Model for action of p factor in termination of transcription p hexamer binds to proteintree RNA and moves along it pdependent site I I l 1 RNA polymerase transcribes along the template and p moves along the RNA RNA polymerase pauses at the pdependent terminator site and p catches up WStructure in RNA that causes pausing ATP p unwinds the RNADNA hybrid and transcription terminates 8 a ADP p EME mu 12mm Chpt u mum numme Temmbm l Tums b bumw b eulmyums I Termmen by RNA Pal II a Na Na madame m adlsue e mum in RNA bbbymm u b 3 and b mRNA 5 ashamed by cleavage and bbbybbenybbbn c 315ml fuxcleavaseand bbbybbenybbbn I AAUAAA shunt ZEI nthefme the 3 end aftbemRNA a Other sebum 3 b cleavage mm d Clea m1 embweubbmmaubb bumbembmma bbayffbbg m 39 mye ApalyApalymemehagheemdem ed y 2 Pa 2 mm mxedfvnenmnaunnh RNApbm bssbb Ebb ba sbyybbeRNAEbbymme y P y z Termmanan by RNA pm In Termmamm amul39s atamnaf T s an the mumbbaaesuana meA sunaunded by com DNA 3 Termmanan by RNA Pu Termmanan lequus an u by binding m m the pmiem Rehlp Wm bausesbbe babymame b pause and 365 b sesmentlaca tgd 5 b LbeRehlS m whchmayhelalunedfvnel seaf pubmm an Reader 1 9o cg 7952753 snubs pausing may he a mmpanznlaf the hansmy an Isltnmaunn pmcess in several RNA babymms Rig 3219 Mm m mnninzl39nn by RNA pulymulgl RNA palymzlase 1 Raw mb Rah 1p mmmgsm mm DNA bs placed mm b hmdng mm m E can m npnssax m npnssax pmum wub mm mrmma nn m an m ybe nauscnpunn naman BMB 400 Part ThreerII Chpt 11 Transcription Promoters and Terminamrs J mRNA structure in bacteria 1 Bacterial mRNA is often polycistronic One transcript can encode the products from several adjacent genes a The set of adjacent genes that are transcribed into one mRNA is an gm b This organization allows for common tJanscriptional control Thus is ti part of the mechanism for coordination of expression of genes whose products are required at the same time E g The lac operon lacZYA encodes three enzymes involved in the uptake and metabolism of lac nose c Production of proteins from polycistronic mRNAs requires initiation at internal AUGs allowing for translation of the part of the mRNA encoding the second third etc proteins Figure 3220 A polycistronic operon in E wh39 lacZ lac Y lacA G enes in tt operon l transcription AUG UAA AUG UAA AUG UAA Poycistronic mRNA 1 translation Bgalactosidase lactose B39QalaCtOSide permease transacetylase BMB 400 Part ThreeII Chpt 11 Tramcription Pmmaels and Terminams 2 The initial uanscn39pt is also tmnslated and subsequently degmded That is transcription lmnslalion and degmdation are all going on simultaneously The inRNAususally is not extensively processed priorlo liarslaion Figure 3223 Tianslation occuis simultaneously with tiansm39ption in baueria laCZ lac Y lacA F Transcription of genes Translation of Nascent ribosome mRNA polypeptide Bgalactosidase BMB 400 Part Three II Chpt 11 Transcription Promoters and Terminators K Polarity The phenomenon of polaritV occurs because of tight linkage between transcription and translation in bacteria 1 Definition Polar mutations are mutations early in the operon that exert a negative effect on the expression of genes later in the operon This is generally a result of some nonsense mutations those that cause premature termination of translation in a gene toward the 5 end of the operon which results in a cessation of transcription before the the subsequent genes are reached Figure 3221 I Polar Effects of Some Nonsense Mutations in Bacteria I lac Z lacY 35 A Wt txn g gt tln C B galactosidase Xpermease x Ac39ase missense mutation X txn X p tln C X permease x Ac39ase 0 no B galactosidase activity Stop tln nonsense mutation X tXn x p dependent tln terminator of txn no B galactosidase no permease no Ac39ase The p dependent terminator of txn functions only on protein free RNA Premature termination of translation will expose the p dependent site for termination of transcription BMB 400 Part Three II Chpt 11 Transcription Promoters and Terminators 2 Model for 9 action can explain why stopping translation can also lead to a cessation of transcription a Suppose a p dependent terminator of transcription is present in the first gene of an operon Normally it does not cause transcription to stop because it is covered by ribosomes translating the mRNA and the subsequent genes in the operon are transcribed Recall that 9 requires protein free RNA to bind to and to move along A nonsense mutation before the cryptic p dependent terminator would cause the ribosomes to dissociate now exposing the cryptic terminator in a protein free stretch of RNA The hexamer p can bind and move along the RNA and when it encounters an RNA polymerase stalled or paused at the terminator it will cause the RNA polymerase to dissociate and the RNA to be released hence preventing transcription of the subsequent genes in the operon Figure 3222 