The central event in transcription is the

Transcription

1 REVIEW: TRANSCRIPTION An Integrated Mdel f the Transcriptin Cmplex in Elngatin, Terminatin, and Editing Peter H. vn Hippel Recent findings nw allw the develpment f an integrated mdel f the thermdynamic, kinetic, and structural prperties f the transcriptin cmplex in the elngatin, terminatin, and editing phases f transcript frmatin. This mdel prvides an peratinal framewrk fr placing knwn facts and can be extended and mdified t incrprate new advances. The mst cmplete infrmatin abut transcriptinal mechanisms and their cntrl cntinues t cme frm the Escherichia cli system, upn which mst f the explicit descriptins prvided here are based. The transcriptinal machinery f higher rganisms, despite its greater inherent cmplexity, appears t use many f the same general principles. Thus, the lessns f E. cli cntinue t be relevant. The central event in transcriptin is the RNA plymerase catalyzed cpying f the sequence f the template strand f a gene int a cmplementary RNA transcript. This transcript may serve as a message fr translatin int prtein, it may cmprise structural RNA that frms the framewrk f a ribsme r f a transfer RNA adaptr mlecule in prtein synthesis, it may frm the genme f an RNA virus, r it may itself serve a regulatry functin. The chemistry f transcript frmatin is straightfrward, but the regulatry mechanisms that have been develped by evlving rganisms t cntrl this synthetic prcess appear almst infinite in number, althugh the basic principles n which they perate are likely t be relatively few. This cmplexity may reflect the fact that transcriptin is the primary event f gene expressin, which is defined at the mlecular level as the transfrmatin f genes (r perns) int the functinal prteins and enzymes that direct and catalyze the events f cellular metablism and differentiatin. As a cnsequence, transcriptin cmprises the first, and thus the mst effective, level at which the reading f the DNA ckbk can be regulated. Cntrl at the Gene Level Frmatin f a transcript has traditinally been divided int three sequential stages, called initiatin, elngatin, and terminatin. All are subject t regulatry cntrl. It may nw be mre apprpriate t divide the verall The authr is at the Institute f Mlecular Bilgy and Department f Chemistry, University f Oregn, Eugene, OR 97403, USA. petevh@mlbi. uregn.edu S CIENCE S C OMPASS REVIEW prcess int tw majr phases: (i) activatin and transcript initiatin and (ii) transcript elngatin, with the latter including terminatin (transcript release) and editing (transcript shrtening and resynthesis with increased fidelity), because bth can be viewed as alternative pathways that, in principle, cmpete with elngatin at every template psitin. At the activatin-initiatin stage, transcriptin cntrl prcesses regulate intergene (r interpern) cmpetitin fr a limiting amunt f RNA plymerase. Each gene, t be cmpetitive, is activated by multiple factrs that increase the relative ability f its prmter t recgnize and bind plymerase and then t facilitate prmter pening, transcript initiatin, and prmter clearance. These activatin prcedures, smetimes cupled with specific repressin events, invlve interactin f the prmter-bund plymerase with prtein subassemblies that may bind at adjacent r mre distant DNA sites and be brught t the prmter by cntrlled DNA lping (1, 2). The activatin-initiatin prcess is cmplete when the nascent transcript becmes sufficiently lng t stabilize the transcriptin cmplex against dissciatin frm the DNA template. Cnfrmatinal changes permit the cre RNA plymerase (defined as the basic enzymatic unit required fr the template-directed synthesis f the transcript; the 2 cmplex in Escherichia cli) t free itself frm mst f the factrs and regulatry subassemblies invlved in the activatin-initiatin prcess (1, 3). These cmpnents are either released int slutin r left behind at the prmter, and the cre plymerase mves int the elngatin phase, which is characterized by multiple and specific pausing events. The duratin f these pauses depends n (i) the specific DNA sequence being transcribed, (ii) interactins with regulatry prteins that either bind directly t the elngating transcriptin cmplex r are brught t it frm prtein-binding sites lcated upstream n the nascent transcript (belw), (iii) the cncentratin f the next required (by the template) nucletide triphsphate (NTP), and (iv) whether a misincrpratin event has ccurred at the 3 end f the chain. These interactins may als change the stability f the cmplex and thus make dissciatin f the plymerase frm the DNA template, with cncmitant release f the nascent transcript, either mre r less likely (4). The final step in transcript frmatin is terminatin, which ccurs when the elngating transcriptin cmplex mves int (r, in sme cases, beynd) ne r mre terminatr sequences alng the DNA template that may serve as transcriptin regulatrs within genes r mark the end f a gene r pern. In E. cli, terminatrs are either intrinsic, meaning that release f a nascent transcript can be brught abut at these terminatrs withut the invlvement f prtein factrs, r rhdependent, in that release requires the participatin f the E. cli transcriptin terminatin factr, rh. The efficiency with which transcripts are released at either type f terminatr is generally regulated by additinal factrs (1, 4). Cntrl at a Specific Template Psitin After transcript synthesis has been initiated, the transcriptin cmplex at any given template psitin can, in principle, prceed by several alternative reactin pathways (Fig. 1). Each f these ptential pathways is characterized by a particular reactin rate, which may vary frm ne template psitin t the next. These alternative reactins are in kinetic cmpetitin with ne anther (5); thus, changes in relative rates can switch the transcriptin cmplex between pathways. This kinetic cmpetitin between ptential reactin pathways can be quantified by frmulating it in terms f free energy f activatin barriers fr the cmpeting reactins (6). Differences in barrier heights can JULY 1998 VOL 281 SCIENCE

