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Information Processing by RNA Polymerase: Recognition of Regulatory Signals during RNA Chain Elongation

Information Processing by RNA Polymerase: Recognition of Regulatory Signals during RNA Chain Elongation
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  J OURNAL OF  B  ACTERIOLOGY ,0021-9193/98/$04.00  0July 1998, p. 3265–3275 Vol. 180, No. 13Copyright © 1998, American Society for Microbiology. All Rights Reserved. MINIREVIEW  Information Processing by RNA Polymerase: Recognition of Regulatory Signals during RNA Chain Elongation RACHEL ANNE MOONEY, IRINA ARTSIMOVITCH,  AND  ROBERT LANDICK*  Department of Bacteriology, University of Wisconsin—Madison, Madison, Wisconsin 53706-1567  INTRODUCTION The primary function of RNA polymerase (RNAP) is toproduce RNA transcripts optimal for a given environmental ordevelopmental state of a cell. RNAP accomplishes this selec-tion by deciding whether or not to initiate transcription of agene and then both how fast and how far to elongate the RNA chains. Because each decision depends on information input toRNAP and can produce results that influence subsequent de-cisions, RNAP’s function in the cell in many ways resembles aninformation processor. In this short review, we will (i) brieflysummarize the transcription cycle from the perspective of RNAP as a cellular information processor, (ii) describe thestructure of the transcription elongation complex (TEC) andthe protein-nucleic acid contacts that function as intrinsic in-puts to its regulatory decisions, and (iii) present a model of pausing and   -independent termination as an example of in-formation processing by RNAP. RNAP AS AN INFORMATION PROCESSOR  Because RNAP moves along a digital tape (the DNA), readsinformation from it (and from the nascent RNA chain), andthen based on this information, either writes new informationto an RNA chain or decides to halt transcription temporarilyor permanently, it exhibits many similarities to the first theo-retical computer (114), which was devised by the British math-ematician Alan Turing and now is called a Turing machine. A Turing machine carries out computation by repeated logicaloperations in which, based on a series of rules and digitalinformation it reads from a tape, it writes information back tothe tape, potentially changes its logical rules for future oper-ations, and moves to a new position on the tape (41). Since newRNAs can feed back on the activity of RNAP (directly or astranslated proteins) and dictate both the selection of new startsites and its response to pause and termination signals, like aTuring machine and the CPU of a computer, RNAP is capableof logical operations. Unlike the digital logic of a computer,however, RNAP’s decisions are governed by competing ratesand equilibria among alternative conformations and complexesand constitute the first step in the conversion of the fundamen-tally digital language of DNA into the analog circuitry of cel-lular metabolism.To understand how RNAP makes regulatory decisions, wemust describe the structures of the competing intermediates,the inputs that influence RNAP’s decision as to which amongthem is formed and the mechanisms and rates by which theseintermediates interconvert in response to the various inputs.Regulatory decisions by RNAP rely on two types of informa-tion input to the TEC: intrinsic and extrinsic. Intrinsic inputsare discrete segments of RNA and DNA that interact withRNAP. These intrinsic interactions generate different TECconformations, which vary in their capacity for RNA chainelongation from rapid nucleotide addition to halted chainelongation to termination. These different conformations arein turn the targets for a variety of extrinsic inputs that canenhance or inhibit chain elongation or termination (Table 1).These inputs are small molecules, proteins, and other RNAsthat interact with the TEC and modulate its activity, either bydirectly contacting RNAP or via contacts to RNA or DNA.The combination of distinct TEC conformations and regula-tory ligands whose affinities and actions can depend on theconformation and the position of the TEC confers enormousregulatory flexibility on the process of transcription. To illus-trate how intrinsic and extrinsic inputs modify regulation byRNAP, we will first describe the transcriptional cycle. We willthen direct our principal attention to the structure of the TECand its response to intrinsic regulatory signals at related RNA hairpin-dependent pause and termination signals. TRANSCRIPTION CYCLE AND INTERMEDIATES The regulatory intermediates of transcription are most easilydepicted in a cycle (12). The cycle can be divided into majorphases: promoter engagement, initiation, RNA chain elonga-tion, and termination (Fig. 1). Each intermediate represents adecision point (or logical operation in our analogy) whereinputs can alter the course of RNA synthesis. Promoter engagement.  