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Downstream DNA Selectively Affects a PausedConformation of Human RNA Polymerase II

Murali Palangat1, Christopher T. Hittinger1,2 and Robert Landick1*

1Department of BacteriologyUniversity ofWisconsin-Madison, MadisonWI 53706, USA

2Laboratory of GeneticsUniversity ofWisconsin-Madison, MadisonWI 53706, USA

Transcriptional pausing by human RNA polymerase II (RNAPII) in theHIV-1 LTR is caused principally by a weak RNA:DNA hybrid that allowsrearrangement of reactive or catalytic groups in the enzyme’s active site.This rearrangement creates a transiently paused state called the unacti-vated intermediate that can backtrack into a more long-lived pausedspecies. We report that three different regions of the not-yet-transcribedDNA just downstream of the pause site affect the duration of the HIV-1pause, and also can influence pause formation. Downstream DNA in atleast one region, a T-tract from þ5 to þ8, increases pause duration byspecifically affecting the unactivated intermediate, without correspondingeffects on the active or backtracked states. We suggest this effect dependson RNAPII-modulated DNA plasticity and speculate it is mediated bythe “trigger loop” thought to participate in RNAP’s catalytic cycle. Thesefindings provide a new framework for understanding downstream DNAeffects on RNAP.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: RNA polymerase; transcription; pausing; HIV-I; DNA bending*Corresponding author

Introduction

Elongation of mRNAs by bacterial and eukary-otic RNA polymerases (RNAP and RNAPII,respectively) is regulated by the interplay of intrin-sic signals in the RNA and DNA and extrinsicmolecules that modulate response to the intrinsicsignals by the transcription elongation complex(TEC).1 – 4 Intrinsic signals can cause transcriptionalpausing (temporary catalytic inactivity), arrest(inactivation due to backtracking of RNAP onRNA and DNA), or termination (release of RNAand DNA). These signals are generally multipar-tite; i.e., separate interactions of discrete portionsof the RNA and DNA with RNAP synergisticallyaffect the behavior of the TEC. For pausing, whichalso is a precursor to arrest and termination, theseinteractions can involve the DNA sequence down-stream from the catalytic site, the template andRNA bases in the catalytic site, the 8 to 9-bpRNA:DNA hybrid immediately upstream of thecatalytic site, and, in some cases, nascent RNAstructures that form in the RNA exit channel.5

At least 14 bp of downstream DNA affects

pausing, arrest, and termination,6 – 11 but themechanistic basis of these effects is not well under-stood. Current explanations involve effects ontranslocation of the enzyme along the DNAduplex, such as the stability of the duplex thatmust be melted,12 static DNA bends from AT-richsequences that could impede RNAP movement,11

or NTP-binding to a melted region of downstreamDNA, which facilitates translocation.13,14 However,at least for a bacterial pause signal, effects of down-stream DNA do not correlate with the ease ofduplex melting.8

Bacterial and eukaryotic RNAPs interact withdownstream DNA in a channel formed by the twolarge subunits (b0-b and RPB1-2, respectively), andalso RPB5 in RNAPII. Contacts within this channelrange from the position of duplex melting 1–3 ntin front of the catalytic center to ,20 bp furtherdownstream. Distinct parts of RNAP contact DNAthroughout this region, most notably the lobe andclamp domains at þ5 to þ10 and the jaw domainat þ12 to þ20 (þ1 is the first nucleotide down-stream of the RNA 30 end;15 – 17 see Discussion). Del-etion of the jaw domain in bacterial RNAP, whichdiffers in structure from the RNAPII jaw, greatlydiminishes pausing.18 These downstream DNAinteractions appear to be largely sequence-non-specific to ensure facile translocation of the duplexduring transcription,16,19 and strongly contribute to

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E-mail address of the corresponding author:[email protected]

Abbreviations used: RNAPII, RNA polymerase II;TEC, transcription elongation complex.

doi:10.1016/j.jmb.2004.06.009 J. Mol. Biol. (2004) 341, 429–442

the salt-stability of bacterial TECs, but not RNAPIITECs.20 – 22

We have dissected the contribution of down-stream DNA to pausing by human RNAPII at theHIV-1 pause site (Figure 1A and B). Pausing atthis site depends on a weak RNA:DNA hybridthat triggers a conformational change in RNAPII’scatalytic center to form a transiently paused statecalled the unactivated intermediate (U62p toU62u, Figure 1C).23,24 Subsequent backtracking ofRNAPII by one bp generates a more long-lived,paused conformation (U62b, Figure 1C). TAR, anascent RNA structure that recruits HIV-1 Tat to

the TEC, first can form at the HIV-1 pause site byout-competing an alternative structure called anti-TAR. Although TAR can reduce pause duration byinhibiting backtracking, neither TAR nor anti-TARplays a direct role in the pause mechanism23,24

(Figure 1A). The possible role of the downstreamDNA at the HIV-I pause has not been investigatedbeyond showing that pausing does not require thewild-type sequence.23

To study the effect of downstream DNA on theHIV-1 pause, we truncated it systematically andvaried its sequence extensively. By measuringeffects on pause formation (efficiency) and duration