2 S CIENCE S C OMPASS be used t predict the rate r stability changes within the transcriptin cmplex that are required t bring abut any particular regulatry cnsequence. This apprach fr the cmpetitin between elngatin and terminatin at a given template psitin is shwn in Fig. 2. Each pathway cnsists f a series f steps, and thus the activatin barriers shwn are versimplified and represent nly the rate-limiting step fr each reactin under the cnditins f the experiment. Changing cnditins can change the relative heights f these barriers, and Fig. 2C shws the terminatin efficiency (TE; defined as the fractin f the ttal transcripts reaching a given template psitin that terminate there) as a functin f the difference in barrier heights ( G ) fr this tw-pathway cmpetitin. Clearly, the transitin frm dminatin f the verall reactin by elngatin t dminatin by terminatin is very abrupt with respect t G, meaning that a very small change in the relative rates f the cmpeting prcesses, under cnditins where the activatin free energy barriers are f cmparable height (at terminatr psitins; Fig. 2B), can result in an effective switch frm ne reactin pathway t the ther. In cntrast, an equivalent change under cnditins f substantially different barrier heights (at elngatin psitins; Fig. 2B) has virtually n effect n TE (Fig. 2C), meaning that the rate f the elngatin prcess and the stability (and thus the dissciatin rate) f the elngatin cmplex can be regulated ver a wide range at these psitins withut risk f transcript release. This quantitative frmulatin f the kinetic cmpetitin mdel will be explited in describing transcriptinal cntrl mechanisms as regulatable switches in what fllws. Fig. 1. Three cmpeting pathways fr the transcriptin cmplex at a given template psitin. The ptins are (i) t mve frward alng the template with the cncmitant elngatin f the RNA chain by ne nucletide residue, (ii) t mve backward alng the template (with r withut shrtening the transcript by ne r mre residues), r (iii) t dissciate frm the template, resulting in release f the nascent RNA. Stability f the Elngatin Cmplex A crucial mechanistic feature f the transcriptin elngatin cmplex is its extreme stability. Thus, elngatin cmplexes can be halted at elngatin psitins (mst easily by mitting the next required NTP frm the transcriptin mix) and can remain bund t the DNA template and the nascent transcript fr lng perids withut dissciatin (7). This stability is dynamic, as well as static, because the cmplex als cannt dissciate while mving frm ne template psitin t the next. Cntrl f the prcessivity f the elngatin prcess, defined in terms f the relative prpensities f the plymerase t extend the transcript r t dissciate, is central t the regulatin f all plymerases. RNA plymerases cntrl prcessivity by frming elngatin cmplexes that are massively stable, perhaps in part as a cnsequence f the clsure f structural cmpnents f the plymerase arund the DNA and the nascent transcript t limit dissciatin (8, 9). The prblem then becmes ne f bringing abut sequence-specific destabilizatin f the elngatin cmplex when terminatr sites are reached, s G (Activatin Free Energy) B A (at elngatin psitins) (at terminatin psitins) General: Terminatin STATE A Elngatin G frward that the nascent transcript (and the cre plymerase) can be released at these template psitins. A Structural Mdel f the Elngatin Cmplex The tw mechanisms used by E. cli t achieve the destabilizatin necessary fr terminatin are mst easily described in the cntext f a verall structural mdel f the prtein and nucleic acid cmpnents f the elngatin cmplex (Fig. 3). A central feature is a transiently pen transcriptin bubble [ 18 base pairs (bp) in length], which mves with the cre RNA plymerase thrugh the therwise duble-stranded DNA while the plymerase catalyzes template-directed transcript elngatin. The mving plymerase prtects a ftprint f abut 30 bp alng the DNA against nuclease digestin, and this ftprint includes the transcriptin bubble as well as sme duble-stranded DNA n either side. Chemical ftprinting f the transcriptin bubble, as well as direct binding measure- STATE B k A B [dntp] k A C STATE C Terminatin Efficiency (e.g., elngatin) 0.6 (e.g., terminatin) 0 0 G G frward release G (kcal/mle) Fig. 2. Cmpetitin between elngatin Reactin Crdinate and terminatin at a given template psitin. (A) General frmulatin f the rate cmpetitin prcess in terms f apparent reactin rate cnstants. The rate f the transcript elngatin prcess (state A t state B) may be dependent n the cncentratin f the next required NTP (as shwn), r it may be pseud first rder in the presence f excess NTP. Bth rates may als depend n the cncentratins f regulatry factrs if these are present at less than saturating