Promoter engagement encompassesseveral steps: promoter location and recognition by the RNAPholoenzyme (core enzyme complexed with one of severalsigma factors), initial and reversible binding of RNAP to du-plex promoter DNA (closed complex formation), and forma-tion of an open complex in which   12 bp of DNA, includingthe transcription start site, are melted (Fig. 1). At least fourimportant intrinsic inputs affect promoter engagement: thehexamer centered at position   10 upstream from the tran-scription start site (  10 region), the hexamer at position   35(  35 region), the region of DNA between these two elements(spacer region), and a region located between   40 and   60(the UP element). The mechanisms by which RNAP recog-nizes these promoter elements and positions itself at a pro-moter sequence were recently reviewed in detail (25). Briefly,RNAP holoenzyme initially binds to one face of double-stranded DNA at the promoter. Footprinting of these closed * Corresponding author. Mailing address: Department of Bacteriol-ogy, 1550 Linden Dr., University of Wisconsin—Madison, Madison,WI 53706-1567. Phone: (608) 265-8475. Fax: (608) 262-9865.   onM a y 1 2  ,2  0 1  6  b  y  g u e s  t  h  t   t   p:  /   /   j   b . a s m. or  g /  D  ownl   o a d  e d f  r  om   complexes indicate a protected region of 75 to 80 bp, extendingfrom   55 to approximately   20 (87). The DNA strands arethen melted, from   10 to   1 (26), forming open complexes.The process of open-complex formation may also be accom-panied by major conformational changes in RNAP (25, 87, 97), which may include the closing of the jaw-like clamp that ap-pears to be open in the holoenzyme but closed in the corepolymerase (86).Multiple closed- and open-complex intermediates form dur-ing promoter engagement (87). For some promoters, however,open-complex formation can be modeled by a simple two-stepmechanism involving equilibrium between free RNAP and theclosed complex followed by a rate-limiting isomerization to anopen complex (71). This model must be applied with caution,though, since it can fail either because another step is ratelimiting or because open complexes reverse to closed com-plexes rapidly.The rates at which these steps occur are dictated by bothextrinsic and intrinsic interactions. Extrinsic inputs includeprotein-protein contacts made by activators and repressorsthat bind in the vicinity of the promoter DNA and can modifythe rates of either closed- or open-complex formation or pro-moter escape. In some cases, a single extrinsic factor can eitherinhibit or accelerate one or another of these steps with rela-tively subtle changes in contacts (63, 74, 107). Intrinsic contactsare made by the    subunit of RNAP to the   10 and   35regions of the DNA (reviewed in reference 36) and sometimesby the   -subunit C-terminal domain to the UP element (98).Open-complex stability can be further affected by the next stepin the pathway, nucleoside triphosphate (NTP) binding, whichin turn is affected by the spacer region between the   10 hex-amer and the start site, as well as by the identity of nucleotideto be incorporated at the 5   end of the RNA (33, 65). FIG. 1. The transcription cycle. Intermediates in the four phases of transcrip-tion (promoter engagement, promoter escape, RNA chain elongation, and tran-script termination) are discussed in the text. Elongation is represented by growthof the RNA chain between two transcription elongation complexes. Terminationand arrest are shown occurring from the paused transcription complex to reflectthe likelihood that a transcriptional pause is the first step in each. However, notall paused complexes involve RNA secondary structures as depicted, especiallythose that lead to arrest, which likely would be blocked by an RNA hairpin thatprevents reverse translocation of the RNA transcript (see the text). TABLE 1. Regulatory inputs to the transcription complex  Input (component of regulatory signal) Target(s) Effect(s) References IntrinsicNascent RNA structures RNAP Pausing, termination 23, 85, 118, 121Upstream DNA (hybrid formation) Replication primer formation, termination 703  -proximal RNA RNAP Pausing (10–11 nt); termination (U-rich 7–9 nt);arrest (U rich)  a 16, 37Template DNA strand 3  -proximal RNA, RNAP ? 