112 Class Note for B M B 400 at PSU
112 Class Note for B M B 400 at PSU
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Date Created: 02/06/15
13MB 400 Part Three 1 Chapter 10 Transcription RNA polymerase B M B 400 Part Three Gene Expression and Protein Synthesis Lecture Notes Overview of Part Three The pathway of gene expression Recall the Central Dogma of molecular biology DNA is transcribed into RNA which is translated into protein We will cover the material in thatorder since that is the direction that information ows However there are additional steps in particular the primary transcript is frequently a precursor molecule that is processed into a mature RNA E g the rRNA genes are transcribed into prerRNA that is cleaved methylated and modi ed to produce mature rRNA Many mRNAs especially in eulrarytoes are derived from prermRNAs hy splicing and otherpmcessiug events This geueml topic will he covered after transcription and before translation Fig31l Pathway for Gene Expression Form transcription ends Splice DNA pre mRNA gt gt mRNA 0 reverse transcription translation replication protein mst translational functional protein modi ca ons BMB 400 Part Three 71 Chapter 10 Transcription RNA polymerase CHAPTER 10 PART THREEI TRANSCRIPTION RNA polymerase A RNA polymerase cabrlyaes the DNAdependent synthesis of RNA 1 RNA polymerase requires DNA as atemplate In duplex DNA the templae strand of DNA is copied into RNA by RNA polymerase The choice of nucleotides during this process is direced by base complementarity sotha the sequence of RNA synthesized is the reverse complement of the DNA template strand It is the same sequence as the nontemplate or top strand except tha US are present instead of Ts This process of RNA synthesis direced by aDNA templae catalyzed by RNA polymerase is called transcription 2 RNA polymerase does not require a primer to initiae transcription 3 RNA polymerase malyzes the sequential addition of aribonucleotide to the 3 end ofa growing RNA chain with the sequence of nucleotides specified by the templae The substrateNTP is added as aNMP with the liberation of pyrophosphae This process occurs cyclically during the elongation phase of transcription NTP NMPn 2 NMPm1 PP templae DNA Mg Fig 312 Sequential addtion of ribonucleo des to growing RNA c a r o a aareoboc hf quot l l r quot4 H Hzo a we och 7 o o g o UFOCWO Mme cyboch ltN MHZ o i H O N NHZ m Pyrophosphate PPi BMB 400 Part Three I 2 Chapter 10 Transcription RNA polymerase 4 The liberated pyrophosphate is cleaved in the cell to 2 Pi an energetically favorable reaction that drives the reaction in the direction of synthesis 5 In the presence of excess PPi the reverse reaction of pyrophosphorolysis can occur 6 Synthesis always proceeds in a 5 to 339 direction with respect to the growing RNA chain The template is read in a 3 to 5 direction B E coli RNA polymerase structure 1 This one RNA polymerase synthesizes all classes of RNA mRNA rRNA tRNA 2 It is composed of four subunits a Core and holoenzyme 0L2l5l5390 Z 0L2l5l539 O Holoenzyme ocg o 2 core O 2 can initiate transcription accurately as the proper site as determined by the promoter Core 2 ocg 2 can elongate a growing RNA chain A promoter can be de ned in two ways a The sequence of DNA required for accurate specific intiation of transcription b The sequence of DNA to which RNA polymerase binds to accurately initiate transcription b Subunits Subunit M Gene Function 539 160 kDa rpoC 5 5 form the catalytic center 5 155 kDa rpoB 5 5 form the catalytic center OL 40 kDa rpoA enzyme assembly also binds UP sequence in the promoter 0 70 kDa general rpoD confers specificity for promoter binds to 10 and 35 sites in the promoter Bacteria have several 0 factors ranging in size from 32 to 92 kDa each of which confers specificity for a different type of promoter BMB 400 Part Three I 2 Chapter 10 Transcription RNA polymerase Fig 313 Diagram of E coli RNA polymerase E coli RNA polymerase 3 Threedimensional structure of E coli RNA polymerase Crystals suitable for X ray diffraction studies have not been obtained yet but the surface topography can be determined from by electron crystallography of two dimensional crystalline arrays Fig 314 Low resolution structure of RNA polymerases from electron crystallography e quot EleCtron microscope Computer workstation Form a 2dimensional crystal Record micrographs from the Average the information from ie 1 molecule thick on a