amunts. k A3B and k A3C are the rate cnstants fr ging frm state A t states B and C, respectively. dntp, dexynucletide triphsphate. (B) The elngatin versus terminatin rate cmpetitin is described as free energy f activatin barriers at terminatr psitins (barriers f abut equal height) and elngatin psitins [terminatin barrier (red curve) much higher than elngatin barrier]. The red circle represents the transcriptin cmplex at the grund state. G frward and G release indicate the heights f the free energy f activatin barriers t terminatin r elngatin, respectively, at terminatin psitins, and G frward represents the standard free energy change f the elngatin reactin. The blue bars crrespnd t the range f differences in barrier heights ver which the terminatin efficiency (TE) ranges frm 0.01 (higher terminatin peak within the blue bar versus the lwer elngatin peak) t 0.99 (lwer terminatin peak within the blue bar versus the higher elngatin peak). (C) TE, defined as k term /(k elng k term ), where k elng and k term are the rate cnstants fr the single nucletide elngatin and terminatin reactins, respectively, is pltted here as a functin f the difference in the heights f the cmpeting free energy f activatin barriers ( G ) accrding t the fllwing equatin: TE [1 exp( G / RT)] 1, where R is the gas cnstant and T is abslute temperature. The regin f the graph between the tw vertical lines crrespnds t the blue bars in (B). C SCIENCE VOL JULY

3 S CIENCE S C OMPASS ments, suggest that specific sectins (Fig. 3) f bth the transiently single-stranded and the duble-stranded DNA, as well as prtins f the RNA-DNA hybrid and a few RNA residues at the 5 end f the hybrid, make stabilizing interactins (r at least cntact) with the plymerase (10). The template side f the transcriptin bubble is hybridized t the 3 end f the nascent RNA, with the terminal 3 -OH lcated very clse t the dwnstream edge f the bubble (11). Current estimates f the average length f the transient RNA-DNA hybrid range frm 9 t 12 bp (8, 9, 12 14). Upstream f the hybrid, the plymerase actively displaces the RNA frm the template strand f the bubble (15), sending the nascent transcript acrss a plymerase-binding site fr single-stranded RNA (8, 9) and then n int slutin, which permits the upstream end f the DNA bubble t reclse. The catalytic site f the plymerase (inset, Fig. 3) cntains bth a substrate-binding subsite, at which the incming NTP is bund t the plymerase and t the cmplementary nucletide residue f the template, and a prduct-binding subsite, at which the 3 terminus f the grwing RNA chain is psitined (16). Elngatin f the chain by a single nucletide residue results in phsphdiester bnd frmatin between the NTP bund at the substrate-binding subsite and the 3 end f the RNA chain bund at the prduct-binding subsite. This psitins the 3 end f the newly extended chain in the substratebinding subsite. Cmpletin f the single nucletide additin cycle thus requires that the chain be released frm the substrate subsite, accmpanied by a shift f the active site f the plymerase by ne psitin alng the template. As a cnsequence, the RNA-DNA DNA (nn-template strand) DNA (template strand) III RNA-DNA Hybrid II I Hybrid separatr RNA Transcript hybrid becmes 1 bp lnger at the dwnstream end, the separatin mechanism perates t make the hybrid 1 bp shrter at the upstream end, and the plymerase mves alng the template by ne psitin while the hybrid retains a cnstant length. A Thermdynamic Mdel f the Elngatin Cmplex A structural mdel f this general srt (17) has been used t frmulate a quantitative hypthesis fr intrinsic terminatin (18) that has withstd the tests f time and experimentatin with reasnable success. This hypthesis, which is generally called the thermdynamic mdel, psits that the interactins between the prtein and nucleic acid cmpnents within an elngatin cmplex rearrange rapidly as the cmplex mves frm ne template psitin t the next, meaning that the cmplex can be cnsidered t be at equilibrium during its dwell time at each template psitin. If this is the case, the free energy f frmatin [frm free cre plymerase and a clsed (duble-stranded) DNA genme] f a stable elngatin cmplex at a particular template psitin can be written as G cmplex G DNA-DNA G RNA-DNA G NA-plymerase (1) where G cmplex is the net free energy that stabilizes the elngatin cmplex against dissciatin, G DNA-DNA is the (unfavrable fr elngatin cmplex frmatin) free energy f pening the DNA-DNA base pairs f the transcriptin bubble, G RNA-DNA is the (smaller but favrable) free energy f frming the base pairs f the RNA-DNA hybrid within the unpaired transcriptin bubble, and G NA-plymerase is the (als favrable) net free energy f interactin f the plymerase Transcriptin bubble IV RNA Plymerase 3'OH Next NTP (Substrate binding subsite) Prduct binding subsite Fig. 3. A schematic representatin f essential structural features f the elngatin cmplex. The dwnstream prtin f the RNA- DNA hybrid (I) is the regin in which the ru da sequence is psitined at intrinsic terminatrs (see text). The upstream prtin f the hybrid (II), as well as the RNA sequence at III, indicate the psitin f the RNA chain within which the terminatin hairpin frms at intrinsic terminatrs. The single-stranded RNA-binding site f the plymerase is lcated at III, and IV indicates the psitin f the thumb that clses arund the duble-stranded DNA and may be invlved in maintaining