Contemporary American Society
Contemporary American Society SOC 125
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Bact 612 Fall 2004 RNA polymerase structure and catalysis Page 1 R Landick Lecture 2 Sept 29 2004 wwwbactwiscedulandick I General outline of lecture 2 A In vitro transcription methods Synchronized transcription 2 Stepwise transcription with immobilized TECs 3 Singlemolecule transcription assays B RNAP inhibitors 1 Rifampicin 2 CBRtype inhibitors 3 Microcin J25 C Abortive initiation and the mechanism and regulation of promoter escape l Abortive initiation detected on different promtoers 2 The scrunching model of promoter escape 3 The energetics of promoter esca e 4 Factors that in uence promoter escape and its regulation Assigned readings Please download from the Cell or Genes Dev web sites 1 Opalka N M Chlenov P Chacon W J Rice W Wriggers and S A Darst 2003 Structure and function of the transcription elongation factor GreB bound to bacterial RNA polymerase Cell 114 33545 Lectures 1 and 2 11 Additional useful references Useful reviews or primary publications 1 Geszvain K M and R Landick 2004 posting date The structure of RNA polymerase This review covers most of the important aspects of RNAP structure for both initiaton and elongation It is available as a pdf for download from my web site wwwbactwiscedulandick You will nd the link in red at the bottom of the home page under latest publications 2 Murakami K S and S A Darst 2003 Bacterial RNA polymerases the wholo story Curr Opin Struct Biol 13 319 3 Conaway R C S E Kong and J W Conaway 2003 TFIIS and GreB two like minded transcription elongation factors with sticky fingers Cell 114 2724 4 Hsu L M 2002 Promoter clearance and escape in prokaryotes Biochim BiophysActa 1577191207 5 Darst S A 2004 New inhibitors targeting bacterial RNA polymerase Trends Biochem Sci 29 159160 6 Landick R 2004 Activesite dynamics in RNA polymerases Cell 116 351353 111 Key concepts These are the key things you should learn from this lecture 1 Three different ways that transcription can be assayed in vitro 2 What abortive paused terminated and productive RNAs look like on a gel 3 The mechanism of three RNAP inhibitors rifampicin CBR and microcin J25 and what they say about the mechanism of nucleotide addition by RNAP 4 How abortive transcripts are generated and why they vary so much among promoters Bact 612 Fall 2004 RNA polymerase structure and catalysis Page 2 R Landick Lecture 2 Sept 29 2004 wwwbactwiscedulandick 5 The roles of sigma region 32 promoter interactions and DNA scrunching in promoter escape 6 Ways that promoter escape may be regulated Bact 612 Fall 2004 RNA polymerase structure and catalysis Page 3 R Landick Lecture 2 Sept 29 2004 wwwbactwiscedulandick IV Study Questions numbering continues from earlier questions 6 The bridge helix has been observed in two conformations in crystal structures One conformation the bent conformation occludes the position that would be occupied by the terminal bp in the RNADNA hybrid if it were in the H site This has led to the suggestion that bridge helix movements may drive translocation Recently however a posttranslocated yeast RNAPH TEC structure was obtained in which the bridge helix is straight Westover et al 2004 Science 303 1014 as was also observed in the ppre translocated TEC structure obtained previously Gnatt et al 2001 Science 292 1876 Do you think this rules out the bridge helixtranslocation model What reasoning supports your answer 7 Suppose a protein bound to the looped out nontemplate DNA strand in the scrunched state of the stressed intermediate Considering the energetics of promoter escape and abortive initiation what effect do you think this would have on the two processes assuming the protein was present in sufficient concentration to fully occupy the binding site Bact 612 Fall 2004 RNA polymerase structure and catalysis Page 4 R Landick Lecture 2 Sept 29 2004 wwwbactwiscedulandick V Lecture notes A In vitro transcription How do we actually study transcript elongation in the laboratory I will tell you about three important methods synchronized transcription stepwise transcription using immobilized RNAP and singlemolecule transcription 1 Synchronized transcription Synchronized transcription is most easily accomplished using linear DNA templates although permutations of it on plasmids are possible This method was worked out by Krummel amp Chamberlin 1992 J Mol Biol 225221 Typically one identi es an initially transcribed sequence that lacks one nucleotide for at least the rst 1011 nt which is the minimum required to generate a stable TEC One way to get around the presence of all 4 bases in this sequence is to use a di tri or tetranucleotide as an initiator provided the sequence between the initiating oligonucleotide and the stop site lacks at least one nt RNAP and DNA template can then be incubated with appropriate buffer and NTPs to generate a halted TEC at the selected location