Model for polar effects of nonsense mutations H Wildtype H Nonsense mutation nonsense pdependent site within mutation a transcripton unit 3 Ribosom es prevent p from catching up with RNA polymerase ribosome nonsense codon Structure in RNA f that causes 7 pausing O i 5 l Ribosomes dissociate at ll that causes Transcription and translation continues past the p 0 L 1 Structurein RNA pausing dependent termination site EME mu Pan mun hpt u mmcnpunn mmamsamnmnams a Mmanans m a nipples yummy afmnsense mmaoam Smce p m langr mcunml mmm nn dnesnmaccura h pdzpemzm 5m ea yxmh wpemn ammhsequmgemsan mmma Sn even manghhanslanun wm sun mmmm m m m gen hanscnptmn and than hamsh nn wm cannmlz m m dnwnsmam gems a h wpemn 1 anNAshmmemzulmymts 1 MnslmRNMmzulnlymtsueuppslalmms mdsud plydmyhmd 1 quotIn 3 nub mm 3223 5mm m eukalyohc mRNAs A USA AAUAAA 5 cap W WW 3 This gemnlsmwmle R mu fuxalmnst memo mRNAs m capsmm R almus mamas A few enmka at mRNAs wmmm paly A a m 3 and have been mm Sam am mastammdammRNAs wnhmn pulyA wand m 1mm Hnwever mns mRNAsdn have m i palyAmJ m paly A an a m 3 emcan he sed m pnnfy mRNAs mm athzr RNA 1mm RNA mmaceu which anamuaa WAamless than m mRNA canhe passed am an mg d rycelh ng cahImn m paly AcamamnngNAs wm mm whales athzr RNA wm alum BMB 400 111 112 Part Three II Chpt 11 Transcription Promoters and Terminators Questions Chapter 11 Transcription Promoters and Terminators Determining the sequences that encode the ends of mRNAs A gene that determines eye color in salamanders called almond is contained within a 2000 bp Kpnl fragment After cloning the Kpnl fragment in a plasmid it was discovered that it has a Bglll site 500 bp from the left Kpnl site and an EcoRI site 300 bp from the right Kpnl site as shown in the map below bp 0 500 1000 1500 2000 Kpnl Bglll EcoRI Kpnl In order to determine the positions that correspond to the 5 and 339 ends of the almond RNA the EcoRI and Bng sites were labeled at the 5 or 339 end The Kpnl to Bglll fragments 500 and 1500 bp and the Kpnl to EcoRI fragments 1700 and 300 bp were isolated hybridized to almond RNA and treated with the single strand specific nuclease 1 The sizes of the probe fragments protected from digestion in the RNA DNA duplex are shown below in nucleotides a 0 means that the probe was not protected by RNA 5 end labeled probe 3 end labeled probe protected protected probe fragment probe fragment KpnI Bglll 500 0 BglII Kpnl 1500 1300 KpnI EcoRI 1700 0 EcoRI Kpnl 300 100 KpnI BglII 500 100 BglII Kpnl 1500 0 KpnI EcoRI 1700 1300 EcoRI Kpnl 300 0 The asterisk denotes the end that was labeled a What is the direction of transcription of the almond gene relative to the map above b What position on the map corresponds to the 5 end of the mRNA c What position on the map corresponds to the 339 end of the mRNA Determining the sequences that encode the ends of mRNAs The gene for histone H2A from armadillo can be isolated as a 1400 bp Pstl fragment The map is shown below the armadillo Pstl fragment is shown by the double dashed line and the vector DNA is denoted by the single dashed lines Sizes are in base pairs The H2A gene clone was cleaved with H indIII treated with alkaline phosphatase and incubated with polynucleotide kinase and 3 2P ATP in an appropriate buffer to introduce a radiolabel at the 5 ends of the DNA fragments The DNA was then extracted with phenol to remove the kinase and then cut again with Pstl The labeled 600 bp and 800 bp PstI Hindlll fragments were separated by gel electrophoresis and isolated The isolated fragments were denatured hybridized to histone mRNA and treated with nuclease 1 The Sl resistant labeled DNA fragments were identified by gel electrophoresis followed by radioautography A 200 nucleotide protected fragment was observed when the 600 bp fragment was used in the 81 protection assay but no protected fragment was observed when the 800 bp fragment was used BMB 400 113 Part Three II Chpt 11 Transcription Promoters and Terminators I 600 800 HindIH 0 500 1000 a What is the direction of transcription of the histone H2A gene relative to the restriction map above b With reference to the numbers below the restriction map what is the position of the 5 end of the histone H2A mRNA c What is the position of the 339 end of the mRNA A 400 bp DNA fragment containing the start site for transcription of the almond gene was investigated to find transcriptional control signals The start site 1 in the coordinate system is 100 bp from the right end The 400 bp fragment is sufficient to drive transcription of a reporter gene for luciferase in an appropriate cell line Two series of 5 and 3 deletions were made in the 400 bp fragment and tested for their ability to drive transcription of the luciferase reporter gene Each fragment in the 5 deletion series has a different 5 end but all are fused to the luciferase gene at 100 see