Honors Course in Greek
Honors Course in Greek GREEK H195
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Chapter 6 Weaver 2e M01 Biol X107A Ch 6 The transcription apparatus of prokaryotes A Introduction 2 B Common abbreviations and symbols 4 C RNA 1 39 Structure 4 D Promoter 5 E Stages of initiation or anything 6 F Methods Footprinting protection and crosslinking experiments 7 G Initiation 7 H Regulation of initiation 8 1 Sigma factors and the speci city of RNA polymerase 9 J The ac subunit 10 K Perspective Elongation and termination states of the polymerase 10 L Elongation 11 M Transcription and r quot39 13 N Supercoiling and transcription 13 0 Termination 14 P Reporters measuring gene activity 16 Q Previews 16 R Further readino 17 S Frratum 21 T Homework 21 U Partial answers 25 Reading notes Also read required 0 Gel electrophoresis p 92 ff especially part on proteins for Sect C Note that this section was assigned for Ch 2 in the context of DNA analysis The basic ideas of gel electrophoresis are the same for both DNA and protein but technical details differ Reporter genes p 121 for Sect P Filter binding p 122 for Sect D DNase footprinting p 125 for Sect F Clark amp Russell Ch 6 Chapter 6 Weaver 2e Mol Biol X107A Page 2 A Introduction Reminder You may want to review the transcription section of Ch 3 in Weaver to serve as an overview of this chapter This chapter discusses the mechanism of transcription It emphasizes the initiation step in bacterial transcription in so doing it presents the role of sigma 6 factors in gene regulation Initiation is the most efficient place to regulate and is thought to be the major site of regulation There is much more on transcription and gene regulation in the rest of Part III along with Parts IV and V and even VI But this chapter is an excellent way to start the story We will focus on bacterial transcription here Although the central ideas are very similar with eukaryotes the details especially for initiation are quite different We will look at this brie y later Ch 1013 highlights Highlights Transcription an overview RNA polymerase General nature The stages of transcription 7 Initiation major topic separate 7 Elongation general biochemistry of the reaction the DNARNA hybrid how the polymerase moves pausing and backing up Is there proofreading 7 Termination Bacterial RNA Pol terminates at fairly specific sites Role of hairpin sequences and accessory proteins in transcription termination pindependent intrinsic and pdependent termination 7 States of the polymerase o Emphasis on key step of initiation The template recognition step is especially important because of its key role in gene function and therefore in regulation Initiation in the broad sense also includes how the polymerase actually starts transcribing once it has found a gene Closed and open complexes promoter clearance 0 RNA Pol accessory factors for initiation 6 factors Chapter 6 Weaver 2e Mol Biol X107A Page 3 o Promoters promoter elements consensus sequences role of DNA microstructure o Footprinting a general method for studying DNAbinding proteins 0 Multiple 6 factors regulatory implications 0 Effect of DNA transcription on supercoiling and vice versa Comment As we get into the core material starting with Ch 6 it may be worthwhile to talk about the level of understanding that we expect It is reasonable to suggest that we can discuss the material at three levels 1 The conclusions For example in this Ch 6 we describe how RNA is made 2 Experimental methods that are useful in studying such matters 3 The historical development of how we learned particular conclusions Level 1 is a basic minimum For some of you not working in the field it may be what you would prefer to focus on However for an upper division course we really should go beyond that Molecular biology has long been driven by the development of methods In general we want to develop some significant A J39 of 39 39 biology quot J 39 On the other hand this is a principles course not a methods course So we want to achieve some balance My general approach will be to present some basic methodology focusing on the logic of the experiments and on how the results are interpreted However I will not try to create a detailed history story It is fun to do so but those of you who are in science know that any textbook presentation of history is oversimplified Consistent with the above tests will generally take it for granted that you know the basic conclusions Few questions will solely address the conclusions Many questions will deal with experimental methods in general including interpretation of data However I do not expect you to remember history For example I would not ask you to remember the history of how a particular point was established but I might present you with some data similar to that shown in the book and ask for your interpretation Weaver has a strong emphasis on describing the experimental basis of the work he presents This is good but I would caution that the emphasis should be on following the ideas not on remembering specific experiments What is true is that the types of experiments that Weaver discusses often have broad applicability Learning molecular biology is like peeling an onion There are layers upon layers of understanding One first learns about the importance of DNA gt RNA then the discovery of RNA polymerase then the discovery of the sigma 6 factor that has a key role in initiation Then one learns about the specific functions of 6 which are due to specific parts of the protein Then one learns that different 6 factors work differently and later that eukaryotes have even more complex variations Remembering that region 24 of 6 binds to the 10 part of Chapter 6 Weaver 2e Mol Biol X107A Page 4 the promoter is a very speci c detail It has little meaning unless you understand all the higher level ideas I would also suggest that this is a level of detail not worth remembering at all because of its lack of generality Remember that you do not have to read and master a chapter straight through Depending on your background you may well want to browse the chapter outline the key conclusions and then go back and ll in some of the experimental development B Common abbreviations and svmbols For RNA polymerase we may say RNA Pol or with appropriate context just Pol RNAP is also used We will use several Greek letters for bacterial RNA Pol subunits most importantly 6 sigma The particular 6 may be denoted by a superscript either a letter which is more or less arbitrary or a number which gives the approximate size in kilodaltons GS and 670 are examples Recall Ch 3 handout Sect C about use of terms such as coding strand C RNA polymerase structure Ch 6 Sect 1 Sect 1 and 2 both introduce some things that get elaborated in the rest of the chapter Major messages from this section Basic subunit composition of E coli RNA polymerase xz 39c We usually ignore the small mysterious and nonessential n subunit mentioned in Fig 639 Fundamentally this composition is of little signi cance until we attach functional signi cance to parts The only functional information revealed in this section is that 6 is involved in choosing speci c initiation sites It is also hinted here that 6 is not always part of the enzyme That is as will be detailed later 6 joins the polymerase for the initiation step then leaves and is not part of the P01 during elongation Thus we introduce the idea that the composition of RNA Pol varies during the transcription cycle One might also speculate at this point that there migh be more than one type of 6 with different speci cities Useful methods PAGE Fig 6 l but the method is introduced in an assigned part of Ch 5 see Also read note at top of handout Gel electrophoresis of proteins As usual in such physical separation methods the migration rate of a particular type of molecule depends on the driving force Chapter 6 Weaver 2e Mol Biol X107A Page 5 electrical in this case and on the shape of the molecule via friction In one common form of PAGE analysis of proteins the detergent sodium dodecyl