124Nontemplate DNA strand Sigma factors, RNAP Pausing 95, 117Bases in active site RNAP Pausing 1, 17Downstream DNA duplex     N-terminal zinc finger,    C-terminalregion (duplex clamp)Pausing, termination 62, 79, 111Extrinsic   Nontemplate DNA Pausing 95   Nascent RNA, RNAP? Termination 84, 93   Unknown Termination 10ppGpp Unknown Pausing 11NusA     C-terminal domain    or   , RNA hairpins?Pausing, termination antitermination  b 35, 68, 93NusB BoxA nascent RNA Antitermination  b 35, 93NusE BoxA nascent RNA Antitermination  b 35, 93NusG RNAP Pausing, antitermination  b 35, 93GreA RNAP, nascent RNA? Transcript cleavage, antiarrest, promoter escape 8, 32, 42GreB RNAP, nascent RNA? Transcript cleavage, antiarrest, promoter escape 8, 32, 42Gene-specific regulators RNAP, DNA or nascent RNA Various 58  a  Arrest signals for RNAP II in eukaryotes are U-rich tracts (or corresponding dA tracts in template DNA). Arrest of bacterial RNAP has been detected in vitro(53) but remains of uncertain regulatory significance.  b Nus factors assemble into an antitermination complex that modifies RNAP to resist termination signals (reviewed in reference 23), in combination with, for instance,   N protein. Alone, Nus factors may exert different effects (e.g., NusA enhances termination). Cellular antiterminators functionally homologous to    N or Q proteinsare hypothesized but remain uncharacterized. 3266 MINIREVIEW J. B  ACTERIOL  .   onM a y 1 2  ,2  0 1  6  b  y  g u e s  t  h  t   t   p:  /   /   j   b . a s m. or  g /  D  ownl   o a d  e d f  r  om   Initiation.  After the open complex has bound the initiatingNTPs, it becomes an initial transcription complex and canfollow several alternative reaction pathways (Fig. 1): (i) thesynthesis and release of short (2 to 8 nucleotides [nt]) RNA transcripts (abortive initiation); (ii) reiterative synthesis result-ing in homopolymer extensions of the initial RNA transcripts(stuttering; not depicted in Fig. 1, see references 13 and 64);and (iii) release of    , translocation away from the promoter,and formation of a TEC with loss of upstream DNA contacts,usually when the transcript is 8 to 9 nt in length (promoterescape). The relative rates of abortive initiation, stuttering, andpromoter escape determine how fast productive RNA chainelongation begins and how soon the promoter is vacated toallow another RNAP molecule to bind. These rates appear tobe determined principally by intrinsic interactions with pro-moter elements and with the initially transcribed 8 to 10 nt of RNA or corresponding DNA, but at least in vitro can also bemodulated by extrinsic factors such as the transcript cleavagefactors GreA and GreB, which stimulate escape from certainpromoters by suppressing abortive initiation (32, 42). A betterunderstanding of initiation will require learning the following:(i) to what extent do abortive initiation and stuttering occur in vivo and are they regulated at some promoters; (ii) what de-termines the relative rates of promoter escape, abortive initi-ation, and stuttering; and (iii) do the GreA and GreB tran-script cleavage factors regulate promoter escape in vivo? Elongation.  Once RNAP converts from initial transcriptioncomplex to TEC (i.e., escapes the promoter), it becomes stablyassociated with the RNA and DNA chains (most TECs resistdissociation in 1 M KCl or at 65°C) and can elongate the RNA chain 30 to 100 nt/s in vivo. These are average, not maximal,rates (see the next section). Two distinct types of translocationsin the TEC must occur to allow this rapid movement: (i)translocation of the RNA 3   end from position  i  1 to  i  in theactive site (modified from reference 30 to use the 3  -terminalnucleotide as the index position) as successive nucleotides areadded (RNA 3  -end translocation; see Fig. 2) and (ii) translo-cation of DNA and RNA chains through RNAP (RNA andDNA translocation). The extent to which these two types of translocations are coupled has been a source of considerablecontroversy in the transcription field (reviewed in reference54). RNAP could translocate the RNA 3   end and RNA tran-script and DNA chains in a monotonic manner, advancing 1 bp with each nucleotide addition (123). Alternatively, the additionof 2 or more nt could occur without the complete translocationof DNA and RNA through