layer of crystalline arrays at three angles the micrographs to determine a positively charged lipid Place on 2 tilted to the incident electron low resolution map eg 27 an electron microscope grid and beam and 1 untilted Angtroms of the surface stain with uranyl acetate topography ie the part outlined by the uranyl acetate EME mu Pan mm 7 x chapm m mmcnpnan RNA palymzlase m 3 5 E to RNA plymmg me am mmm my I Images mm ways by Sam Dam m mum m m plasma uf a hnhenzymz 1 an m gm mm m apen chame fur mm hmmng m mm mm ahseme Ufa m cm emymz 15aan 1m Nam thank channzhsmwclased as m ugusamthnmhsafahammwclusedm make mm This smug swimmammal smug mm acculs Wm B msmm s mugm m Canter hlgh pmcesnvnyan m RNA palymzme m 3 5 gm n iumles m5 cal m Wm Yummymz muenzyme hr RNA wwwemse my E um um 272mmquot wwwmm w mm mm m WWW Mmaw Ennm w mm mum mm 2 mm mm m an vam m muv m m m w m um um Dams A WWW mmwmmmm m m Dgt WW m EME mu Pan mm 7 chapm m mmcnpnan RNA palymzlase o Anm y ms to RNA Wm m usnhnnnhas Mu mm damn m Man damn MNm s mvalved m mumAmman m farm W and mnhzr assembly a hz RNA palymzlase m 0 mmmaldnmamhas mmmmm hemgwedmthz hmmngm m upnm seqmnce m pmmnms in mm moan gems and m cammammm many hm mm all manpmmxacnvam ng3l7 Rnbmmzsumnnmasmnhlyuldmnn mmms Linker arNTD arCTD rDmIE RIZATION VDDJERIZATION mam ASSEVIELY EEN39D 11M 2 7 131m ACTIVATORS quot l 7 POST TRANSLATIDNAL 0 2 P gt 29 MUDEFICATXON SITE 0154 3quot az h39 MW gt azBD39o cam RNAplymnu mm 1 Mummmgm m plasma a hz mama m RNA pulymuase hnhenzymz a m selzcuve mammgm me afmmmmn This s accamphshzdpnmanly thlmghe e san m dlssmmnun m mum palymzlase mm mm 3 Cal has 5 an a m y in 3mm mm sequnces m mm mssacmmn at m camplzx uf mam x mm 5n mm This x um um m elnngaunnphase hm um um mmaonn n Hulaenzymz has a nduced ammty m gemnl mm n s dzcnased mm m rum m mm mssacmmn afhnlnemymz mm gemnl mm s nduced m sham c Hulaenzymz has a gully mmagdaf mty fmpmmnmxsequmes m 2mmssacmmnarmaem mmpmmnmxsequmesls afthz am at hauls BMB 400 Part Three I 2 Chapter 10 Transcription RNA polymerase 2 Events at initiation of transcription a RNA polymerase holoenzyme binds to the promoter to form a closed complex at this stage there is no unwinding of DNA The polymerase promoter complex undergoes the closed to open transition which is a melting or unwinding of about 12 bp The initiating nucleotides can bind to the enzyme as directed by their complementary nucleotides in the DNA template strand and the enzyme will catalyze formation of a phosphodiester bond between them This polymerase DNA RNA complex is referred to as the ternary complex During abortive initiation the polymerase catalyzes synthesis of short transcripts about 6 or so nucleotides long and then releases them This phase ends when the nascent RNA of 6 nucleotides binds to a second RNA binding site on the enzyme this second site is distinct from the catalytic center This binding is associated with quotresettingquot the catalytic center so that the enzyme will now catalyze the synthesis of oligonucleotides 7 12 long The enzyme now translocates to an new position on the template During this process sigma leaves the complex A conformational change in the enzyme associated with sigma leaving the complex lets the quotthumbquot wrap around the DNA template locking in processiVity Thus the core enzyme catalyzes RNA synthesis during elongation which continues until quotsignalsquot are encountered which indicate temlination EMB Ann Part Three 71 Chapmr 1n quotnammpuun RNA pulymerase Figure 313 mm at initiation promoter DNA homenzyme l core polymerase emngation complex 9 swgma dissociates T transloca e make RNA 712 m closed comp ex open oomp ex RNA abomve ini a on BMB 400 Part Three 71 Chapter 10 Tmnscn39ption RNA polymerase 3 Tmnscrip on cycle a Initiation RNA polymerase holoenzyme binds at the promoter unwinds DNA open complex and form phosphodiester links between the initiating nucleotides b Elongation odissociales and core elongales Perhaps other factors bind to enhance the processivity maybe NusA Z c Termination At a termination signal RNA polymerase dissociates from the DNA template and the newly synthesized RNA is released The factor p is required at many terminators Hgnm 319 Diagram of Transcription Cycle in Bacteria Start Stop Nontemplate FINA pol Template hoioenzyme wpolcore G k 1 T Stan Closled complex Sigma I Terminating complex Hhow Open complex NusA 5 quot J W d3g Elongating complex BMB 400 Part Three I 2 Chapter 