sulfate SDS is added This denatures most proteins It also coats the protein rather completely therefore the amount of charge is mainly due to the SDS Considering all these effects proteins generally separate by size in SDSPAGE analysis Hybridization Fig 62 and recall Ch 2 In a simple experiment Fig 62a one simply measures how much of a sample can hybridize to a reference DNA In a competition analysis Fig 62b one tests two samples to see whether they compete with each other Ignore Discussion of phage T4 life cycle and classes phases of RNA D Promoter Ch 6 Sect 2 Major messages from this section The main message is the identification of a specific region where RNA polymerase binds to DNA and initiates transcription We call this the promoter In bacteria a major type of promoter is characterized by regions shown in Figs 610 amp 6 11 The 10 and 35 elements are most commonly discussed The optional UP element is particularly important for extremely high levels of expression Caution The term promoter is clearly defined in a broad conceptual sense but its precise definition is about as clear as the definition of a gene recall Ch 3 handout Sect J The broad concept relates to RNA Pol binding Some specifics are well characterized for bacterial promoters but it turns out that not all bacterial promoters follow exactly that plan And then with eukaryotes the concept is fine but the details are very different Ch 10 Consensus seguences Introduced here for RNA Pol and the promoter but generalizable Promoters that are recognized by a particular RNA polymerase or rather by a particular 6 tend to have similar sequences In E coli the major recognition areas for RNA polymerase with 670 are the 35 and 10 regions l is the start site Weaver shows the E coli 670 consensus promoter in Fig 610 The quality of the consensus at 10 the Pribnow box can be summarized by T80A95T45A50A50T95 Chapter 6 Weaver 2e Mol Biol X107A Page 6 The term T80 means that T is found at that position 80 of the time 25 would be random Consensus sequences are often derived simply from statistical analysis of what is found in Nature that is they describe the most common base at each site In some cases as in this one the closer a specific sequence is to the consensus the stronger the binding Mutations that make a specific promoter closer to consensus usually make initiation more likely up mutations etc p 140 A third optional promoter element termed the UP element is now recognized It is upstream of the main promoter Fig 6 l 1 It is needed for the high level function of the rRNA genes among others and is contacted by the ac subunit of RNA Pol Fig 630 Lohrke et al 1999 is an example of regulation at the level of the ac subunit Bartlett et al 2000 discuss one role of FIS shown in Fig 611 Schaumburg amp Tan 2000 show an additional complexity of Chlamydia promoters Meima et al 2001 show how to isolate promoters Stages Fig 67 See next section Useful method Filter binding See Fig 535 and associated text see Also read note at top ofhandout Note that this method is strictly empirical E Stages of initiation or an hing Fig 67 shows stages of RNA Pol binding to the promoter The remaining sections of the Ch discuss general stages of the transcription process initiation etc It is very useful for us to divide complex processes into stages for ease of investigation and discussion The caution is that these distinctions are for our convenience and do not necessarily represent clear natural divisions So be exible when dealing with such manmade categories We recognize fairly distinct steps in initiation of RNA synthesis by RNA polymerase 1 initial binding to DNA nonspecific 2 promoter search 3 promoter recognition which leads directly to formation of the closed complex 4 formation of the open complex 5 6 beginning synthesis of a chain clearance of the promoter Chapter 6 Weaver 2e Mol Biol X107A Page 7 The third step may be considered promoter binding per se formation of the open complex involves opening up melting the DNA We will focus on promoter recognition Weaver introduces the first four steps in Fig 67 Then Fig 6 l3 overlaps with this and continues F Methods Footnrinting quot and cm Iinkinoe neriments The following sections involve many experiments with the general theme of trying to identify where a protein interacts with DNA There are variations but several involve some type of interference Footprinting See Fig 537 see Also read note at top of handout The key idea is that the bound protein the one we are studying interferes with DNase digestion of the DNA Where this interference occurs is analyzed by gel electrophoresis The result is that some lengths don t appear because the protein protected the DNA from being cleaved at specific sites Chemical footprinting or protection Binding of the protein to DNA may affect chemical reactions we perform on the DNA On the other hand specific chemical modifications of the DNA may affect protein binding Fig 538 introduces one type of such experiment and Fig 6 17 is an application In these experiments the only bases that get methylated by the chemical reagent DMS are those that have been exposed as a result of a protein binding to the DNA Crosslinking experiments can be thought of as a variation of chemical footprinting or protection experiments The key difference is that the crosslinking experiment preserves the bound state of something for further analysis p 159 amp Fig 637 3 You should be able to follow the basic logic of these experiments However I would not expect you to remember the specific reactions involved or the details of any particular analysis G Initiation Sect 63 RNA polymerase recognizes a specific recognition site called a promoter near the beginning of a gene or operon group of genes transcribed together introduced in Sect D above With bacterial RNA polymerase this base sequence recognition is due largely to a transient RNA polymerase subunit called 6 The cpromoted binding leads to the closed complex this isomerizes to the open complex and then RNA synthesis can proceed Open complex formation involves opening up the DNA Chapter 6 Weaver 2e Mol Biol X107A Page 8 Figs 61719 It also involves a conformational change of the core Pol itself allowing the Pol to hold the DNA Weaver introduced this conformational change back in Sect 2 Figs 68 amp 9 Much of Section 3 describes evidence that 6 binds to the promoter and defines the specific regions of each that are involved Much of the early evidence was from footprinting and protection experiments Sect F More recently evidence from genetic analysis and protein structures has contributed Ultimately we achieve the rather detailed information summarized in Fig 623 Guthold et al 1999 observe RNA Pol sliding along DNA this onedimensional diffusion following nonspecific binding presumably aids in finding the promoter Narysth et al 2000 discuss recent work on the open complex using crosslinking analysis The key property of 6 is recognizing the promoter Along the way we learn other things about it 6 alone cannot bind to promoters p 148 Callaci et al 1999 and Young et al 2001 explore this 6 reduces nonspecific binding p 151 After RNA synthesis has started 6 dissociates from the polymerase Fig 615 summarized in Fig 616 This weakens the strong Polpromoter contact and allows Pol to transcribe away from the start site The relationship between 6 release and abortive initiations Fig 612 is not clear Weaver recognizes open complex formation e g Fig 617 and promoter clearance eg Fig 613 but does not try to explain them That is fine for now we simply note that we can recognize those steps Method Rifampicin rifamycin is an antibiotic that specifically inhibits the initiation step of bacterial RNA synthesis p 143 Rifampicinresistant mutants can be easily isolated The resistance is associated with the 5 subunit you should be able to follow the logic of the mixandmatch experiment of Fig 632 which shows this Later p 163 Weaver explains that Rif blocks the exit channel for the nascent RNA Rifampicin is a major drug in TB treatment but resistance