As samples are removed from the reaction they are mixed with denaturing stop buffer final concentrations of 25 mM EDTA and 6 M urea in this case and heated to at least 65 OC prior to loading on a gel to prevent further transcription and to denature both the RNA and RNAP The samples are then electrophoresed on a standard denaturing polyacrylamide gel TBE buffer with 8 M urea 525 nal polyacrylamide depending on what size RNAs are to be examined In the example shown Fig 11 in handout l RNAP halts at A29 because there is no UTP in the reaction and no U except at position 2 which is supplied by the ApU dinucleotide cc32PCTP is included to label the initial RNA transcript typically at a low concentration to allow ef cient labeling Actually all NTPs except the dinucleotide are kept at relatively low concentration to avoid traces of contaminating NTPs we don t want any UTP in this case and to prevent misincorporation when the TEC sits at A29 As you can see the A29 RNA accumulates over the rst 2 min of the reaction this is at 37 OC The predominant abortive transcript is ApUpC in these conditions and you can see this accumulate also At the end of the initial synthesis period heparin or rifampicin are added to prevent further productive initiation and the NTP concentrations are adjusted to those desired in the elongation reaction including the addition of UTP On this template one then observes a strong pause which goes away with time and longer RNAs coming from a terminator near the end of the template and from the runoff transcript the latter is barely distinguishable from the top of the gel There are other ways to synchronize transcription by starting directly from the promoter but these are much inferior owing the slow kinetics of promoter escape and the resulting heterogeneity that is introduced as a consequence of these slow escape kinetics 2 Step wise transcription A modi cation of the synchronized transcription procedure allows isolation of TECs at virtually any template position Fig 12 in handout 1 This modi cation is to immobilize the TEC on a solid support so that the transcription solution can be changed at will The two best ways to accomplish immobilization are either to use a modi ed RNAP containing a hexahistidine tag typically attached at the Cterminus of the 539 subunit but also works at the Nterminus of 5 and the Cterminus of CC or to attach biotin to the end of the DNA template typically by using biotinylated oligonucleotides in a PCR reaction Bact 612 Fall 2004 RNA polymerase structure and catalysis Page 5 R Landick Lecture 2 Sept 29 2004 wwwbactwiscedulandick but can be done in a variety of ways The DNA or RNAP can be immobilized to an appropriate matrix NiZNTA agarose or streptavidinagarose and TECs formed on the beads by adding the other reaction components or initially halted TECs can first be formed and then immobilized Regardless of how they are made the immobilized TECs are then subjected to a series of washes and incubations with limited sets of NTPs to move them to the desired position see example gel in Fig 12 This process is sometimes referred to as TEC walking Two things are especially important in making this procedure work First low NTP concentrations and scrupulous washing are needed to prevent low levels of unwanted NTPs from reacting when they are not desired Second TECs halted at some positions don t restart well 7 in other words they arrest probably by the backtracking events we ve already discussed An example of this in Fig 12 is seen at C34 where about 20 ofthe TECs do not start up when incubated with the NTP mix designed to move them to C44 Thus some care must be taken to pick an order of walking steps that avoids halting the TEC at such arrestprone locations Stepwise transcription was invented about 10 years ago principally by workers in Alex Goldfarb s lab with some improvements from others see Kashlev et al 1993 Gene 13091 Nudler et al 1994 Science 265793 Kashlev et al 1996 Meth Enzymol 274326 Wang et al 1995 Cell 81341 It was adapted to human RNAPII shortly thereafter see Palangat et al 1998Mol Cell 11033 3 Single molecule transcription One of the most remarkable and informative ways to look at transcription elongation is to observe the activity of a single RNAP molecule This is important because despite the best efforts at synchronization a fundamental character of transcription elongation is that RNAP molecules quickly diverge to different states and different template positions This precludes detailed study the elongation behavior from the ensemble averages that can be detected on gels detecting a band on a gel requires the products of at least ten million RNAP molecules A method to observe elongation by single RNAP molecules was first worked out almost 15 years ago Schafer et al 1991 Nature 352444 and has now been refined to the point that the spatial resolution is nearing that of a single bp or nucleotide addition cycle when that resolution is