10 Transcription RNA polymerase 4 Sites on RNA Polymerase core a The enzyme covers about 60 bp of DNA with a transcription bubble of about 17 bp unwound b The duplex DNA being transcribed is unwound at one active site on the enzyme thereby separating the two strands Fig 3110 The two strands are rewound at another active site regenerating duplex DNA c Within the unwound region bubble the 3 terminus of the growing RNA chain is bound to its complement on the template strand via H bonding The DNA strand whose sequence is the same as the RNA except for T s instead of Us is displaced This displaced strand can be called quottopquot quotnontemplatequot or quotmessage synonymous strandquot The template strand can also be called bottom antisense or message complementary strand Fig 3110 RNA polymerase Nontemplate strand 8 lt 5 Template strand d The incoming nucleotide NTP that will be added to the growing RNA chain binds adjacent to the 3 end of the growing RNA chain as directed by the template at the active site for polymerization e The incoming nucleotide is linked to the growing RNA chain by nucleophilic attack of the 3 OH on the X phosphoryl of the NTP with liberation of pyrophosphate BMB 400 f Part Three I 2 Chapter 10 Transcription RNA polymerase The reaction progresses the enzyme moves about 50 nts per sec This is much slower than the rate of replication about 1000 nts per sec If the template is topologically constrained the DNA ahead of the RNA polymerase becomes overwound positive superhelical turns and the DNA behind the RNA polymerase becomes underwound negative superhelical turns The effect of the unwinding of the DNA template by RNA polymerase is to decrease T by 1 for every 10 bp unwound Thus AT 2 1 and since AL 2 0 then AW 2 1 for every 10 bp unwound This effect of the increase in W will be exerted in the DNA ahead of the polymerase The effect of rewinding the DNA template by RNA polymerase is just the opposite of course T will increase by 1 for every 10 bp rewound Thus AT 1 and since AL 2 0 then AW 2 1 for every 10 bp rewound This effect of the decrease in W will be exerted in the DNA behind the polymerase since that is where the rewinding is occurring 5 Inhibitors useful reagents and clues to function a b Rifamycins eg rifampicin bind the 5 subunit to block initiation The drug prevents addition of the 3rd or 4th nucleotide hence the initiation process cannot be completed How do we know the site of rifampicin action is the 5 subunit Mutations that confer resistance to rifampicin map to the 11703 gene Streptolydigins bind to the 5 subunit to inhibit chain elongation These effects of rifamycins and streptolydigins and the fact that they act on the 5 subunit argue C that the 5 subunit is required for nucleotide addition to the growing chain Heparin a polyanion binds to the 5 subunit to prevent binding to DNA in vitro D Eukaryotic RNA polymerases 1 Eukaryotes have 3 different RNA polymerases in their nuclei a Each nuclear RNA polymerase is a large protein with about 8 to 14 subunits V W is approximately 500000 for each b Each polymerase has a different function RNA polvmerase localization svnthesizes effect of oc amanitin RNA polymerase I nucleolus pre rRNA none RNA polymerase II nucleoplasm pre mRNA inhibited by low concentrations some snRNAs 003 ugml RNA polymerase III nucleoplasm pre tRNA other inhibited by high concentrations small RNAs some snRNAs 100 rigml BMB 400 Part Three I 2 Chapter 10 Transcription RNA polymerase 2 Subunit structures a The genes and encoded proteins for the subunits of the yeast RNA polymerases have been isolated and the sequences determined and some functional analysis has been done b Some of the subunits are homologous to bacterial RNA polymerases The largest two subunits are homologs of 5 and SE The roughly 40 kDa subunit is the homolog of 0L 0 Some subunits are common to all three RNA polymerases d Example of yeast RNA polymerase II Approximate size kDa subunits role comment per polymerase 220 1 related to 5 catalytic 130 1 related to 5 catalytic 40 2 related to CL assembly 35 lt 1 30 2 common to all 3 27 1 common to all 3 24 lt 1 20 1 common to all 3 14 2 10 1 e The largest subunit has a carboxyterminal domain CTD with an unusual structure tandem repeats of the sequence Tyr Ser Pro Thr Ser Pro Thr The yeast enzyme has 26 tandem repeats and the mammalian enzyme has about 50 These can be phophorylated on Ser and Thr to give a highly