to it is becoming a problem H Regulation of initiation End of Sect 63 and misc Chapter 6 Weaver 2e Mol Biol X107A Page 9 The most efficient place to regulate a process is at the start In fact transcription is regulated at every conceivable place but regulation of initiation does seem to be most common Regulation is the explicit topic of other chapters but we note some aspects here in passing Anything that modulates the quality of the Polpromoter interaction will regulate We have already noted that there are promoters of various strengths Weaver also notes brie y that there may be multiple 6 s for the same cell each with a different type of promoter speci city More in Sect I Weaver notes the role of the UP promoter element and its interaction with the ac subunit of Pol Fig 630 has an interesting implication In frame a if another protein bound nearby and was able to tether at it might also enhance transcription In fact it is known that some activator proteins proteins that stimulate transcription work precisely that way Peek ahead to Fig 717 for an example Also see Lohrke et al 1999 More generally The promoter where RNA polymerase recognizes the gene and binds is a good target for gene regulation A protein tightly bound near the promoter can prevent the polymerase from binding or functioning properly that s the idea of arepressor Or a protein might help the polymerase bind to the promoter thus enhancing gene expression that s the idea of a transcriptional activator see previous paragraph The role of an activator protein is particularly important if the promoter is intrinsically weak Some genes have more than one promoter to allow for complex regulation The promoters may use different 6 factors next section or different activator proteins to respond to different signals Note the title of Sect 84 but don t worry about that section I Sigma factors and the specificity of RNA polymerase The 6 subunit of bacterial RNA polymerase is required for initiation of RNA synthesis 6 recognizes the promoter One can imagine having different 6 factors that recognize different promoters thus conferring different specificities on the polymerase Weaver brie y notes this on p 147 He discusses some examples of multiple 6 factors in the upcoming chapters on gene regulation For now some brief comments Multiple 6 factors were first recognized in viral systems where the virus introduces a new 6 to change the transcription specificity from host genes to viral genes Bacillus subtilis a bacterium which sporulates changes 6 factors for the sporulation phase of growth In fact there is a cascade of 6 factors during sporulation Sect 83 Qiu amp Helmann 2001 discuss two of the seventeen B subtilis 6 factors Chapter 6 Weaver 2e Mol Biol X107A Page 10 What about our old friend E coli For a long time we recognized only one 6 factor in E coli However it has become clear that there are several others with special roles It is likely that there are at least seven Maeda et al 2000 Interestingly as information accumulates similar types of 6 factors are being found in quite diverse bacteria Not all 6 factors work exactly the same way As an example Weaver introduces the B subtilis 5 protein on p 149 The details of this are not important but there is a general idea that the various functions that are required may occur in different arrangements in different cases 654 which transcribes the glnA gene is an interesting 6 variation Ch 8 Sect 4 More about regulation and 6 factors in later chapters Jishage et al 2001 discuss an antisigma factor J The ac subunit In Sect D we noted how the ac subunit may make DNA contacts and may be subject to 39 quot We also quot J the 39 quot via 0c in Sect H In Sect 64 middle subsection p 159 Weaver discusses the role of 0c in the overall structure of the Pol Arguably this might be better placed with the earlier sections on initiation rather than with elongation In any case I won t pay much attention to this little subsection K Per nective39 Plnmmtinn and termination39 states ofthe 39 This is a new section to try to provide some perspective for a couple of messy sections that follow Weaver 64 amp 65 L amp 0 here Comments and suggestions welcomed You may wonder why all the detailed discussion of the mechanism of elongation and termination in sections 64 amp 65 There may seem to be an excessive concern with the most intimate details of the Pol which amino acid is binding what It might seem to you that elongation involves adding nucleotides and that is done until there is a signal to stop Why all the detail Unfortunately that simple view of elongation and termination begins to break down almost as soon as any attempt is made to understand how Pol moves My purpose in this section is to give you a brief overview of what emerges from all the discussion of elongation and termination so you will have a sense why the rest follows The simple view would suggest that Pol moves down the template at some constant rate which is in fact easy enough to measure However closer examination reveals that the rate is very uneven In particular some template sequences are easier to transcribe than others You might be tempted to say so wha to this So long as the template is properly transcribed who cares about the detailed kinetics Chapter 6 Weaver 2e Mol Biol X107A Page 11 The simple view would suggest perhaps even seem to demand that there are welldefined stop signals for transcription However closer examination reveals that is not really so Sites characterized as stop signals increase the probability of stopping but it is an oversimplification to suggest that a given sequence supports either elongation or termination As part of that we have learned that there are proteins that modulate termination For example in the presence of antiterminator proteins Pol will elongate through sites that might have been stop sites It turns out that the elongation and termination complexities hinted at above are related As a brief overview we may now think of the Pol moving along the template somewhat irregularly At its worst it stalls or pauses as is usually said A paused Pol may actually be in a different conformation than an active Pol And we can expand that may of the previous sentence to suggest that there may be multiple paused states Some of them may be not serious but some may be more serious the Pol may even be in a state where it cannot proceed a state some will call arrested A paused Pol may even back up and then try again Or it may terminate In fact it is now a reasonable view that termination is a special case of pausing that termination can occur only from the paused state Finally remember those proteins that modulate termination they may help the P01 deal with pauses With all this you should see that there is interest then in exactly how Pol moves and how it behaves when it has trouble moving This is a messy field with complicated experiments sometimes subject to artifacts Models for elongation and termination have changed considerably over my years of X107A I do not know how close we are to a final truth But the broad perspective of trying to see how these processes occur are related and are modulated by proteins is worth attention L Elongation Ch 6 Sect 4 first and last subsections The basic biochemistry of elongation is straightforward Nucleoside triphosphate NTP precursors donate NMP to the 339 OH of the growing chain Fig 313 RNAn NTP gt RNAn1 PPi RNAn means an RNA chain of length n nucleotides NTP is a general notation for any ucleotide IriEhosphate Note that we might write RNAn as NMP PPi pyrophosphate The incoming nucleotide is activated at the 539 end it reacts with the 339OH of the growing chain This is called growth at the 339 end or growth from 539 to 339 Recall Fig 313 Pyrophosphate PE is released and later hydrolyzed Mentioned in Ch 2 handout Sect F This PP hydrolysis step is important it provides additional energy to drive the reaction toward polymerization In fact without PPi hydrolysis the energy change AG for the Chapter 6 Weaver 2e Mol Biol X107A Page 12 polymerization