the prcessivity f the plymerase (21). Plymerase cntacts (and perhaps interactins), defined by ftprinting reactins, als ccur at ther psitins alng the nntemplate strand f the transcriptin bubble and elsewhere. The inset shws the active (catalytic) site f the plymerase at the dwnstream end f the transcriptin cmplex, including the substrate-binding subsite fr the next NTP (green square) and the prduct-binding subsite fr the 3 end f the nascent transcript (black circle). with the varius parts f the nucleic acid (NA) framewrk f the cmplex. The magnitudes f tw f these thermdynamic terms ( G DNA-DNA and G RNA-DNA ) can be calculated frm knwn nucleic acid stability basis sets (18) at any particular template psitin, if we assume equilibrium and the abve (r any ther structurally defined) mdel fr the nucleic acid cmpnents f the cmplex at a given template psitin. Because the net free energy f cmplex frmatin ( G cmplex ) can be estimated independently (18), the net (favrable) magnitude f the multipartite G NA-plymerase term can be calculated by difference. The experimentally demnstrated stability (with respect t dissciatin) f the transcriptin cmplex at elngatin psitins alng the template indicates that the unfavrable G DNA-DNA (transcriptin bubble frmatin) term is mre than ffset by the sum f the favrable G RNA-DNA and G NA-plymerase terms. Destabilizatin Is Required fr Terminatin T make the terminatin pathway kinetically accessible (Fig. 2, B and C), any mdel that purprts t explain intrinsic terminatin must prvide a mechanism fr the massive destabilizatin f the transcriptin cmplex that ccurs as it mves thrugh these terminatrs alng the DNA template (19). The extent t which terminatin actually des ccur at any particular template psitin within an intrinsic terminatr depends n the interactins f the transcriptin cmplex with the lcal DNA sequence and with factrs bund t the plymerase and may als be regulated by decreasing the cncentratin f the next required NTP. Cmplexes that emerge frm the terminatr sequence withut having dissciated resume their riginal stability characteristics (19). Intrinsic terminatrs, which represent abut ne-half f the terminatin sites f E. cli, are defined by a template DNA sequence that cdes fr a stable terminatin hairpin in the nascent RNA, fllwed by a run f riburidylate (ru) residues at the 3 terminus f the transcript (17, 19). A plausible quantitative mdel (18) fr intrinsic terminatin has been develped n the basis that the RNA- DNA hybrid is substantially destabilized as the cmplex passes thrugh an intrinsic terminatr sequence, thus als destabilizing the elngatin cmplex by reducing the (favrable) cntributin f the G RNA-DNA term t G cmplex. The run f dexyadenylate (da) ru base pairs that is psitined at the dwnstream end f the hybrid when the cmplex is lcated at template psitins within an intrinsic terminatr (Fig. 3, regin I) is particularly unstable relative t its DNA-DNA r RNA- RNA cgnates (20). In additin, the frmatin (in cmpetitin with the upstream prtin f the RNA-DNA hybrid; Fig. 3, regin JULY 1998 VOL 281 SCIENCE

4 S CIENCE S C OMPASS II) f the stable terminatr hairpin within the RNA at intrinsic terminatrs als destabilizes the RNA-DNA hybrid [see als (21)]. The sum f these sequence-dependent destabilizatin events suffices t lwer the height f the activatin barrier t terminatin t abut the same level as the barrier t elngatin (Fig. 2B), primarily by decreasing the (favrable) magnitude f the G RNA-DNA term f Eq. 1. This general mdel is cnsistent with the results f experiments n apprpriately mutated intrinsic terminatrs (19). Rh-dependent terminatrs, which are respnsible fr the ther half f the transcriptin terminatin events in E. cli, d nt share the destabilizing sequence features f intrinsic terminatrs. Instead, the required destabilizatin f the transcriptin cmplex at these terminatrs is prvided by the RNA- DNA helicase activity f the rh prtein (22). There are tw imprtant sequence requirements fr rh-dependent terminatrs. First, the (rather variable) DNA sequences at the terminatrs must prduce substantial pausing f the transcriptin cmplex within these sites. In additin, DNA sequences that cde fr an extended ( 70 nucletides) site alng the transcript that is essentially devid f RNA secndary structure must be present upstream f these terminatrs. This unstructured RNA sequence serves as a binding site that permits the hexameric rh helicase t be laded nt the transcript, thus activating the cryptic RNA-dependent adensine triphsphatase f the prtein t prvide the chemical free energy needed t drive the bund rh prcessively and directinally (5 3 3 ) alng the nascent RNA. When the translcating rh helicase catches up with a paused transcriptin cmplex at a rh-dependent terminatr, it triggers the release f the nascent transcript, presumably by separating the RNA-DNA hybrid within the transcriptin bubble, thus again destabilizing the cmplex sufficiently t permit terminatin (22). Fine-Tuning f Terminatin Efficiency by Regulatry Factrs A grss destabilizatin f transcriptin cmplexes at intrinsic r rh-dependent terminatrs is required t make the heights f the cmpetitive free energy f activatin barriers fr elngatin and terminatin cmparable, thus making terminatin pssible. The actual terminatin efficiencies that are bserved at individual template psitins within terminatrs are ften fine-tuned by terminatin