charged CTD RNA Pol Ha is not phosphorylated in the CTD RNA P01 110 is phosphorylated in the CTD Model Phosphorylation of Pol Ha to make Pol Ho is needed to release the polymerase from the initiation complex and allow it to start elongation Figure 3111 Eukaryotic RNA polymerase II kinase ATP gt Pol Ila Pol Ilo phosphatase CTD of large subunit of Pol II CTD of large subunit of Pol II EME mu Pan Thleer chapm m mmcnpnan RNA palymzlase a mlmmnlsmulmtlmn hyuisNAplylnnullsm nmmlm RNApnlymzlu mnEw1L m 3112 m 3113 DgxmmyugRNAplymmg n mmguln tnnscnpmn mls RN pdymmunHmevnul munmmuuunumnmnmn imam mm pm m mum mm mzawmmunmm BMB 400 Part Three I 2 Chapter 10 Transcription RNA polymerase 4 RNA polymerases in chloroplasts plastids and mitochondria a The RNA polymerase found in plastids is encoded on the plastid chromosome In some species the mitochondrial RNA polymerase is encoded by the mitochondrial DNA b These organellar RNA polymerases are much more related to the bacterial RNA polymerases than to the nuclear RNA polymerases This is a strong argument in favor of the origins of these organelles being bacterial supporting the endo symbiotnt model for acquisition of these organelles in eukaryotes c These RNA polymerases catalyze specific transcription of organellar genes E General transcription factors for eukaryotic RNA polymerase II 1 Definition a The general transcription factors GTFs are proteins required for accurate and efficient transcription that are not subunits of purified RNA polymerase We will focus primarily on the general transcription initiation factors GTIFs which are proteins needed for accurate initiation of transcription They are required for RNA polymerase to bind avidly and specifically to normal sites for transcription initiation thereby generating specific transcripts of genes see Fig 3114 Other transcription factors are needed for elongation In living cells RNA polymerases usually start transcription at the beginning of genes The segment of DNA required for specific initiation of transcription by RNA polymerase is called a promoter it is commonly adjacent to the 5 end of a gene Promoters will be covered in more detail in the next chapter Purified preparations of eukaryotic RNA polymerases can transcribe a DNA template containin g a promoter but not with specificity The purified polymerase starts at many different sites on the DNA template not just at the promoter Thus some factors required for specific initiation are missing from purified eukaryotic polymerases These specificity factors are present in crude nuclear extracts because when such crude extracts were added to the purified polymerases speci c initiation at promoters was observed Biochemists purified several transcription initiation factors by fractionating nuclear extracts and assaying for this ability to confer specificity on the RNA polymerase Several different general transcription initiation factors have been defined for each of the three eukayotic RNA polymerases The GTFs for RNA polymerase II are named TFIIx where x A B D E F H etc These originally designated a particular chromatographic fraction that is required for accurate in vitro transcription and now the active protein components of each fraction have been purified TFII stands for transcription factors for RNA Pol II TheGTFs are for RNA polymerase III are called TFIIIA TFIIIB and TFIIIC BMB 400 Part Three I 2 Chapter 10 Transcription RNA polymerase Fig 3114 General transcription factors for RNA polymerase II helicase protein kinase CTD of large subunit of Pol II 2 TFIID is a complex of many subunits It includes the protein that binds speci cally to the TATA box called TATA binding protein TBP plus several IBP associated factors or TAFs TBP binds in the narrow groove minor grove of DNA at the TATA box and bends the DNA Fig 3115 Ribbon diagram of TBP bound to DNA Human TBP TATA Complex 7 min 210mm b ant DNA Image from crystal structures provided by Dr T Nixon BMB 400 Part Three I 2 Chapter 10 Transcription RNA polymerase It is not known if the same set of TAFs are in the TFIID for all promoters transcribed by RNA polymerase II or if some are used only for certain types of promoters TFlID is the only sequence specific general transcription factor so far characterized and it binds in the minor groove of the DNA It is also used at TATA less promoters so the role of the sequence specific binding is still under investigation 3 Summary of general