reaction is about 0 making it freely reversible There is no change in the number of phosphate bonds the NMP is transferred from the PPi to the growing chain Coupling the transfer reaction with PE hydrolysis hydrolyzes net one high energy bond thus the overall set of reactions liberates N 7 kcalmole For initiation of an RNA chain the first NTP is simply laid down Thus the presence of a triphosphate on the 539 end is a hallmark of a biosynthetic RNA end as distinct from a cleaved RNA end or as we shall see later from a DNA end The work on elongation reveals that Pol subunits l and 539 are the workhorse subunits for the core functions of elongation in fact these are the most highly conserved subunits from one organism to another Several Figs such as 642 show the basic transcription bubble Weaver introduces the melting of the template DNA and various measurements of the size of the melted region in the open complex in Figs 6 1719 During elongation the melted region remains as a transcription bubble The size of the DNARNA hybrid in this bubble long debated is now generally accepted as about 89 bases In the abstract the size of the hybrid has no particular significance although it may have implications for important mechanistic details For example it is now fairly clear that the DNARNA hybrid is not the key contributor to maintaining the P01 bound Much more important is the question of what happens when Pol is stalled While the details are still under debate it is clear that Pol can back up allowing the recently incorporated nucleotides to be removed thus allowing Pol to proceed again The argument is that the DNA RNA hybrid senses mispairing which may stimulate backing up and error correction It is debatable whether the reverse reaction provides careful proofreading as occurs with DNA synthesis Sect 202 More likely it merely deals with gross stalls Mair amp Roberts 2000 discuss the GreA protein an accessory factor involved in this process The idea is that Pol backs up thus exposing the newly synthesized region for cleavage It is also possible that the process is coupled to template repair Thomas et al 1998 argue that careful proofreading may occur There are three important contact areas in the transcription complex Two of these are protein nucleic acid in front of and behind the growing chain end Fig 636 The third is the 89 base RNADNA hybrid in the bubble The proteinDNA contacts maintain the basic positioning of the Pol the hybrid region may well be sensitive to mismatches that promote backing up Nudler 1999 reviews recent work on how Pol moves much of the work is from his lab Korzheva et al 2000 use a combination of techniques to explore the structure of the transcription complex with an attempt to understand the structural transitions that occur during elongation Cheetham amp Steitz 1999 discuss the specialized RNAP of phage T7 Chapter 6 Weaver 2e Mol Biol X107A Page 13 M Transcription and quot39 Ch 6 Sect 4 nal subsection on Topology p 164 Supercoiling of DNA relates to stress on the DNA including unwinding Ch 2 handout Sect H Thus it should not be surprising that supercoiling interacts with transcription Transcription affects supercoiling in two ways During initiation formation of the open complex opens up the DNA this introduces positive supercoiling if the DNA resists Fig 619 makes use of this to measure the amount of DNA melting during open complex formation During elongation the P01 moves along the DNA with the DNA unwinding in front and rewinding behind the Pol There is no net change in DNA winding as a constant size bubble moves along the template but there are local effects The DNA ahead of the polymerase becomes overwound gains positive supercoils and the DNA behind the polymerase becomes underwound gains negative supercoils Fig 643 lays the groundwork for how the moving Pol affects supercoiling I will bring a model to show this The effect is greatest if the DNA is anchored so that it cannot freely rotate You should be able to predict the direction of the effects as stated above from what you know about transcription and DNA structure Harada et al 2001 observe the DNA rotation that occurs along with transcription if the DNA is free to move N a quot39 and transcription not in book Supercoiling and transcription can interact in another way supercoiling can affect transcription Initiation of transcription requires a protein to recognize a specific DNA site and requires the DNA to melt Supercoiling affects the appearance of DNA thus it might reasonably affect recognition events for better or worse Further the common form of supercoiling found in vivo provides a stress that helps opens up the DNA The most common result is that increased negative supercoiling increases promoter function Remember that negative r quot39 is r 39 39 quotJ 1 to strand unwinding and that unwinding is required for RNA polymerase to act Also remember that bacterial DNA typically is negatively supercoiled in vivo 39 I A counterexample is the gene for the enzyme that adds negative supercoils to DNA topoisomerase I commonly known as DNA gyrase Since gyrase is the enzyme that Chapter 6 Weaver 2e Mol Biol X107A Page 14 provides the negative supercoils it is logical that the cell interprets a de ciency in negative supercoiling as a signal to make more gyrase This is seen in vivo using mutants with varying levels of supercoiling Further adding a drug that inhibits gyrase and therefore reduces supercoiling enhances gyrase synthesis These results describe the phenomenon but do not make clear the mechanism In one case the mechanism is clear In vitro experiments with DNA carrying the gyrase gene provided a simple answer DNA with varying degrees of supercoiling was used to make message The amount of gyrase message made depended on the amount of supercoiling Thus the effect appears to be a direct effect of the supercoiling on the efficiency of the gyrase promoter No other proteins are involved in this in vitro system The bigger story here is to emphasize that the exact DNA structure including effects of base sequence and supercoiling affects exactly what the P01 especially6 sees Of course this also applies to any other protein that recognizes DNA and to any other variable that affects the DNA microstructure Van Komen et al 2000 show how supercoiling can affect recombination Recall Ch 2 handout Sect I Fang amp Wu 1998 describe an example of how the effects of transcription and supercoiling on each other may play out 0 Termination Ch 6 Sect 5 RNA synthesis stops at more or less specific sites In bacteria there are two general modes of termination intrinsic and pdependent p rho is an accessory protein factor Intrinsic termination is also referred to as pindependent termination Hairpin sequences in the RNA transcript tend to cause the RNA polymerase to pause The hairpin intrastrand RNARNA helix tends to pull the nascent RNA chain away from the polymeraseDNA complex Hairpins come in a variety of strengths and this is surely a place where RNA synthesis is regulated Recall the cruciform structure for DNA Ch 2 handout Sect G The hairpin is the SS equivalent p 166 The stories of pausing and then of termination relate to the nature of the elongation complex Sect L Active doing elongation paused or arrested and terminating may all be states of RNA Pol Recall Sect K above where we introduced this interrelationship At intrinsic Qindependent termination sites Fig 645 the hairpin feature is further enhanced by a polyU region which follows immediately RiboUdeoxyriboA base pairs are unusually weak p 166 Thus the intrastrand RNA hairpin and the weak DNARNA hybrid that follows both serve to drive termination Chapter 6 Weaver 2e Mol Biol X107A Page 15 Gusarov amp Nudler 1999 explore details of the intrinsic termination reaction Davenport et al 2000 explore transcription and termination by single RNA polymerase molecules Toulokhonov et al 2001 offer a more complex interpretation of how a pausetermination site works pdependent termination Here is a model for how p may work Fig 650 Probable stages of p action