RNAP and be followed by chaintranslocation of 2 or more bp (discontinuous movement, some-times called inchworming [14, 18]). Evidence for this modelcame from variably sized footprints of halted complexes (53,80). New evidence allows the reinterpretation of these foot-prints within the context of a monotonic model by demonstrat-ing that a potentially rigid RNAP can sometimes slide freelyalong the RNA and DNA chains and displace the 3   end fromthe active site, resulting in larger or smaller footprints depend-ing on the extent and frequency of sliding relative to cutting byfootprinting reagents (49, 82, 88). Thus, a recent model fortranslocation postulates that the RNAP is distributed amongall accessible positions by rapid sliding, with the occupancy of each template position dependent on its relative free energy(positional equilibrium [38, 50, 54]). In this view, the energy fordirectional translocation along DNA is derived when nucleo-tide addition shifts the positional equilibrium of the RNAPsliding back and forth along the RNA and DNA chains towardsthe forwardly translocated conformation (known as a thermalratchet mechanism; reviewed in reference 34). The alternativepossibility is that RNAP is a mechanoenzyme that usuallymaintains tight contacts to RNA and DNA and uses the energyof phosphodiester bond hydrolysis to generate internal move-ments (active locomotion or power-stroke mechanism; re- viewed in reference 34). Although the rigid RNAP-sliding model is appealing, it can-not account for all the behaviors of RNAP; in particular, someinternal flexibility of RNAP must exist to allow it to extendinitial transcripts that are 5   end cross-linked to the    subunitup to 8 nt (76). Three questions are particularly important toanswer. (i) Are the two translocation cycles of RNAP tightlycoupled, or can nucleotide addition occur without RNAPmovement along DNA to produce significantly different con-formations of the transcription complex at different templatepositions? (ii) If different conformations are possible, can theyexist at a single template position; that is, can RNAP transcribeDNA in different phases such that only a fraction of elongatingmolecules reach a given template position in a particular con-formation (see reference 18)? (iii) Does RNAP move by athermal ratchet or power-stroke mechanism? Whether RNAPsenses information as a rigid body that slides along the RNA and DNA chains nearly isoenergetically or undergoes signifi-cant internal movements between nucleic acid contacts or pro-tein domains and requires energy input for translocation is acentral issue for understanding how it processes the informa-tion in the nucleic acid chains.  Arrest, pausing, and termination.  RNA chain elongation ispunctuated by certain sites where nucleotide addition is slowedby pause, arrest, and termination signals. Pause signals causeRNAP to isomerize from the rapidly elongating TEC to alter-native conformations in which RNA chain extension is revers-ibly inhibited (by factors of 10 2 to 10 4 ). Arrest signals blockelongation irreversibly in vitro; arrest has not yet been dem-onstrated to occur in vivo. Termination signals cause the re-lease of RNA and DNA and can be positively or negativelyregulated by a variety of extrinsic inputs (Table 1). At someterminators, these inputs can instruct the enzyme to readthrough the signal and continue transcription (antitermina-tion).Pausing and arrest both occur because the RNA 3   OH andNTP substrate fail to maintain alignment in the active site, butmay reflect different changes in TEC structure. In arrestedtranscription complexes, the RNA transcript appears to bethreaded through the active site so that NTP binding is FIG. 2. Structure and regulatory inputs of a transcription complex. V OL  . 180, 1998 MINIREVIEW 3267   onM a y 1 2  ,2  0 1  6  b  y  g u e s  t  h  t   t   p:  /   /   j   b . a s m. or  g /  D  ownl   o a d  e d f  r  om   blocked, until 5 to 10 nt of 3  -proximal RNA is removed byhydrolytic cleavage. This cleavage reaction is catalyzed byRNAP’s active site and greatly stimulated by the GreA orGreB transcript cleavage factor (8, 83). Key questions aboutarrest are (i) does transcriptional arrest occur in vivo, and is itinvolved in the slow growth phenotype of     greA    greB  strains;and (ii) how do the GreA and GreB cleavage factors, whichcontact both RNAP and the nascent RNA, stimulate transcriptcleavage of arrested complexes?There are several types of paused transcription complexesmodulated