transcription factors for RNA polymerase II Factors for RNA polymerase 11 human cells Functions to Recruit TFIIB No of Molecular Factor subunits mass kDa Functions TFIID TBP 1 38 Recognize core promoter Stablhze TBRDNA binding Anti repression Target RNA PolII to promoter destabilize non specific interactions between PolII and DNA Modulate TFlIH helicase ATPase and kinase activities Directly enhance promoter Helicase to melt promoter CTD kinase promoter clearance Roeder RG l996 TIBS 21 327 335 BMB 400 Part Three I 2 Chapter 10 Transcription RNA polymerase 4 TFIIH is a multisubunit transcription factor also involved in DNA repair Subunits of the human factor Gene Molec mass Proposed Role XPB of protein kDa Function Structure 5 helicase tracks 339 to 5 89 Unwind duplex for ranscription Repair quotCDK7M015 CDK assembly factor Protem kmase Svejstrup JQ P Vichi amp J M Egly 1996 TIBS 21 346350 TFHH is a kinase that can phosphorylate the CTD of the large subunit of RNA polymerase II to form P01 110 This step may be required to release PolII from the initiation complex so that it will begin elongation The general transcription factors and RNA Pol II can be assembled progressively into a preinitiation transcription complex in vitro Experiments using purified GTIFs and RNA polymerase 11 examined the ability of these proteins to assemble a speci c active complex on a particular DNA segment containing a promoter and template for transcription The complex was formed most ef ciently by adding the GTIFs and polymerase in the order shown in Fig 3116 The complex of proteins and DNA could be demonstrated to be speci c and active because when NTPs were added specific transcription from the promoter was observed We call the assembled protein DNA complex that is capable of specific initiation of transcription at a promoter a preinitiation complex As indicated in Fig 3116 the preinitiation complex has the polymerase and GTIFs assembled on the promoter and template The DNA is still a duplex An early step in initiation is melting of the duplex at the start site for transcription The complex in which this has occurred can be called an activated preinitiation complex Once the polymerase has begun catalyzing phosphodiester bond formation then the complex is an initiation complex The experiments showing stepwise formation of a preinitiation complex in vitro have led to the notion that binding of several of the general transcription initiation factors to DNA establishes the structure that the RNA Pol II TFIIF complex will bind thereby establishing the initiation site for transcription According to this model the transcription factors bind to DNA in a preferred order TFHD then TFHA then TFIIB then RNA Pol II TFHF then TFHE BMB 400 Part Three I 2 Chapter 10 Transcription RNA polymerase These and other factors are still being characterized Binding of earlier factors may assist in the binding of later factors E g TFIIE aids in binding of TFIIH See Maxon Goodrich Tjian 1994 GampD 8515 524 Fig 3116 RNA polymerase II initiation Sequentin Binding Model 30 1 ITATA I Ilnr I orTBP TBP mm a Eukaryotic RNA polymerase II A r A c x 39 N DNA melted at Inr Polymerization of 151 few NTPs and phosphorylation of CTD leads to promoter clearance TFIIB TFIIE and TFIIH dissociate PoF elongates and TFIID TFIIA stays at TATA BMB 400 Part Three I 2 Chapter 10 Transcription RNA polymerase 6 RNA polymerase II holoenyme contains the classic RNA polymerase 11 some general transcription factors and other transcriptional regulators Genetic analysis largely in yeast has shown that many other proteins in addition to RNA polymerase II and GTFs are involved in regulated transcription Some were discovered by effects of mutations that alter regulation of genes in one or a few metabolic pathways For instance Ga111 is needed for regulation of the GAL operon encoding enzymes needed for breakdown and utilization of the disaccharide galactose Rgr1 is required for resistance to glucose repression Note that these are the minimal roles for these proteins they were discovered by their roles in these pathways but could be involved in others as well Another class of transcriptional regulatory proteins was isolated as suppressors of alterations in RNA polymerase Yeast strains carrying truncations in the CTD of the large subunit of RNA polymerase II fail to grow at low temperature this is a coldsensitive phenotype Mutation of some other genes can