p gets on On what On the nascent RNA pbinding sites on RNA are not well de ned However they seem to include a large region 100 bases or so without secondary structure Crich Gpoor or other tightly bound proteins or ribosomes That is p enters onto a large section of free RNA Bogden et al 1999 explore how p nds it target The mechanism of p action may well be to pull the nascent RNA out of the PolRNA DNA complex by a mechanism reminiscent of the ATPdependent helicases which unwind duplex DNA Ch 20 However p achieves its goal only when the polymerase slows down at a pause site Once p enters the transcription complex it apparently follows the polymerase until the polymerase falters Then p keeps pulling causing termination This model supports the role of a hairpin which may cause polymerase to slow and also aid p in releasing the RNA Because p acts in part via paused or stalled Pol complexes it follows that anything that affects pausing or the strength of the paused complex may affect pdependent termination More about termination and antitermination in Ch 8 For example Fig 821b shows how protein N from phage 7t helps to form a complex that can prevent termination This complex involves other proteins and thus analysis of N and p helped to reveal the role of several proteins in the transcription complex I encourage you to glance at Fig 821 at this point just to see alternative transcription complexes with different termination properties However we probably will not cover this part of Ch 8 and you are not responsible for the content of the Fig such as understanding the roles of the proteins Weaver introduces the term processivity at Fig 821 Loosely the term refers to the ability of the enzyme the Pol to continue down the chain without falling off His Glossary entry is good We will look at processivity of DNA Pol in Ch 20 good word Termination by eukaryotic RNA Pols is rather different we will brie y note one part of the story in Ch 15 Palangat et al 1998 discuss the role of pausing in allowing an antiterminator protein to bind during the HIV life cycle Chapter 6 Weaver 2e Mol Biol X107A Page 16 The purpose of RNA synthesis is to make a certain piece of RNA Thus we expect that there are speci c places to start and to stop Actually these places need not be too precise Protein synthesis has its own start and stop signals Part VI mRNAs commonly contain untranslated sequences at both the front and back ends Further those RNAs that are not translated eg tRNA and rRNA are trimmed to proper size from the original transcript Ch 16 Sect 12 However even if termination itself is less important than we might have expected the story of termination reveals aspects of the transcription elongation complex It is a reasonable view that the transcription apparatus has the ability to switch between elongation and termination complexes Again recall Sect K P Reporters measuring gene activity Read pp 1212 in Ch 5 see the Also read note at the top ofthe handout How do we measure how active a gene is There are many ways For example we might measure the amount of mRNA made or we might measure the amount of protein product made What if the protein product is hard to measure One approach is to use a reporter gene A reporter gene has the normal regulatory elements including promoter but the proteincoding part has been replaced by the sequence for a protein that is easy to measure Weaver gives galactosidase chloramphenicol acetyl transferase CAT and luciferase as examples of reporter genes Ch 5 assigned part We will learn more about the enzyme 5 galactosidase in Ch 7 Silhavy 2000 discusses the history of lacbased reporters Other popular reporter systems include alkaline phosphatase green uorescent protein GFP and a similar red uorescent protein Terskikh et al 2000 present a fancy variation of a uorescent protein Sussman 2001 discusses a variety of reporter systems We will use a reporter gene in the homework set and from time to time later 3 2 Previews In general outline transcription in eukaryotes is about the same as in prokaryotes However the details are different re ecting the greater complexity of the eukaryotic system especially for initiation Part IV Ch 1013 deals with this we will look at those chapters brie y in sequence In some cases RNA is processed after transcription and before use We hinted at this above in noting that some RNAs are trimmed to proper size Sect 0 Part V discusses these post transcriptional modifications we may discuss this brie y at the end of the course As mentioned regulation of transcription is a key part of the story of gene regulation More about this in various later chapters Chapter 6 Weaver 2e Mol Biol X107A Page 17 R Further reading M Fang amp HY Wu A promoter relay mechanism for sequential gene activation J Bact 180626 2 98 Transcription affects supercoiling which then affects transcription of nearby genes M J Thomas et al Transcriptional delity and proofreading by RNA polymerase II Cell 93627 51598 From in vitro work with a eukaryotic RNA Pol they argue that proofreading to correct errors of base insertion may actually occur during transcription M Palangat et al Transcriptional pausing at 62 of the HIV1 nascent RNA modulates formation of the TAR RNA structure Mol Cell 11033 698 An example of regulated termination of RNA synthesis occurs with HIV The Tat protein binds to the nascent RNA to prevent termination Here they show that the actual secondary structure to which Tat binds called TAR is formed only after the RNA Pol pauses S Callaci et al Core RNA polymerase from E coli induces a major change in the domain arrangement of the 670 subunit Mol Cell 3229 299 Weaver notes that free 6 does not bind to DNA This is due to region 1 serving as an inhibitor of DNA binding until 6 is bound to the core The present work extends that and suggests that core binding to 6 not only moves region 1 but also alters the spacing of regions 24 and 42 to make them more compatible with the spacing of the promoter elements A Viswanathan et al Phenotypic change caused by transcriptional bypass of uracil in nondividing cells Science 284 159 4299 News Bridges p 62 One common type of DNA damage is deamination of C changing it to U This damage is often repaired Ch 20 but what if it is not U is like T and codes for A Here they show that transcription of U containing DNA does indeed lead to A being inserted into the RNA That is an incorrect RNA and therefore an incorrect protein can be made from damaged DNA prior to being repaired The word bypass in the title is misleading The effect is caused by the U in the DNA being transcribed rather than repaired E Nudler Transcription elongation structural basis and mechanisms J Mol Biol 288lll2 42399 Review of recent work on how RNA Pol moves Weaver presents some of the work In general they use carefully controlled in vitro reactions to explore the details of elongation steps C E Bogden et al The structural basis for terminator recognition by the rho transcription termination factor Mol Cell 3487 499 Xray analysis of a crystal of the RNAbinding domain of p complexed with RNA The analysis suggests why p binds pyrimidinerich sequences but seems not to explain why it prefers C I Gusarov amp E Nudler The mechanism of intrinsic transcription termination Mol Cell 3495 499 A detailed analysis of how hairpin formation weakens the elongation complex The A rich template region that follows pauses the Pol enough to allow termination to win Chapter 6 Weaver 2e Mol Biol X107A Page 18 S M Lohrke et al Transcriptional activation of Agrobacterium tumefaciens virulence gene promoters in Escherichia coli requires the A tumefaciens rpoA gene encoding the alpha subunit of RNA polymerase J Bact 1814533 899 An example of the role of the or subunit of RNA Pol in gene regulation The activator protein makes speci c contacts with or Therefore the Agrobacterium activator protein requires the Agrobacterium 0c M Guthold et al Direct observation of onedimensional diffusion and transcription by Escherichia coli RNA polymerase Biophys J 77 22842294 1099 Use of