by different intrinsic or extrinsic interactions. At somepause sites, elongation is slowed at unfavorable sequences, without mediating specific regulatory decisions. Other pausesare specific regulatory intermediates that both prevent tran-scription beyond a region where regulatory input is effectiveand put RNAP in the proper conformation to interact withregulatory molecules. An example of a regulatory pause isthe class of hairpin-dependent pause sites that occur midwaythrough the leader regions of all  Escherichia coli  or  Salmonella amino acid biosynthetic operons that are regulated by attenu-ation (58). Pausing at these sites halts RNAP until a ribosomeinitiates synthesis of the leader peptide, which can cause at-tenuator readthrough during amino acid limitation, or until iteventually escapes spontaneously, which leads to superattenu-ation (58). The ribosome releases RNAP from the pause eitherby melting the RNA hairpin or through direct interaction withthe enzyme. By slowing chain elongation to allow further re-arrangements of the TEC, pausing likely is an initial step inarrest and termination (96). Finally, pausing allows surveil-lance for defective mRNAs by slowing RNAP at pause sitesuntil it is released either by a translating ribosome or, if theRNA is not translatable, by the  termination factor (55). Thus,in our comparison of RNAP to an informational processor,pause signals instruct RNAP to halt and await regulatory input.When RNAP encounters a termination signal, RNAP stopsadding nucleotides to the RNA, separates the DNA-RNA hy-brid, releases the newly synthesized transcript, and dissociatesfrom the DNA template. Three types of termination signals forbacterial RNAP have been described to date: (i) intrinsic ter-minators (  -independent terminators) which require a stableRNA hairpin formed 7 to 9 nt from the terminated RNA 3  end and immediately followed by at least 3 U residues, butno extrinsic factors (recently reviewed in references 85, 93, 96,and 116); (ii)   -dependent terminators, which depend on thepresence of     factor, a hexameric RNA-binding protein with ATPase activity (recently reviewed in references 85 and 92);and (iii) persistent RNA-DNA hybrid terminators at whichpairing of nascent RNA to the template just upstream from theTEC dissociates a complex containing 3  -proximal U-rich RNA (113).We will return to a discussion of pausing and terminationlater by describing a model for the related mechanisms of hairpin-dependent pausing and   -independent termination. STRUCTURE OF THE TEC Both intrinsic and extrinsic inputs alter the behavior of RNAP (and thus its RNA transcript output) by modulatingeither the alignment of nucleotides and catalytic moieties inthe active site or the contacts of RNAP to the DNA or RNA.We must first understand the fundamental mechanisms thatgovern nucleotide addition and TEC stability before we canmeaningfully address how extrinsic inputs modify them. We will focus the remainder of this short review on the intrinsicinputs that govern RNA chain elongation, pausing, and termi-nation by first describing relevant features of RNAP’s three-dimensional structure and RNAP’s two large subunits,    and  , where most intrinsic interactions during elongation occur.We will then detail the critical intrinsic inputs, how theseinteractions program RNAP for hairpin-dependent pausingand   -independent termination, and briefly describe how anextrinsic factor, NusA, can modify these intrinsic interactions. Three-dimensional structure of RNAP.  Electron crystallo-graphic methods have been used by Kornberg, Darst, andothers to deduce low-resolution (16- to 25-Å resolution) struc-tures of   E. coli  holo- and core RNAPs and  Saccharomyces cerevisiae  RNAPs I and II (21, 22, 86, 100). These structuresreveal common features likely shared by all multisubunitRNAPs: (i) a large,   25-Å channel surrounded by “jaws” thathave been suggested to close around the downstream duplex DNA in the transition from RNAP complexed with initiatorproteins (   in bacteria) to TEC (5, 86); (ii) other features thatmay accommodate single-stranded RNA (ssRNA) or DNA;and (iii) two tunnels that run through the enzyme from nearthe channel to the opposite side, one of which may function asthe RNA exit tunnel (21, 48). This overall structure resemblesthe hand-like motif of DNA polymerases whose X-ray crystalstructures have been determined to atomic resolution (for arecent review see reference 46). The correlation of RNAP’sstructure with its