restore the ability to grow at low temperature These second site mutations that restore the wild phenotype are called suppressor mutations Proteins identified by the ability of mutations in their genes to suppress the cold sensitive phenotype of CTD truncations are called Srb proteins since they are suppressors of mutations in RNA polymerase B The ability of mutations in Srb proteins to compensate for the effects of altering RNA polymerase argues that the Srb proteins are associated with RNA polymerase in a functional complex and this has been verified biochemically Hengartner et al 1995 Genes amp Devel 9 897 910 RNA polymerase II the GTFs SRB proteins and other regulatory proteins have now been shown to interact in large complexes in the nucleus Table 316 A complex called the mediator was isolated as a nuclear component needed for a response to activator proteins Assays for in vitro transcription of DNA using purified RNA polymerase II and GTFs failed to increase the amount of transcription when transcriptional activators were added However a component in nuclear extracts would confer the ability to respond this was called the mediator of activation When purified it was discovered to contain several Srb proteins Gall 1 Rgr1 and other transcriptional regulators In a separate line of investigation an RNA polymerase 11 holoenzyme was discovered by isolating the complexes containing Srb proteins This complex contains RNA polymerase II and GTFs unlike mediator plus many of the same proteins found in mediator such as Srbs Rgr1 and Gall 1 This complex was shown to direct correct initiation of transcription in the presence of TBP or TFHD and to be capable of responding to transcriptional activators Fig 3117 BMB 400 Part Three 7 I Chapter 10 Transcription RNA polymerase Table 316 RNA polymerase 11 holoenzyme and mediator Holoenzyme RNA polymerase II TFIIB E F H Srb2 4 5 6 Rgr1 Gal11 others Correct initiation in presence of TBP TFIID Responds to transcriptional activators Mediator 7 Complex needed for a response to transcriptional activators by purified RNA Pol II plus GTFs Yeast Mediator has 20 subunits including Srb2 4 5 6 Srb7 Rgr1 Gal11 Med1 2 6 7 Pgd1 Nut1 2 and others RNA Pol II Mediator some GTFs Holoenzyme These studies show that RNA polymerase II can exist in seVeral different states or complexes One is in a Very large holocomplex containing the mediator In this state it will accurately initiate transcription when directed by TFIID and respond to activators Table 316 The mediator subcomplex appears to be able to dissociate and reassociate with RNA polymerase II and GTFs Indeed this reassociation could be the step that was assayed in the identification of mediator Without mediator RNA polymerase II plus GTFs can initiate transcription at the correct place as directed by TFIID but they do not respond to actiVators In the absence of GTFs RNA polymerase II is capable of transcribing DNA templates but it will not begin transcription at the correct site Hence it is competent for elongation but not initiation Table 317 Expanding the functions of RNA polymerase II Polymerase Trans Startat Respondto cribe Promoter Activator RNA Pol II Yes No No RNA Pol II Yes Yes No GTFs RNA Pol II Yes Yes Yes holoenzyme GTFS BMB 400 Part Three I 2 Chapter 10 Transcription RNA polymerase Fig3117 RNA polymerase II Holoenzyme in initiation Direct Binding of Holoenzyme Model 30 1 TATA I ulnr 39 Holoenzyme activator preinitiation complex DNA melted at Inr Polymerization of 1st few NTPs and phosphorylation of CTD leads to promoter clearance TFIIB TFIIE and TFIIH dissociate PoF elongates and TFIID TFA stays at TATA If the holoenzyme is the primary enzyme involved in transcription initiation in eukaryotic cells then the progressive assembly pathway observed in vitro see section d above may be of little relevance in viva Perhaps the holoenzyme will bind to promoters simply marked by binding of TBP or TFIID to the TATA box in contrast to the progressive assembly model BMB 400 Part Three I 2 Chapter 10 Transcription RNA polymerase that has a more extensive ordered assembly mechanism In both models TBP or TFIID binding is the initial step in assembly of the preinitiation complex However at this point one cannot rule out the possibility that the holoenzyme is used at some promoters and progressive assembly occurs at others 7 Targets for the activator proteins The targets for transcriptional activator