achieved likely in the next 5 years it will be as exciting as the first observation of a single transcribing RNAP molecule These methods rely on attaching a bead that is detectable in a microscope to either the DNA RNA or RNAP to the RNAP in the example shown in Fig 13 Another attachment is made to the surface of the microscope slide and in the example here the bead is trapped into a laser beam that is focused near the surface of the slide The interaction of photons with the bead pulls the bead toward the center of the trap this is the widely used laser tweezer trick Once everything is set up NTPs are owed into the system and transcirption is monitored by the change in the length of the DNA or RNA In this example the bead is kept at a constant position relative to the center of the laser trap and thus experiences a constant force that either assists or opposes transcription by moving the stage with a computer feedback loop The motion of the stage gives a readout of the rate of transcription which can then be plotted As you can see in the example transcription pauses are readily detectable from Neuman et al 2003 Cell 115437 Transcription termination also has been studied by related methods Yin et al 1999 Proc Natl Acad Sci U S A 96 13124 We will return to the use of single molecule methods in our consideration of transcriptional pausing Bact 612 Fall 2004 RNA polymerase structure and catalysis Page 6 R Landick Lecture 2 Sept 29 2004 wwwbactwiscedulandick B Inhibitors ofRNAP RNAP is a fertile target for antibiotic design since it is obviously essential to life yet the sequence variations on the surface of the enzyme that have evolved for species speci c regulation afford binding sites that may be specific for a given kingdom e g bacteria vs eukaryotes or even for a given bacterial lineage e g grampositives vs gram negatives The spectrum of RNAP inhibitors is expanding significantly right now These inhibitors are also especially useful for basic research as tools to block particular steps in the transcription cycle Several important examples are shown in Fig 14 of handout 1 We will consider three in a little detail riifampicin microcin J25 and the CBR703 series of inhibitors 1 Rifampicin Rifampicin is in widespread use as an antibiotic against tuberculosis M ycobacterium tuberculosis It and related compounds Fig 14 bind to RNAP in a pocket that is occupied by the nascent RNA in TECs Fig 15A handout 1 As a result rifampicin blocks the transition from initiation to elongation and forces RNAP into a mode of only making 23 nt long abortive transcripts Fig 15B handout 1 Conversely once a TEC forms rifampicin can no longer bind which makes it a particularly useful experimental tool A crystal structure of RNAP containing bound rifampicin has been obtained and reveals that all known rifampicinresistance substitutions in RNAP occur within the rifampicinbinding pocket Campbell et al 2001 Cell 104901 2 Microcin J25 Microcin J25 is peptide antibiotic made by an E coli plasmid to inhibit the growth of other bacteria the plasmid is only present in certain wildtype isolates of E coli not in laboratory strains Microcin J25 has an unusual structure in which the Cterminal end is stuck into a loop formed between the amino terminus and the side chain of Glu8 Fig 14 called a lassoed tail Wilson et al 2003 JAm Chem Soc 125 12475 Bayro et al 2003 J Am Chem Soc 12512382 Rosengren et al 2003 JAm Chem Soc 125 12464 The peptide binds in the secondary channel of RNAP Fig 15A where it interferes with elongation Fig 15D Mukhopadhyay et al 2004 Mol Cell 14739 Adelman et al 2004 Mol Cell 14753 This inhibition is at least partially competitive with NTPs consistent with the passage of NTPs through the secondary channel to the active site but the possibility exists that microcin J25 also may interfere with movements of the trigger loop the bridge helix or both that are required for nucleotide addition Such a mechanism has been suggested for the eukaryotic RNAPII inhibitor ocamanitin which binds in a similar location Bushnell et al 2002 Proc Natl Acad Sci U S A 991218 3 CBR703 type inhibitors CBR703 type inhibitors are synthetic compounds isolated in a highthroughput screen Artsimovitch et al 2003 Science 302650 They are particularly interesting because like microcin J25 they inhibit the nucleotide addition reaction Fig 15C but the location of resistance substitutitons suggest they bind to the outside of RNAP where the bridge helix emerges brie y on the surface of the 5 subunit Fig 15A Thus the only obvious explanation for their action is an effect on the bridge helix that is transduced to the active site As such the CBR703 inhibitors offer the best evidence yet that movements of the bridge helix are likely to be involved in the nucleotide addition cycle Bact 612 Fall 2004 RNA polymerase structure and catalysis Page 7 R Landick Lecture 2 Sept 29 2004 wwwbactwiscedulandick C Abortive initiation and the mechanism of promoter