diagram below Each fragment in the 3 deletion series has a common 5 end at 300 but each is fused to the luciferase gene at the designated 3 position The amount of luciferase a measure of the level of transcription for each construct is shown in the first two pairs of columns in the table The intact reporter construct with almond DNA the horizontal line fused to the luciferase gene is diagrammed immediately below 300 250 200 150 100 50 1 50 100 Luciferasegt starlgt To further investigate the function of different regions sub fragments of the almond DNA fragment were added to a construct in which the reporter gene was driven by a different promoter as diagrammed below The effects of the almond DNA fragments on this heterologous promoter are shown in the third pair of columns in the table Test fragment from almond DNA heterologous promoter Lucif erase genegt gt 5 deletion Amount of 3 deletion Amount of Test fragment Amount of endpoints expression endpoints expression of almond expression 300 100 200 0 300 to 250 100 250 100 150 0 250 to 200 500 200 50 100 0 200 to 150 100 150 50 50 0 150 to 100 300 100 25 1 100 100 to 50 300 50 10 50 100 50 to 1 100 1 0 100 100 none 100 a What do you conclude is the role of the 250 to 200 fragment BMB 400 Part Three II Chpt 11 Transcription Promoters and Terminators b What do you conclude is the role of the 200 to 150 fragment c What do you conclude is the role of the 150 to 100 fragment d What is the role of the 50 to 1 fragment of the almond gene 114 An electrophoretic mobility shift assay was used to test for the ability of a short restriction fragment to bind to proteins from the nuclei of kidney cells The restriction fragment was labeled at one end mixed with an extract containing the nuclear proteins and run on a non denaturing polyacrylamide gel Lane 1 below shows the free probe and lane 2 shows the the probe plus extract electrophoresis is from the top to the bottom Complexes between proteins and the labeled DNA probe move more slowly on the gel than does the free probe Further tests of specificity are shown in the competition lanes in which the labeled probe was mixed with an increasing excess of other DNA before mixing with the nuclear proteins to test for binding Competitor DNAs included the unlabeled probe self competition lanes 3 5 the triangle above the lanes indicates that an increasing amount of competitor is used in successive lanes a completely different DNA sheared E coli DNA as a nonspecific competitor lanes 6 8 and two different duplex oligonucleotides one containing the binding site for Sp1 lanes 9 11 and the other containing the binding site for Octl lanes 12 14 Thinner less densely filled boxes denote bands of less intensity than the darker thicker bands Competitor E coli Spl OCtl EXtraCt Complex A m ma Complex B m Free Probe mm mmm mm mmm mmm Lane1234567891011121314 a How many protein DNA complexes are formed between the labeled DNA probe and the nuclear extract b What do lanes 3 8 tell you about the protein DNA complexes c What do lanes 9 14 tell you about the protein DNA complexes 115 In order to determine the contact points between a regulatory protein and its binding site on the DNA a small fragment of duplex DNA was end labeled at the 5 terminus of the left end as written below and treated with dimethyl sulfate so that each molecule on average has one G nucleotide methylated The regulatory protein was mixed with the preparation of partially methylated DNA and protein bound DNA was separated from unbound DNA After cleaving the DNA at the methylated sites the resultant fragments were resolved on a quotsequencing gelquot An autoradiogram of the results showed bands corresponding to all the GS in the labeled fragment for the unbound DNA but the protein bound DNA did not have bands corresponding to the GS at positions 14 and 16 below When the left end of the fragment was labeled at the 3 terminus no band corresponding to the G bottom strand at BMB 400 116 position 18 same numbering system as for top strand was seen in the preparation of protein bound DNA 5 10 15 20 25 3O 5 39 GATCCGCATGGATGAGTCACGTAACGTGTA 3 39 GCGTACCTACTCAGTGCATTGCACAT What is the binding site for the regulatory protein Are the following statements about 9 and polar effects of some mutations in operons in E coli true or false a Nonsense mutations terminating translation in the first gene of an operon can have no effect on the transcription of subsequent gene in the operon b Mutations in the gene for p rho gene can suppress polarity c The hexameric protein 9 binds to protein free RNA and moves along the RNA when it encounters a stalled RNA polymerase it promoters termination of transcription d The protein 9 is an RNA dependent ATPase Part Three II Chpt 11 Transcription Promoters and Terminators
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