function will require answers to the followingquestions: (i) what are the paths of the RNA and DNA chains within the RNAP structure; (ii) where are the   ,   ,   , and   subunits of RNAP located, particularly the conserved seg-ments (see below); and (iii) which parts of the structure, if any,move during different stages of the transcription cycle?The first question deserves particular attention. Althoughpassage of the downstream duplex DNA through the jaws of the channel offers a compelling physical picture for how RNAPcould become clamped onto the DNA during RNA chain elon-gation (79), it offers no ready explanation for establishmentand maintainence of the separation of the DNA strands duringopen-complex formation and RNA chain elongation. Otherarrangements of the nucleic acid chains in the existing struc-tures are possible. For instance, the downstream DNA duplex could be positioned in one of the grooves that runs across thefront face of the yeast RNAP structure (see Fig. 4 in reference21) and the RNA-DNA hybrid held between the jaws in theprominent central channel. This would offer a ready explana-tion for DNA strand separation if the nontemplate strandpassed outside of the jaws. One version of such a model issuggested by Kim et al. (48). Learning if any of these ideas iscorrect must await higher-resolution crystals and extensivestructural work on ternary transcription complexes, whichshould be possible given their great stability. In the meantime, we caution against overinterpreting the features evident instructures of RNAPs and have deliberately depicted two majorchannels in models of transcription complexes to avoid imply-ing a particular hypothesis (see Fig. 1, 2, and 4). Contacts in RNAP subunits.  The elongating form of RNAPconsists of the    and    subunits and a dimer of     subunits.   and    appear to make most of the important DNA and RNA contacts during elongation and are responsible for RNAP’scatalytic activity (Fig. 3).    and    homologs are evident in allmultisubunit RNAPs based on colinear sequence similarities,designated A-I for    and A-H for    (Fig. 3) (2, 45, 126). As aframework for describing RNAP’s intrinsic inputs, we will sum-marize the known contacts of RNA and DNA to RNAP’s   and    subunits (Fig. 3). Many of these contacts, which werededuced from cross-linking or genetic analysis, correspond tosequences within both the    and    subunits of RNAP that areknown to influence pausing and termination (44, 57, 110, 120).RNAP also makes contacts to several metal ions, including 3268 MINIREVIEW J. B  ACTERIOL  .   onM a y 1 2  ,2  0 1  6  b  y  g u e s  t  h  t   t   p:  /   /   j   b . a s m. or  g /  D  ownl   o a d  e d f  r  om   two Zn 2  ions, one bound to    and one bound to    (122), andtwo or more Mg 2  ions that are required for full activity(reference 77 and references therein; 127, 128). The Mg 2  ionscatalyze nucleotide addition (see the next section) and may beinvolved in DNA melting (109). The    Zn 2  in bacteria andboth the    and    Zn 2  ions in eukaryotes are complexed byCys residues in an apparent Zn-finger-like arrangement (Fig.3). The Zn 2  -finger-like motifs, which are near the N terminusof     and the C terminus of     (in eukaryotes), were shown tointeract genetically (125) and may be responsible for closingRNAP’s clamp around DNA (56, 79). The site of Zn 2  bindingto    in bacteria is unknown.Interestingly, downstream-to-upstream contacts in the TECmostly move C to N terminal in    and N to C terminal in   (Fig. 3). Additionally, the similar locations of some mutationsand split sites, the likely genesis of   and  by gene duplication(120), and the viability of a strain in which the C terminus of    is fused to the N terminus of     (103) all are consistent with amodel in which key contacts in the TEC may occur at theinterface of these two weakly homologous but oppositely ori-ented large subunits (56). Surprisingly, essentially nothing isknown about the secondary or tertiary structures of any con-served segment of     and   ; so far they have defied the in-creasingly powerful structure prediction and folding algorithmsbased on sequence similarities. Thus, improving understandingof structure in the conserved regions is a key objective forcurrent study of the    and    subunits. INTRINSIC INPUTS Multiple intrinsic inputs govern RNA chain elongation: (i)an active site that aligns the RNA 3   end and incoming NTP,(ii) 10 to 18 bp of duplex DNA held by RNAP in a clamp-likestructure at the front edge of the complex, (iii) an   18 bpmelted region of DNA within which the nontemplate strandappears to be channeled to the outside of the TEC, (iv) an  8-bp RNA-DNA hybrid that positions the RNA 3  end in theactive site 1 to 3 nt from the point of DNA strand separation,(v) at least one region of contact to single-stranded RNA upstream from the hybrid (an exit tunnel), and (vi) an inter-action site for nascent RNA secondary structures.  