scanning force 39 r J to study 39 J39 39J 39 39 39 Of particular note they observe RNA Pol sliding along the DNA in a process that can be considered as onedimensional diffusion It is likely that Pol makes contact with DNA originally via nonspecific binding then slides along the DNA and finds a promoter Onedimensional diffusion speeds up the process of finding promoters G M T Cheetham amp T A Steitz Structure of a transcribing T7 RNA polymerase initiation complex Science 2862305 121799 The phage T7 RNA Pol is simple compared to regular Pols mainly because it only recognizes one fairly specific promoter Otherwise it carries out the same basic functions Weaver introduces this phage RNA P01 in Sect 82 T7 Pol along with its corresponding phage promoter is often used in special gene constructs H Maeda et al Two extracytoplasmic function sigma subunits 6E and GFCCI of Escherichia coli promoter selectivity and intracellular levels J Bacteriol 182 1 1811184 200 A brief note about the two least familiar of the seven 6 factors of E coli These 6 factors typically transcribe genes for extracellular functions including periplasmic and outer membrane proteins R J Davenport et al Singlemolecule study of transcriptional pausing and arrest by E coli RNA polymerase Science 2872497 33100 News Buc p 2437 Another example of studies of single molecules using optical tweezers Recall Allemand et al 1998 Ch 2 handout Previous work had established that RNA Pol is a quite powerful motor protein exerting a force of 14 pN similar to or greater than that from motor proteins such as those associated with muscle or microtubules Here they study the transcription rate of individual RNA Pol molecules they focus on pause events Their key findings so far are that Pols move at different rates and have varying susceptibility to pausing M S Bartlett et al Regulation of rRNA transcription is remarkably robust FIS compensates for altered nucleoside triphosphate sensing by mutant RNA polymerases at Escherichia coli nn P1 promoters J Bacteriol 18219691977 400 Weaver notes the role ofthe FIS protein in activating rRNA synthesis Fig 611 Another regulator of rRNA synthesis is the concentration of the initiating nucleotide More NTP leads to more synthesis They have mutants with an altered response to NTP level they require a higher level of NTP for efficient transcription Surprisingly these mutants grow and make rRNA fine Why Apparently because the role of the activator FIS protein is increased The work shows mechanisms of rRNA regulation but also shows how the mechanisms interact N Naryshkin et al Structural organization of the RNA polymerasepromoter open complex Cell 1016601 6900 Analysis of proteinDNA interaction by crosslinking Chapter 6 Weaver 2e Mol Biol X107A Page 19 N Korzheva et al A structural model of transcription elongation Science 289619 72800 They use a combination of xray structure analysis and crosslinking to explore the contacts between the nucleic acids template and product and the Pol during transcription As a result they deduce features of the elongation process R Wooster Cancer classification with DNA microarrays is less more TIG 168327 800 A gene chip contains samples of DNA from thousands of genes RNA samples prepared from various conditionstissuespeople or whatever is being studied are hybridized to the chip and computer analyzed to show the pattern of gene expression This paper discusses use of gene chips for analyzing patterns of gene expression in cancer The idea is that such patterns may distinguish classes of cancer that are not otherwise distinguishable and this may have implications for proper therapy Also see Hamadeh amp Afshari 2000 below C S Schaumburg amp M Tan A positive cisacting DNA element is required for highlevel transcription in Chlamydia J Bacteriol 18251675171 900 Chlamydia promoters and RNA Pol seem similar to those in E coli but now they show that an ATrich region in the spacer near the 35 element greatly stimulates transcription They do not know whether this is due to contact with one or another protein or to an effect on DNA shape If nothing else the work is a caution that our usual description of the promoter region is oversimplified S Van Komen et al Superhelicitydriven homologous DNA pairing by yeast recombination factors Rad51 and Rad54 Molecular Cell 63563 900 Negative supercoiling can affect recombination in ways similar to what we discussed for transcription Same basic ideas A Terskikh et al Fluorescent timer Protein that changes color with time Science 2901585 11 2400 News Chicurel p 1478 They start with a red uorescent protein and make a mutant whose color changes with time As a result they can measure not only the level of transcription but its timing H Hamadeh amp C A Afshari Gene chips and functional genomics Amer Sci 88508 1100 Good introductory article on the use of gene chips or arrays for analyzing expression levels of very large numbers of genes For more see the X107 web page section on Gene chips Also see Wooster 2000 above T J Silhavy Gene fusions J Bacteriol 18259355938 1100 Commentary We introduced reporter genes in Sect P Reporter genes are a modern application of gene fusions and Silhavy tells some of the history of this broader story Much of it makes use of the lac system Nice reading M T Marr amp J W Roberts Function of transcription cleavage factors GreA and GreB at a regulatory pause site Molecular Cell 66 127585 1200 GreA and GreB are part ofthe machinery for cleaving stalled transcripts so Pol can try again The details of the mechanism remains unclear This paper shows that Greinduced cleavage is necessary for normal progression through wellidentified pause sites Y Harada et al Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase Nature 409113 1401 Use of single molecule biophysics Chapter 6 Weaver 2e Mol Biol X107A Page 20 Y Cheng et al A long T A tract in the upp initially transcribed region is required for regulation of upp expression by UTPdependent reiterative transcription in Escherichia coli J Bacteriol 183221228 101 This paper illustrates a complexity of initiation it relates to choice of start site promoter clearance and regulation The key phenomenon is RNA Pol stuttering transcribing the same bases over and over again This occurs in repeated sequences and is presumably due to template slippage What makes this case interesting is that the stuttering varies with conditions in a way that is useful The gene in question involves pyrimidine metabolism and reasonably its function depends on the level of pyrimidines But the mechanism is odd The level of UTP affects the choice of transcriptional start site And the site chosen when UTP is high is in a position where stuttering is a problem and few transcripts get made Thus high UTP serves to reduce gene function due to altered start site selection which enhances stuttering J Qiu amp J D Helmann The 10 region is a key promoter specificity determinant for the Bacillus subtilis extracytoplasmicfunction 6 factors 6X and SW J Bacteriol 18319211927 301 Bacillus subtilis has long been a model system for studying 6 factors and was known to have many of them Current count is 17 7 of which were uncovered by the genome sequencing Here they define a very specific recognition difference between 6X and SW Interestingly the recognition information led them to postulate which amino acids of the 6 made critical contacts but changing these the kind of experiment Weaver discusses on p 149 did not lead to the expected changes I Toulokhonov et al Allosteric control of RNA polymerase by a site that contacts nascent RNA hairpins Science 292730 42701 They show a tripartite interaction of a hairpin in nascent RNA a particular region of the P01 that can block the RNA exit channel and a regulatory protein that can promote termination They interpret all this in terms of control of the state of the Pol The major point may be their suggestion that the primary effect of hairpin formation is to cause an allosteric change in the Pol inhibiting addition of the next nucleotide to the growing chain J