proteins may be some component of the initiation complex One line of investigation is pointing to the TAFs in TFIID as well as TFIIB as targets for the activators Thus the activators may facilitate the ordered assembly of the intiation complex by recruiting GTFs However the holoenzyme contains the quotmediatorquot or SRB complex that can mediate response to activators Thus the activators may serve to recruit the holoenzyme to the promoter Further studies are required to establish whether one or the other is correct or if these are separate paths to activation F General transcription factors for eukaryotic RNA polymerases I and III 1 General transcription factors for RNA polymerase I a Core promoter covers the start site of transcription plus an upstream control element located about 70 bp further 539 The factor UBF1 binds to a GC rich sequence in both the upstream control element and in the core promoter A multisubunit complex called SL1 binds to the UBFl DNA complex again at both the upstream and core elements One of the subuntis of SL1 is TBP the TATA binding protein from TFIID RNA polymerase I then binds to this complex of DNAUBF1SL1 to initiate transcription at the correct nucleotide and the elongate to make pre rRNA 2 General transcription factors for RNA Pol III a Internal control sequences are characteristic of genes transcribed by RNA Pol b III see below TFIIIA binds to the internal control region of genes that encode SS RNA type 1 internal promoter TFIIIC binds to internal control regions of genes for SS RNA alongside TFIIIA and for tRNAs type 2 internal promoters TFIIIB The binding of TFIIIC directs TFIIIB to bind to sequences 40 to 11 that overlap the start site for transcription One subunit of TFIIIB is TBP even though no TATA box is required for transcription TFIIIA and TFIIIC can now be removed without affecting the ability of RNA BMB 400 Part Three I 2 Chapter 10 Transcription RNA polymerase polymerase III to initiate transcription Thus THIIA and TFIIIC are assembly factors and TFIIIB is the initiation factor Figure 3118 75 50 25 1 5 0 75 A v r k RNA TAFS olymerase TBP TFIID Pol Eukaryotlc RNA polymerase I lt UBF1 gt SL1 Eukaryotic RNA polymerase III PO39 I IIIA IC TFIIIB TBP TBP e RNA polymerase III binds to the complex of TFIIIBDNA to accurately and efficiently initiated transcription 3Transcripti0n factor used by all 3 RNA Pol ases TBP TBP seems to play a common role in directing RNA polymerase I II and III to initiate at the correct place The multisubunit factors that contain TBP TFHD SL1 and TFIIIB may serve as positioning factors for their respective polymerases BMB 400 101 102 103 104 105 106 Part Three I 2 Chapter 10 Transcription RNA polymerase Questions for Chapter 10 Transcription RNA polymerases What is the role of the sigma factor in transcription and how does it accomplish this Specific binding of E coli RNA polymerase to a promoter 1 completely envelopes the DNA duplex both sides 2 requires sigma factor to be part of the holoenzyme 3 is enhanced by methylation of purine bases 4 results in a temperature dependent unwinding of about 10 base pairs Which statements are correct FOB RNA polymerase How long would it take for the E coli RNA polymerase to synthesize the primary transcript for E coli rRNAs 6500 bases given that the rate of RNA chain growth is 50 nucleotides per second What is the maximum rate of initiation at a promoter assuming that the diameter of RNA polymerase is about 204 Angstroms and the rate of RNA chain growth is 50 nucleotides per second Although three different eukaryotic RNA polymerases are used to transcribe nuclear genes the enzymes and their promoters show several features in common Are the following statements about common features of the polymerases and their mechanisms of initiation true or false a All three purified polymerases need additional transcription factors for accurate initiation at promoter sequences b All three polymerases catalyze the addition of a nucleotide quotcapquot to the 5 end of the RNA 0 For all three polymerases the TATA bindin g protein is a subunit of a transcription factor required for initiation not necessarily the same factor for each polymerase d All three polymerases are composed of multiple subunits What is common and what is distinctive to the reactions catalyzed by DNA polymerase RNA polymerase reverse transcriptase and telomerase
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