r antiterminatin factrs that bind t the transcriptin cmplex, either directly r as a cnsequence f RNA lping (23). These factrs can functin by altering the rate at which the transcribing elngatin cmplex mves alng the template (raising r lwering the height f the elngatin barrier), by changing the rate f dissciatin f the transcriptin cmplex (raising r lwering the height f the terminatin barrier), r by a cmbinatin f bth mechanisms (4, 6). As Fig. 2C shws, nly relatively small changes in these parameters are required t adjust TE acrss its entire range. G (Activatin Free Energy) At Misincrpratin Psitins A Editing G editing Misincrpratin G frward Reactin Crdinate Elngatin G frward Fig. 4. Prpsed sliding and fidelity (editing) mechanisms. (A) Cmpeting free energy f activatin barriers t nrmal elngatin (lwer barrier n right), t elngatin f a chain cntaining a misincrprated residue B Translcatin f the Transcriptin Cmplex Alng the Template In additin t describing elngatin and terminatin in the cntext f the equilibrium prperties f the transcriptin cmplex at a particular template psitin, we must als cnsider the kinetic mechanisms respnsible fr the translcatin f the cmplex alng the template. Hw des frward translcatin ccur within the single nucletide additin cycle (24)? There is n direct evidence with respect t RNA plymerases n this pint, but a minimal prpsal can be put frward that is cmpatible with ideas generated fr DNA plymerases (24) and can als accmmdate the backward mvement f the transcriptin cmplex (belw). This prpsal suggests that phsphdiester bnd frmatin [tgether with inrganic pyrphsphate (PP i ) release; Eq. 2] triggers the release f the 3 terminus f the newly extended RNA frm the substrate-binding subsite f the plymerase (Fig. 3). This puts the plymerase int a sliding mde (Fig. 4 and belw) relative t the nucleic acid framewrk f the transcriptin cmplex, permitting the next required NTP t bind t bth the substrate-binding subsite f the plymerase and, by cmplementary hydrgen-bnding, the next psitin n the DNA template. This dual binding f the next NTP serves t relck the plymerase t the nucleic acid framewrk, with the 3 terminus f the extended RNA chain nw psitined in the prduct-binding subsite f the plymerase in preparatin fr the next phsphdiester bnd frmatin event (25). Regardless f detailed mechanism, this repeating single-nucletide additin cycle drives the plymerase directinally alng the template DNA in much the same fashin as a cytplasmic mlecular mtr prtein (dynein, kinesin, r mysin) mves directinally alng a micrtubule r actin filament r a nucleic acid helicase mves directinally alng DNA r RNA (26). Of curse, the mvement f an elngating plymerase differs frm these ther mlecular mtrs in that plymerase translcatin results in the cncmitant extensin f the nascent transcript, whereas cytplasmic mtrs and helicases generate nly heat, adensine diphsphate, and inrganic phsphate t mark their passage. The elngatin-dependent mvement f RNA plymerase alng the template has recently been bserved directly in single-mlecule experiments with a laser-trapping prcedure, and the frce generated by this m- 3' 3' 3' Elngating cmplex Backsliding cmplex Elngating cmplex after backsliding and cleavage (Cleavage activated by Gre Factrs) (higher barrier n right), and t the editing (backward) reactin. Under nrmal synthesis cnditins, the frward pathway is favred, whereas under misincrpratin cnditins the backward pathway is favred. The red circle represents the transcriptin cmplex in the grund state. (B) ( Tp) The transcriptin cmplex in the elngatin mde. The square marked 3 represents the substratebinding subsite cntaining the next required NTP. (Middle) The transcriptin cmplex in the backsliding mde. The green transcript sequence shwn within the RNA-DNA hybrid in the tp panel has been extruded frm the frnt f the cmplex by backsliding (see text). (Bttm) The transcriptin cmplex again in the elngatin mde after backsliding and Gre factr activated cleavage f the sectin f transcript extruded in the middle panel. The plymerase and the transcriptin bubble mve alng the transcript RNA and thrugh the duble-stranded DNA in register, with the RNA-DNA hybrid rlling (r zippering ) alng the transcript while maintaining base pair cmplementarity and cnstant hybrid length (see text). SCIENCE VOL JULY

5 lecular mtr has been measured (27). The chemical reactin that underlies transcript elngatin may be written as RNA n NTP N RNA n 1 PP i (2) with incrpratin f the next NTP extending the RNA chain by ne residue and releasing ne mlecule f PP i. This chemical reactin is readily reversible, with a measured equilibrium cnstant (K a [PP i ]/ [NTP]) 100 fr template-directed RNA synthesis catalyzed by E. cli RNA plymerase (28). Althugh the free energy balance in the presence f 1 mm cncentratins f NTPs and PP i [the estimated physilgical cncentratins f these species (28)] favrs elngatin, the chemical reactin can be driven backward in the presence f excess f pyrphsphate, resulting in bth the shrtening the nascent transcript and the mvement f the transcriptin cmplex backward alng the template (Fig. 1) (28, 29). RNA plymerase can als mve backward alng the template by a prcess that is akin t that driven by the exnuclease editing reactin used by DNA plymerases (30). Misincrpratin at the 3 terminus f an RNA chain is favred by mitting the next required NTP. This