Active site.  RNAP’s active site catalyzes an S N2 -type nucleo-philic displacement of the NTP   -   pyrophosphate moiety bythe RNA 3   oxygen (30). It closely resembles the active sites of DNA polymerase, reverse transcriptases, and T7 RNA poly-merase, all of which catalyze the same reaction using two Mg 2  ions that are positioned by coordination bonds to the carboxy-lates of Asp or Glu residues and that direct the 3   OH and   phosphate into a trigonal bipyramidyl transition state (46, 108).In RNAP, Asp side chains in the highly conserved sequenceDFDGD at    positions 460 to 464 (  460–464) are proposedto chelate the Mg 2  ions (27, 128), whereas the    phosphate onthe RNA 3   terminal nucleotide is near   K1065 and   H1237(75). The 3   base is near   515–660 (the “Rif” region),   1100,and   460 in the reactive conformation, but relocates to near  940 after arrest (7, 69, 75). Two observations may indicatethe involvement of additional metal ions in positioning nucle-otides in the active site: (i) the [Mg 2  ] dependence of DNA strand melting is consistent with binding of three rather thantwo Mg 2  ions (109), and (ii) nuclear magnetic resonancemeasurements suggest that the   -bound Zn 2  ion may contactthe 3   base in the active site (6, 29). The major mechanisticimplications of these results make confirmation by alternativemethods critical objectives for future study.Ultimately, positioning of the RNA 3   end and NTP in theactive site determines most regulatory decisions made by theTEC. Several sets of interactions may connect this event todifferent parts of RNAP. The catalytic Mg 2  ions are con-nected through a coordination network with the 3   oxygen andNTP phosphates in the transition state (46). Thus, NTP bind-ing and proper location of the RNA 3  OH may be cooperativebecause the coordination bond network stabilizes their reactivealignment; improper positioning may inhibit nucleotide addi-tion. Further, contacts to the bases, the ribose ring, the reactivegroups (3   OH and    phosphate), and the complementarytemplate nucleotides may be over 15 Å apart and involveseveral different portions of     and   , based both on cross-linking (Fig. 3) and the recent mapping of multiple free-radicalcleavages in both subunits resulting from Fe 2  being substi-tuted for the active-site Mg 2  (77). Thus, regulatory interac-tions outside the active site may ultimately be transduced to FIG. 3. Sites of regulatory inputs to RNAP large subunits. The    and    subunits are shown in opposite orientation to simplify depiction of known contacts to RNA and DNA. Conserved regions (A to I for    and A to H for   ) are shaded in blue (2, 45, 126). Regions that tolerate deletion of disruption are indicated by wavy lines(104, 105). Rifampin (rifampicin) and streptolydigin resistance regions, represented by orange and yellow, respectively, are based on the locations of known amino acidchanges that allow growth on the antibiotics (40, 43, 102, 106). Other features shown are (i) the location of the catalytic Mg 2  ions that bind to the Asp side chainsin the highly conserved sequence DFDGD at   460–464 (27, 128); (ii) a Cys 4  Zn-finger-like-motif in    possibly involved in forming the downstream DNA clamp (79)and in antitermination (20); and (iii) sites of contact to 3  -proximal RNA or DNA, exiting RNA or RNA hairpins, active-site bases, and the downstream DNA duplex that are described in the text (69, 75–79, 82, 118). The pause hairpin and upstream RNA contacts are depicted in a single block because the same bases may contactthe    or    subunit depending on whether or not a hairpin has formed (118). The active-site contact to   -region G is indicated with a dotted line because the RNA 3   end makes this contact only in an arrested transcription complex (69). V OL  . 180, 1998 MINIREVIEW 3269   onM a y 1 2  ,2  0 1  6  b  y  g u e s  t  h  t   t   p:  /   /   j   b . a s m. or  g /  D  ownl   o a d  e d f  r  om 

Konsultasi work

Dec 27, 2018

Client Copy

Dec 27, 2018
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