escape The first step in transcript elongation in many ways is among the most interesting because it involves the opposing properties of the promoterRNAP complex which is designed to recruit RNAP to a specific location on DNA and the TEC which is designed to move freely along the DNA Thus to escape a promoter RNAP must break the promoter contacts either by disrupting the sigmaRNAP interface or releasing the sigma factor from RNAP In addition there is a further inhibitory effect on initial RNA chain elongation imposed by sigma region 32 which lies directly in the path of the nascent RNA both in the RNAP main cleft the hybrid binding cavity and in the RNA exit channel Sigma region 32 must be removed from this location in order for a mature TEC to be established These opposing forces result in RNAP sometimes failing to escape the promoter and instead releasing the initially formed short RNA transcript This process is called abortive initiation The balance between abortive and productive initiation varies at different promoters 1 Abortive initiation detected on different in Vitro Let s look first at some examples of promoter escape using transcription gels Fig 1A handout 2 The extent of abortive initiation varies from not very much for the T7 Al promoter to quite a bit for the N25 promoter to a lot for the N25 anti promoter these are promoters from two different bacteriophage T7 and N25 In most cases promoter escape is thought to occur by the time the transcript reaches 8 nt the size of a full RNADNA hybrid and this is true for T7 Al and N25 However for the N25anti promoter abortive products as long as 1315 are detectable Since this is the size of RNAs able to create a stable TEC at some promoters it suggests that at N25anti all of the RNA contacts of the TEC are established but the RNAP is still unable to let go of the promoter An extensive study of abortive initiation at different promoter variants was recently published by Chamberlin and coworkers Vo et al 2003 Biochemistry 423787 Vo et al 2003 Biochemistry 423798 Hsu et al 2003 Biochemistry 423777 They characterize many of the interactions that govern abortive initiation and promoter escape The results are consistent with a stressedintermediate model of promoter escape that dates back more than 15 years Krummel amp Chamberlin 1989 Biochemistry 287829 but that is described in the context of the RNAP structure only recently Hsu 2002 Biochim Biophys Acta 1577191 2 The scrunching model of promoter escape This model explains how an initiating RNAP can maintain promoter contacts while extending the RNA chain and allowing downstream DNA to enter the active site without actually translocating along the entire DNA molecule Fig 1B handout 2 In the scrunching model the DNA strands loop out of the surface of RNAP as the template DNA enters the active site for directing nucleotide addition Recall that the nontemplate DNA is already extruded onto the surface of the open complex so that it can easily accommodate more looped out DNA strand between the point of melting in front of the active site and the contact of sigma to the 10 element The template DNA strand must loop out upstream of the hybrid region since it must stay engaged in the hybrid within the main channel and before it repairs with the nontemplate strand and interacts with sigma region 4 as duplex DNA The template DNA comes out of the main channel through a small tunnel that is well illustrated in Fig 6 of Murakami et al 2002 Science 2961285 which you were assigned as reading for the Gourse lectures Thus as RNAP Bact 612 Fall 2004 RNA polymerase structure and catalysis Page 8 R Landick Lecture 2 Sept 29 2004 wwwbactwiscedulandick begins RNA synthesis and draws downstream DNA into the enzyme it maintains promoter contacts to upstream DNA by looping out the template and nontemplate DNA strands scrunching at opportune positions There are at least two other important dynamics that in uence abortive initiation and promoter escape The first is the movement of the sigma region 32 loop When RNA synthesis commences the sigma region 32 loop lies through the RNA exit channel and in the main channel near the active site its exact trajectory in the open complex is unknown As the RNA chain grows the RNA must push the region 32 loop out of the exit channel One unresolved possibility is the 32 loop actually interacts with the 5 end of the transcript which will contain a triphosphate whenever RNAP initiates with an NTP which is how it works in vivo Such a triphosphate is likely to be chelated to a Mg2 ion and the 32 loop contains two acidic residues that might also contact this Mg2 ion The second dynamic is the generation of the RNADNA hybrid As the RNA grows the hybrid will become more stable and also generate stabilizing interactions of the hybrid with the RNAP main cavity However it also is likely that the short initial RNA is prone to backtracking because there are no upstream RNA interactions to