greatly slws the elngatin reactin, because bth the additin f an incrrect nucletide residue and the subsequent extensin f the RNA chain beynd such a misincrprated residue are slw relative t nrmal elngatin (31). During such enfrced pausing at misincrpratin sites, the transcriptin cmplex can g int an unactivated state frm which elngatin is nt readily resumed when the next required NTP is again added. Hwever, this recalcitrant state can be vercme and elngatin resumed, if factrs that activate transcript editing are added (31, 32). Stalled transcriptin cmplexes ccasinally appear t suffer spntaneus transcript cleavage reactins in which a 3 terminal lignucletide f variable length is remved frm the transcript, fllwed by resumptin f elngatin frm the newly created 3 end f the shrtened RNA (33). Subsequent wrk has shwn that transcriptin factrs GreA and GreB in E. cli, and the equivalent factr SII in eukarytes, activate RNA plymerase t cleave a nascent transcript at variable (1 t 11 nt) distances back alng the chain (12, 32, 34), suggesting that these prcesses culd accunt fr such spntaneus chain cleavage. What is likely t be happening in such paused r arrested cmplexes is that the 3 end f the chain is ccasinally released frm the prduct-binding subsite f the plymerase, presumably by much the same mechanism that applies under active elngatin cnditins. At this pint, if a terminal misincrpratin has ccurred r if (perhaps at an arrest S CIENCE S C OMPASS site ) the plymerase has adpted an altered cnfrmatin that des nt immediately permit the next required NTP t relck the plymerase at the next template psitin, the cmplex may cntinue t slide backward alng the template DNA in a ne-dimensinal diffusin (randm walk) prcess. This sliding is accmpanied by the extrusin f the 3 end f the nascent RNA frm the transcriptin cmplex, and when GreA r GreB (r SII) factrs are available t activate chain cleavage, a new 3 terminus is frmed that can rebind the shrtened transcript t the plymerase at the apprpriate template psitin and permit renewed synthesis in the presence f the next required NTP (Fig. 4) (12, 34, 35). This backward sliding pathway results in the editing f the nascent RNA and an increase in the fidelity f transcriptin (35). Transcripts that cannt be extended, either because the next required NTP is missing frm the transcriptin mix r as a cnsequence f the misincrpratin f an incrrect terminal nucletide residue, are stalled n the template. As a result, the prbability f release f the 3 terminus frm the plymerase active site at such template psitins is increased, favring the backsliding mde f the plymerase with subsequent GreA- r GreB-dependent chain cleavage and release f the 3 -lignucletide cntaining the incrrect base, fllwed by resynthesis. In cntrast, chain shrtening by pyrphsphrlysis invlves the direct reversal f the nrmal elngatin mechanism (Eq. 2). Because a misincrpratin event that inhibits elngatin wuld als inhibit transcript shrtening by this means, pyrphsphrlysis shuld nt result in effective RNA editing. Hw Des Backward Sliding Occur? The definitive answer t this questin is nt knwn, but a plausible mechanism can be frmulated in the cntext f the structural mdel fr the transcriptin cmplex presented in Fig. 3. The sliding prcess appears t be diffusinal in nature, in that it des nt require the hydrlysis f adensine triphsphate r ther surces f chemical free energy. This suggests that the interactins that hld the template and the nntemplate DNA strands f the transcriptin bubble and the single-stranded sectin f the nascent RNA t the plymerase are likely t be nn sequence-specific (electrstatic?) in nature, permitting the plymerase t diffuse alng these strands as alng an isptential surface, with the upstream end f the bubble pening and the dwnstream end clsing t maintain cnstant bubble size as the cmplex slides backward and the reverse prcess ccurring when the cmplex again slides frward (12, 34 36). Figure 3 suggests that the plymerase is held t the nucleic acid framewrk f the transcriptin cmplex nt nly by nn sequence-specific prtein-dna and prtein- RNA interactins that can permit sliding as described abve but als by cmplementary Watsn-Crick hydrgen-bnding between the nascent RNA and the template DNA in the transient RNA-DNA hybrid. This latter specific and cmplementary interactin certainly cannt slide in the same way. Hwever, if the nrmal prcesses that maintain the hybrid at a cnstant (9 t 12 bp) length in elngatin als perate in the backward sliding prcess, then the hybrid can essentially rll alng the DNA template, shifting the sequence that is base-paired within the hybrid backward r frward alng the template and the transcript in cncert with the ne-dimensinal diffusin f the plymerase. Thus, the maintenance f the transient hybrid at a fixed length als serves t make it a part f the isptential ne-dimensinal diffusin surface, in that whenever a base pair at ne end f the hybrid clses, a cmpensating base pair pens at the ther end. Furthermre, as shwn experimentally (12, 34) and as expected in terms f this mdel, the plymerase cannt diffuse t within less than 12 bp f the 5 terminus f the transcript (r t within less than 12 bp f the end f a blcking cmplementary lignucletide that has been hybridized t the nascent RNA), nr can it diffuse beynd the 3 end f the transcript, because either f these prcesses wuld result in a net pening (shrtening) f the RNA-DNA hybrid and therefre cst mre free energy than is available thrugh a diffusinal mechanism. This prpsed mechanism fr the mvement f the plymerase alng the nucleic acid framewrk f the transcriptin cmplex is illustrated in Fig. 4. Cnclusins and Perspectives Althugh the integrated mdel f the elngatin cmplex that has been presented here can ratinalize mst f what we knw abut the behavir f this entity t date, it is still shrt f bth structural and mechanistic detail. Thus, we d nt yet knw the mlecular structure f any multisubunit RNA plymerase, althugh the cmmn features f the varius DNA plymerase and reverse transcriptase structures that have been slved prvide useful hints (37) and available lw-reslutin images f transcriptin cmplexes and bichemical crss-linking studies prvide insight int sme details f structure and tplgy (8, 9). Hwever, we d nt knw the exact path taken thrugh the plymerase by the DNA and RNA framewrk f the transcriptin cmplex, hw varius transcriptin factrs change the rates f mvement r the stability prperties f the transcriptin cmplex, r hw these changes are further JULY 1998 VOL 281 SCIENCE

6 mdulated by the lcal sequences f the template and nntemplate DNA and the nascent transcript. Additinal facts, as they cme in, will further define r mdify the cnceptual framewrk that has been presented here. References and Ntes 1. S. L. McKnight and K. R. Yamamt, Transcriptinal Regulatin (Cld Spring Harbr Labratry Press, Cld Spring Harbr, NY, 1992). 2. D. Herschlag and F. B. Jhnsn, Genes Dev. 7, 173 (1993); K. Rippe, P. H. vn Hippel, J. Langwski, Trends Bichem. Sci. 20, 500 (1995). 3. A. A. Travers and R. R. Burgess, Nature 222, 537 (1969); J. Greenblatt and J. Li, Cell 24, 421 (1981); S. C. Gill, S. E. Weitzel, P. H. vn Hippel, J. Ml. Bil. 220, 307 (1991). 4. P. H. vn Hippel, W. A. Rees, K. Rippe, K. S. Wilsn, Biphys. Chem. 59, 231 (1996); M. R. Van Gilst, W. A. Rees, A. Das, P. H. vn Hippel, Bichemistry 36, 1514 (1997). 5. K. M. Arndt and M. J. Chamberlin, J. Ml. Bil. 213,79 (1990); R. R. Reynlds, C. R. M. Bermudez, M. J. Chamberlin, ibid. 224, 31 (1992); K. S. Wilsn and P. H. vn Hippel, ibid. 244, 36 (1994); W. A. Rees, S. E. Weitzel, A. Das, P. H. vn Hippel, ibid. 273, 797 (1997). 6. P. H. vn Hippel and T. D. Yager, Prc. Natl. Acad. Sci. U.S.A. 88, 2307 (1991); Science 255, 809 (1992). 7. Althugh elngatin cmplexes are stable with respect t dissciatin under such lengthy incubatin cnditins, slw reactins may alter the kinetic r cnfrmatinal prperties f such halted cmplexes during extended dwell times at a given template psitin (see discussin f editing and sliding reactins in text). 8. A. Plyakv, E. Severinva, S. A. Darst, Cell 83, 365 (1995); E. Nudler, E. Avetissva, V. Markvtsv, A. Gldfarb, Science 273, 211 (1996); F. J. Asturias, G. D. Meredith, C. L. Pglitsch, R. D. Krnberg, J. Ml. Bil. 272, 536 (1997); fr earlier references, see (18). 9. E. Nudler, I. Gusarv, E. Avetissva, M. Kzlv, A. Gldfarb, Science 281, 424 (1998). 10. M. Kainz and J. Rberts, Science 255, 838 (1992); see als references in (19) and (21). A mre three-dimensinally crrect and cmplete mdel f the elngatin cmplex [fr example, Fig. 1 f (9)] wuld shw that the duble-stranded DNA is sharply bent in passing thrugh the transcriptin cmplex [W. A. Rees, R. W. Keller, J. P. Vesenka, G. Yang, C. Bustamante, Science 260, 1646 (1993)]. This bending culd facilitate the pening f the transcriptin bubble and the displacement f the nascent RNA and perhaps als imprve access fr regulatry factrs t specific prtins f the elngatin cmplex. 11. Y. Shi, H. Gamper, B. Van Huten, J. E. Hearst, J. Ml. Bil. 199, 277 (1988). 12. N. Kmissarva and M. Kashlev, Prc. Natl. Acad. Sci. U.S.A. 94, 1755 (1997). 13. I. Sidrenkv, N. Kmmissarva, M. Kashlev, Ml. Cell, in press. 14. K. S. Wilsn, C. Cnant, P. H. vn Hippel, unpublished data. 15. J. P. Richardsn, J. Ml. Bil. 98, 565 (1975). 16. J. S. Krakw and E. Frnk, J. Bil. Chem. 244, 5988 (1969). 17. H. B. Gamper and J. E. Hearst, Cell 29, 81 (1982); T. D. Yager and P. H. vn Hippel, in Escherichia cli and S CIENCE S C OMPASS Salmnella typhimurium: Cellular and Mlecular Bilgy, F. Neidhardt, Ed. (American Sciety f Micrbilgy, Washingtn, DC, 1987), pp T. D. Yager and P. H. vn Hippel, Bichemistry 30, 1097 (1991). 19. K. S. Wilsn and P. H. vn Hippel, J. Ml. Bil. 244,36 (1994); Prc. Natl. Acad. Sci. U.S.A. 92, 8793 (1995). 20. F. H. Martin and I. Tinc Jr., Nucleic Acids Res. 8, 2295 (1980). 21. Bth the run f ru resides and the cntiguus terminatr hairpin within the RNA are required fr effective intrinsic terminatin (19). The frmatin f the terminatin hairpin culd als partially destabilize the elngatin cmplex by cmpeting with the binding f a single-stranded RNA segment f the nascent transcript t the RNA-binding site (Fig. 3, regin III) lcated just upstream f the hybrid (19) [see als C. R. Altmann, D. E. Slw-Crder, M. J. Chamberlin, Prc. Natl. Acad. Sci. U.S.A. 91, 3784 (1994); (9, 14)], resulting in an additinal partial destabilizatin f the cmplex by decreasing the (net favrable) cntributin f the G NA-plymerase term (Eq. 1) t the stability f the cmplex. 22. W. D. Mrgan, D. G. Bear, B. L. Litchman, P. H. vn Hippel, Nucleic Acids Res. 13, 3739 (1985); C. A. Brennan, A. J. Dmbrski, T. Platt, Cell 48, 945 (1987); D. Jin, R. R. Burgess, J. P. Richardsn, C. A. Grss, Prc. Natl. Acad. Sci. U.S.A. 89, 1453 (1992); J. Geiselmann, Y. Wang, S. E. Seifried, P. H. vn Hippel, ibid. 90, 7754 (1993); J. P. Richardsn, J. Bil. Chem. 271, 1251 (1996); K. M. Walstrm, J. M. Dzn, S. Rbic, P. H. vn Hippel, Bichemistry 36, 7980 (1997); A. Q. Zhu and P. H. vn Hippel, ibid., in press. 