Xu et al Functionbased selection and characterization of basepair polymorphisms in a promoter of Escherichia coli RNA polymerase670 J Bacteriol 18328662873 501 See hw M Jishage et al Mapping of the de contact site on the sigma 70 subunit of Escherichia coli RNA polymerase J Bacteriol 18329522956 501 de is an antisigma factor it is a protein that binds to 6 and inhibits it An increasing number of antisigmas are being found apparently turning 6 off is an important part of gene control Here they show that de interacts with 6 in region 4 and probably prevents 6 from making critical contacts with core Pol andor regulatory proteins R Meima et al Promoter cloning in the radioresistant bacterium Deinococcus radiodurans J Bacteriol 18331693175 501 Cloning promoters is a way to find out what promoters are like in a new organism The basic logic is to use a vector in which a reporter gene fails to function only because it lacks a promoter Cloned sequences that allow reporter functioning are by definition promoters Cf other cloning vectors Sect 43 Chapter 6 Weaver 2e Mol Biol X107A Page 21 B A Young et al A coiledcoil from the RNA polymerase 539 subunit allosterically induces selective nontemplate strand binding by 670 Cell 1057935944 62901 They identify a particular region of the P01 539 subunit that is responsible for allowing 6 to bind the promoter H E Sussman Choosing the best reporter assay The Scientist 1515 72301 p 25 Discussion of reporter systems with pro and con features Includes information on commercial availability More extensive information is at the online version httpwwwthescientistcom G BarNahum amp E Nudler Isolation and characterization of Smretaining transcription elongation complexes from Escherichia coli Cell 1064443451 82401 accompanying article by Mukhopadhyay et al p 453 Ch 6 hw 13 points you to a new article from Nudler I finally read it and also the accompanying one Both papers report that 6 does not always dissociate from Pol for elongation They even suggest that the nondissociation is useful in making initiation of the next round more efficient This new idea goes against the long held view of the 6 cycle which includes 6 dissociation as a key part of the transition from initiation to elongation Even if the new work is correct I suspect that there is some functional change of the cPol interaction that allows elongation perhaps complete dissociation is not necessary but it seems likely that some change is necessary Time will tell S Erratum p 156 Fig 633 part b In both structures there is an extra 0 between the adenosine and the first phosphate T Homework Weaver provides a long and useful problem set I would like for you to go through this though not necessarily doing everything Some questions go more into experimental detail than we need as discussed above We can discuss some ofthese as needed It may be good to reread the Comment at the end of Sect A above for some perspective on what your goals are as you go through all these problems 1 RNA is easily hydrolyzed by mild base between the 539 position of the sugar and the 339 phosphate of the adjacent nucleotide Thus after complete hydrolysis all internal nucleotides have been converted to 339NMP 339NMP means 339 nucleoside monophosphate the nucleoside with one phosphate group on the 339 end We might also write it as Np where the p on the right side of the N refers to the 339 side What is the 539 nucleotide of the RNA chain converted to by this kind of hydrolysis The 339 nucleotide Make a diagram using the shorthand shown in Fig 313 as a guide 2 A simple statement 6 is designed to bind to promoters as strongly as possible What s wrong with this simple view For each issue you raise explain how 6 deals with it Chapter 6 Weaver 2e Mol Biol X107A Page 22 3 This question explores one at a time possible ways for supercoiling to in uence the level of transcription of a gene a Consider for now only the effect of supercoiling on the polymerase opening up the DNA What effect would you expect increased negative supercoiling to have on transcription Explain b Now consider only the effect on the polymerase recognizing and binding to the promoter What effect would you expect increased negative supercoiling to have on transcription Explain 4 Gyrase topoisomerase II is the enzyme that adds negative supercoils to bacterial DNA The gyrase promoter is more active as negative supercoiling decreases an unusual but logical effect A special construction is made with the promoter for gyrase in front of the galK gene which codes for the easily assayed reporter enzyme galactokinase For the nature of reporters see Sect P a Sketch the construction b With this construction what effect would you expect addition of the drug nalidixic acid Nal a gyrase inhibitor which inhibits the activity of the enzyme to have on the synthesis of galactokinase Explain 5 The level of supercoiling of bacterial DNA in the cell is due to the balance between the activities of gyrase topoisomerase II which puts in negative supercoils andor removes positive supercoils and topoisomerase I which relaxes negative supercoils In Ch 2 we noted that DNA is commonly underwound in the cell Let s look at the effect of transcription on the level of supercoiling Consider a small plasmid carrying a highly transcribed gene It is in a mutant host that is highly deficient in gyrase What would happen to the supercoiling of the plasmid DNA Explain Assume that the proposed effect of transcription on supercoiling plays a dominant role in determining the supercoiling For simplicity in thinking about it you can assume that you start with a plasmid with no supercoiling the direction of the effect is what is interesting 6 Consider a piece of DS DNA with a l2base bubble where the two strands are not complementary to each other Next bind a short piece of RNA which is complementary to one strand in the bubble Finally add RNA polymerase and nucleotides etc a Sketch the nucleic acid structure with the Pol b Would you expect that the RNA polymerase would extend the RNA chain Explain the basis of your prediction 7 An operational definition of the open complex is that it is resistant to adding heparin a polyanion mentioned p 141 In recent work a lab reported exploration of the importance of sequences in the 10 region for formation of the open complex In each of two parts of the experiment they made all possible single base changes in the 10 region and then asked which sequences worked the best In one part of the experiment Part B in the Figure below chapter 6 Weaver 2e Mol Blol X107A Page 23 y r mall ofRNA Pol a The followmg Flg shows Lhelr results A b What ls the major dlfference they found quot under these condlnons7 59 m M I Ll E m vlrtually rdenueal for the two seleetrons7 2n 7 7 V H Why do you thlnkthlsls 507 FI ts d found forPart A T nsensus you wrote above d does 3 not agree Wth the 710 eonsensus Weaver tells you Why7 1s Weaverwrong7 f Ifsomeone else ddd a slrnllar enpenrnent ls rt plauslble that they mlgnt nd dlfferent e e results7 Try to offer sgeclflc reasons for your answer 8 Look at the enpenrnents of Flgs 5 37 and o 20 What ls the key dlfference tn enpenrnental dlscussedln Seet F above7 9 UN r n h w You plasmld Restneuon analysls lnvolves treatment of the DNA wth an enzyme that euts DNA 44quot FRVTwwrmn r tt n test below The gel eleetrophoresls results forthe punfled DNA atter restneuon analyses are Chapter 6 Weaver 2e Mol Biol X107A Page 24 Test Control Size kb 30 20 15 05 Your conclusions Explain 10 In Sect 0 above I explain that a hairpin and polyA rich template are required for intrinsic pindependent termination In the first full paragraph p 168 right hand column Together these results show Weaver says quite to the contrary Please resolve our little disagreement Try to do this by reading over the section in text and handout and not by focusing on his summary That is try to develop your own understanding from reading the evidence rather than just reading his understanding In the answer section I offer some more speci c thoughts on how to approach this