restrain it and also because the sigma region 32 loop may oppose forward translocation of the RNA Backtracking would force the short RNA into the secondary channel It seems likely that this is the route by which abortive RNAs are lost from the initiating complex 3 The energetics of promoter escape One of the most useful ways to envisage the processes of abortive initiation vs promoter escape is as an energy pro le of the two pathways Fig 1C The initiating complex is equivalently stable to the open complex at the beginning of the process although less stable than the TEC The rates at which the complex aborts or escapes are determined by the heights of activation energy barriers AGaI for abortive initiation AGeI for escape The height of the barrier to escape will be determined in large part by the strength of promoter contacts that must be broken As the RNA grows DNA scrunching will destabilize the complex because the number of DNA basepairs that must be melting increases and movement of the 32 loop will destabilize the complex as the 32RNAP interactions are disrupted At the same time the growth on the RNADNA hybrid will somewhat mitigate this destabilization but the net effect will still be a less stable complex Thus the overall stability of the initiating complex will be decreased corresponding to a rising level of the complex on the energy diagram This increases the rates of both the abortive and escape pathways rates are determined by the height of the energy barrier above the ground state in simple transition state theory of reaction rates However the generation of the RNADNA hybrid also will raise the transition state energy for abortive release because each new RNADNA bp will have to be broken to accomplish release The net effect of both trends is to make the escape pathway more favorable However the extent to which the two pathways compete kinetically will depend on the energetics of particular promoters I should caution that this energy diagram probably oversimplifies the actual processes but it offers a useful paradigm to understand what occurs during this interesting phase of transcription 4 Factors that in uence promoter escape and its regulation Multiple factors will change the energetics of promoter escape vs abortive initiation Fig 2 Some if not all of these are used for regulation of transcription in bacteria Bact 612 Fall 2004 RNA polymerase structure and catalysis Page 9 R Landick Lecture 2 Sept 29 2004 wwwbactwiscedulandick As we ve already discussed strong promoter contacts can restrain RNAP during initiation Fig 2A This is probably why one does not nd natural promoters in which all three elements extended 10 35 and UP are consensus At least one report exists of strong UP elements creating barriers to promoter escape although the interaction of these sequences with the OL CTD was unknown at the time the work was done Ellinger et al 1994 JMolBiol 239466 Strong interaction of sigma with RNAP also may create barriers to promoter escape Fig 2B The most obvious ofthese is the sigma 32 loop Some amino acid substitutions in this region of sigma affect promoter escape Cashel et al 2003 J Biol Chem 2785539 The energetics of the scrunched DNA is likely to in uence the stability of the stressed intermediate Fig 2C Although little work has been done to investigate this certain DNA sequences are known to form extremely stable secondary structures Nakano et al 2002 Biochemistry 4114281 Such structures would increase the stability of the stressed intermediate and thus lower the rates of abortive initiation and promoter escape perhaps allowing synthesis of longer RNAs before collapse of the stressed intermediate The initially transcribed sequence is likely to in uence the energetics of abortive and productive initiation Fig 2D Two highly aborting promoters N25anti and a T7 A1 variant share the same initial sequence 5 AUCCC Feng et al 1994 J Biol Chem 26922282 Hsu et al 1995 Proc Natl Acaal Sci USA 9211588 This may stabilize the stressed intermediate making productive initiation slower while perhaps still allowing abortive release DNA binding proteins activators and repressors can inhibit promoter escape or promote it depending on how they interact with the stressed intermediate Fig 2E One well characterized example of each effect is published the phage CD29 P4 protein Monsalve et al 1996 EMBO J 15383 which can inhibit promoter escape through interactions with the OL CTD and the P22 Arc protein which can promote escape by competing for promoter contacts Smith amp Sauer 1996 Proc Natl Acaal Sci USA 938868 Finally the Gre transcript cleavage factor can promote promoter escape Fig 2F Feng et al 1994 J Biol Chem 26922282 Hsu et al 1995 Proc Natl Acaal Sci USA 92 11588 Gre factors either inhibit release of abortive RNAs out the secondary channel or allow multiple tries at successful initiation by cleaving the initial RNA if it backtracks
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