23. Tethering the regulatry prteins t the nascent RNA, and thus cntrlling the effective cncentratin f these prteins at the transcriptin cmplex by RNA lping, permit the intrductin f terminatr specificity int the regulatry prcess (4). Thus, nly terminatrs lcated fairly clsely dwnstream f the sequence that cdes fr the specific site n the transcript that binds the regulatry prtein will be affected. In the same way, the requirement fr an unstructured rh-lading site n the nascent RNA transcript intrduces terminatr specificity int rhdependent terminatin (22). 24. D. Erie, T. D. Yager, P. H. vn Hippel, Annu. Rev. Biphys. Biphys. Chem. 21, 379 (1992); F. R. Fairfield, J. W. Newprt, M. K. Dlejsi, P. H. vn Hippel, J. Biml. Struct. Dyn. 1, 715 (1983); T. A. Steitz, Curr. Opin. Struct. Bil. 3, 31 (1993). 25. The simplest versin f this mdel suggests that the transcriptin cmplex as a whle mves mntnically alng the template, maintaining register between the 3 end f the elngating transcript and the active site f the plymerase. A mre elabrate scheme, based largely n nuclease ftprinting bservatins f halted transcriptin cmplexes, suggested that the plymerase might mve (at least in part) by an inchwrming mechanism [M. J. Chamberlin, Harvey Lect. 88, 1 (1995)]. In this scheme, the plymerase was thught t be flexible, and althugh the active site mved mntnically with the elngating transcript, it was suggested that ther parts f the plymerase might remain fixed and then mve alng the template in larger, multi nucletideresidue jumps, reflecting a cycling between strained and relaxed plymerase cnfrmatins. This alternative hypthesis has generated much valuable experimentatin that has advanced the field, but it nw appears (12, 34) that sme f the evidence n which it was based may have invlved dynamic ftprinting artifacts assciated with mvement (during ftprinting) f the halted and back-sliding transcriptin cmplexes. 26. T. M. Lhman, K. Thrn, R. D. Vale, Cell 93, 9 (1998). 27. H. Yin et al., Science 270, 1653 (1995); H.-Y. Wang, T. Elstn, A. Mgilner, G. Oster, Biphys. J. 74, 1186 (1998); J. Gelles and R. Landick, Cell 93, 13 (1998). 28. T. D. Yager, J. Krte, and P. H. vn Hippel, unpublished, cited in D. A. Erie et al. (31); see als K. I. Kat, J. M. Gncalves, G. E. Huts, F. J. Bllum, J. Bil. Chem. 242, 2780 (1967). 29. T. A. Rzskaya, A. A. Chenchik, R. S. Bibilashvilli, FEBS Lett. 137, 100 (1982); G. A. Kassevetis, P. G. Zenner, E. P Geiduschek, J. Bil. Chem. 261, (1986). 30. H. Echls and M. F. Gdman, Annu. Rev. Bichem. 60, 477 (1991). 31. D. A. Erie, O. Hajiseyedjavadi, M. C. Yung, P. H. vn Hippel, Science 262, 867 (1993). 32. M. G. Izban and D. S. Luse, Genes Dev. 6, 1342 (1992); S. Brukhv, V. Sagitv, A. Gldfarb, Cell 72, 459 (1993). The analgy t editing by DNA plymerase is nt cmplete, because these transcriptin factrs d nt themselves seem t have any exnuclelytic r endnuclelytic activity. Rather, they appear t activate chain cleavage by an as yet undefined mechanism, prbably invlving the same catalytic subsites f the plymerase that functin in transcript elngatin. 33. C. K. Surratt, S. C. Milan, M. J. Chamberlin, Prc. Natl. Acad. Sci. U.S.A. 88, 7983 (1991). 34. T. Reeder and D. K. Hawley, Cell 87, 767 (1996); N. Kmissarva and M. Kashlev, J. Bil. Chem. 272, (1997); E. Nudler, A. Mustaev, E. Lukjtanv, A. Gldfarb, Cell 89, 33 (1997); R. Landick, ibid. 88, 741 (1997). 35. M. J. Thmas, A. A. Platas, D. K. Hawley, Cell 93, 627 (1998). 36. This prpsed sliding mechanism fr plymerase relative t the nucleic acid framewrk f the transcriptin cmplex is similar t the ne-dimensinal diffusin f electrstatically (and nnspecifically) bund prteins alng duble-stranded DNA in lcating their regulatry targets [see P. H. vn Hippel and O. G. Berg, J. Bil. Chem. 264, 675 (1989)]. Hwever, the zippering mvement f the transcriptin bubble and the RNA-DNA hybrid alng template DNA is nt entirely isptential, because translcatin f the cmplex by ne base pair may result in the pening (fr example) f tw mre stable G C base pairs and the clsing f tw less stable A T (r A U) base pairs. The resulting small differences in the net stability f the transcriptin cmplex as a functin f template psitin may help t define the pausing psitins f elngatin cmplexes mving bth backward and frward alng the template. 37. Fr example, see H. Pelletier, M. R. Sawaya, A. Kumar, S. H. Wilsn, J. Kraut, Science 264, 1891 (1994); S. Dublié, S. Tabr, A. M. Lng, C. C. Richardsn, T. Ellenberger, Nature 391, 251 (1998); J. R. Kiefer, C. Ma, J. C. Braman, L. S. Beese, ibid., p I thank my labratry clleagues, whse experimental and theretical wrk has led t many f the ideas develped here, as well as many clleagues at the University f Oregn and elsewhere fr stimulating discussins. References included in this article can nly scratch the surface in acknwledging the wrk f clleagues in ther labratries and f thse wh came befre. The preparatin f this article was supprted in part by U.S. Public Health Service research grants GM and GM The authr is an American Cancer Sciety Research Prfessr f Chemistry. 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