But again please give it a serious try yourself before reading that Neither Weaver s summary nor my answer need be the way you go 11 Improve the chapter summary 12 Internet question Go to the Univ Akron site listed in the Ch 3 handout under Computer Resources Look at the animation showing transcription In what ways is the animation accurate inaccurate I may show this in class and then we can discuss it If we don t feel free to turn in your comments The purpose of the question is to explore your understanding of transcription not to criticize the animation 13 Internet question Much of the work on elongation was done by E Nudler s group e g Fig 637 and Nudler 1999 A reasonable question would be to ask whether he has published more recent work To check this you want to do a literature search See my Library page for how to search Medline Go to the MedlinePubMed site and enter Nudler as the author search term See what you get and perhaps browse the most recent item In the answer section below I brie y note the expected output Those who are experienced searchers can skip this you are not responsible for the output The point is to get those who are not familiar with Medline to get started Searching on an author with an uncommon name is a simple search Chapter 6 Weaver 2e Mol Biol X107A Page 25 U Partial answers 1 The 539 end is converted to pppr the 339 end is converted to the nucleoside The 239OH group is key to this basecatalyzed hydrolysis which is why it occurs with RNA but not with DNA The rst intermediate involves the P group being attached to both the 239 and 339 OH groups breaking the P bond with the next nucleotide This cyclic phosphodiester is then hydrolyzed to a mixture of 239 and 339NMP The statement in the question that the internal nucleotides become 339NMP is not exactly correct They actually become a mixture of 239 and 339NMP 2 For example Binding of free 6 to DNA would not seem to be so useful Solution 6 binds to DNA only when bound to Pol Comment One might consider the possibility that 6 selects a promoter site then recruits Pol to it This does not occur for bacterial 6 but this sequence of events probably does occur in eukaryotes Another issue 3 a Negative supercoiling is topologically equivalent to unwinding the primary helix Thus the DNA is already partially unwound and it would be easier for polymerase to open up the negatively supercoiled DNA b The polymerase makes speci c contacts with speci c parts of the DNA We know that there are promoters of various strengths which allow various quality of contact with the polymerase One might expect that the quality of the contact in a speci c case would be sensitive to supercoiling of the DNA since that does affect its three dimensional structure However it is just as reasonable that the effect might be in either direction depending on the speci c case The supercoiling might affect any regulatory protein that makes speci c contacts with the DNA structure Depending on whether the protein acts positively or negatively the effect of increased binding would be to increase or decrease gene function respectively Further the same point could be made for any other DNA modi cation such as methylation or binding of some other protein The net result from parts a amp b is that supercoiling can affect transcription by multiple mechanisms at least some of the mechanisms can act in either direction The big message is the importance of the exact DNA microstructure Chapter 6 Weaver 2e Mol Biol X107A Page 26 4 a 1 where 1 marks the transcriptional start site pigyr lt galK gene gt b Nal inhibits gyrase thus reduces the degree of negative supercoiling This would stimulate the gyrase promoter and therefore the production of galactokinase which is being made from that promoter in this case 5 The DNA would be positively supercoiled Transcription causes regions of positive supercoiling and negative supercoiling Topo I would relax the negative supercoils However in the absence of Topo II gyrase the transcriptioninduced positive supercoils would accumulate This question summarizes a key experiment that showed the effect of transcription on supercoiling It s necessary to use a small plasmid with only one active gene or else the effect is likely to be diluted out Indeed positively supercoiled DNA can be found when Topo II is reduced either by mutation or drugs such as Nal see question 4 The positively supercoiled DNA is found only when the gene is being actively transcribed p 165 6 The first point is to recognize that the assembled nucleic acid structure does look like an RNA elongation complex eg Fig 642 except of course it lacks polymerase So can Pol add into an elongation complex What do you think Why Sidorenkov et al 1998 in Weaver s list used this methodology which had been developed a few years earlier by von Hippel s lab 7 a One part simply selects for rate for fast formation of open complex The other part selects for a high affinity of the promoter for the P01 because of the low concentration of Pol In fact they label Part A as af nityselected and Part B as rateselected b Position l2 A strong preference for G in Part A vs a strong preference for T in Part B c 7 9 11 e The work here shows that the best promoter sequence depends on the conditions Part A vs Part B of the Figure The consensus in Weaver is merely a statistical compilation of which bases are found most commonly It is commonly thought that the consensus promoter is very strong Consensus is a general term one needs to make sure you know the basis of any particular statement of a consensus sequence f What are some specific reasons that would result in different best sequences This question is based on Xu et al 2001 The Fig given is slightly modified from their Fig 2 8 Look at the properties of the nuclease used to digest the DNA in the two experiments If you haven t already do that before reading further Chapter 6 Weaver 2e Mol Biol X107A Page 27 The nuclease of Fig 537 is an endonuclease which nicks the DNA at random sites within the chain It thus creates a range of fragments of all lengths except those protected by the bound protein The nuclease of Fig 620 is an wnuclease which degrades only from the 339 end It degrades from that end to the block creating one speci c fragment that fragment is defined by one end of the binding site of the protein on the DNA Note then that the first experiment helps to define both ends of the protein binding site whereas the second only defines one end of it 9 First let s explicitly state the observation The presence of the replication protein causes two fragments to now appear as one It thus seems that the added protein binds to the plasmid close enough to one of the EcoRl sites that it blocks that site from being restricted 10 Some things to think about you may have more 7 In what order did we learn the various pieces of evidence 7 Did later evidence negate or elaborate on the original model 7 Which model is higher level 7 Which model is most relevant 13 The output will vary depending on when you do the search At my last check the PubMed site gave a new article in the Aug 24 2001 issue of Cell Here is a brief comment about that article which I added later to this handout G BarNahum amp E Nudler Isolation and characterization of Smretaining transcription elongation complexes from Escherichia coli Cell 1064443451 82401 accompanying article by Mukhopadhyay et al p 453 Both papers report that 6 does not always dissociate from Pol for elongation They even suggest that the nondissociation is useful in making initiation of the next round more efficient This new idea goes against the long held view of the 6 cycle which includes 6 dissociation as a key part of the transition from initiation to elongation Even if the new work is correct I suspect that there is some functional change of the 6POl interaction that allows elongation perhaps complete dissociation is not necessary but it seems likely that some change is necessary Time will tell x107awv6h 91801 corrections 8708
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