Figure 1. HIV-1 pausing and effect of downstream DNA truncations. A, Structure of the HIV-1 paused TEC. Pausingis caused by the sequences of the RNA:DNA hybrid and the downstream DNA. Two RNA secondary structures com-pete at the pause site: anti-TAR, which dominates prior to TEC arrival at the pause site, and TAR, which is the targetfor Tat-binding and promotes pause escape. B, Sequence of the HIV-1 transcript. Positions of U14 (halt) and U62(pause) are indicated. Colored bars indicate RNA segments in TAR and anti-TAR (A). C, Mechanism of pausing andempirical parameters used to quantify pausing. Translocation and NTP binding are depicted as a single coupledequilibrium;54 chemistry and PPi release are also combined (U62p ! G63p). D, Transcription from the full length andtruncated (with 36 bp of downstream DNA) templates (Materials and Methods). U14, TECs halted by ATP omission.RO, run-off transcript. Samples were taken at seven-second intervals after addition of NTPs to U14 TECs; lane C,sample was incubated for an additional five minutes after increasing the GTP to 5 mM. Emax and ke

obs were obtainedas the intercept and slope, respectively, of ln[pause RNA] versus time by non-linear regression (plot; Materials andMethods). Full-length template, open circles; 36-bp downstream truncated template, filled circles. E, Emax and t1/2 fortemplates with different lengths of DNA downstream of the HIV-1 pause site (Materials and Methods). RNAPII addsAMP on the þ3 template, rather than GMP, but substitution of A for G did not affect pausing on longer templates(data not shown).

430 Downstream DNA Selectively Affects Paused RNAPII

(half-life), we could distinguish effects on differentpause intermediates.

Results

Experimental framework

The distribution of RNAP among the threekinetically distinct TEC conformations that form atthe HIV-1 pause site (Figure 1C) depends on sub-strate concentration and the nucleic acid sequencesin the TEC.24 However, the elementary rate con-stants for interconversion among these confor-mations, as well as for the steps of NTP-binding,catalysis, pyrophosphate release, and translocation,are unknown. Two empirical parameters, t1/2 andEmax, can be used instead to characterize pausingfor a given sequence, substrate concentration, andset of reaction conditions (Figure 1C). t1/2, thepause half life, is a measure of pause duration andis inversely proportional to the overall rate atwhich paused TECs resume transcription (typicallya single-exponential; assigned composite constantkobs

e ; Figure 1C). Emax is the maximal fraction ofTECs that enter the paused state, obtained fromthe y-intercept of ln[pause RNA] versus time(Figure 1D). Emax is an upper limit to the pause effi-ciency (E), which can be expressed as a functionof the overall rates of pause bypass and isomeriza-tion to the paused state (corresponding to com-

posite rate constants, kobsb and ki

obs, respectively;Figure 1C).

To measure Emax and t1/2 for a particular tem-plate, we first formed and purified halted U14TECs using templates attached to agarose beadsand incubated in HeLa nuclear extracts with GTP,CTP, [a-32P]UTP and 20dATP (U14, Figure 1B andD; Materials and Methods). Upon addition of allfour NTPs (1 mM each final) to U14 TECs, RNAPIItranscribed to the pause site, where ,50% of thetranscribing molecules paused. Emax and t1/2 wereobtained from measurements of [U62 RNA] versustime (Figure 1D).

Downstream DNA preferentially affectspaused TECs

A previous report suggests RNAPII is unusuallysusceptible to backtracking and transcriptionalarrest near the end of a DNA template.25 Therefore,we tested a series of templates with successivelyshorter segments of DNA downstream from thepause site to ask how much downstream DNA isrequired for wild-type pausing. Emax and t1/2 wereindistinguishable when measured on templatescontaining 36 bp or hundreds of bp of downstreamDNA (compare truncated 36DS to full-length,Figure 1D). However, when the downstream DNAwas reduced to 18 bp or less, both Emax and t1/2

were affected (Figure 1E). As the length of down-stream DNA was decreased, t1/2 increased, and

Figure 2. Effect of downstream DNA sequence on pausing at U62. Sequence changes are shown in bold face for thenon-template DNA strand. Pausing was assayed as illustrated in Figure 1 and described in Materials and Methods.Errors (s. e. m.) with cross bars are shown for templates tested in three or more experiments; data for other templatesare averages of two independent measurements that varied by less than 15%, yielding the estimated errors shownwithout cross bars.

Downstream DNA Selectively Affects Paused RNAPII 431

was ninefold higher than for wild-type when only3 bp of downstream DNA were present. We con-clude that 19–24 bp of downstream DNA arerequired for normal behavior of RNAPII at theHIV-1 pause site. This matches the apparentfootprint of RNAPII on downstream duplexDNA.16,22,26

These results also suggest that downstreamDNA affects paused and active TECs differently.Except for the þ3-truncated template, downstreamDNA truncation increased pause dwell time (t1/2)while decreasing efficiency ðEmaxÞ. If DNA trunca-tion also increased dwell time of the active TEC atU62, then it should increase, not decrease, pauseefficiency by allowing more time for paused TECformation prior to nucleotide addition. Thus,downstream DNA appears to preferentially affectthe paused states (the unactivated intermediate,the backtracked TEC, or both).

Pause escape is inhibited by T-tracts indownstream DNA

We next asked if pause duration was affectedby the sequence of downstream DNA. We firsttested the effects of homopolymer or alternatingcopolymer tracts placed þ6 to þ15 after the pausesite. Alternating-AT, alternating-GC, oligo-G, or arandom sequence did not significantly alter eitherpause duration or efficiency (#1–#4, Figure 2).However, both oligo-A and oligo-T tracts increasedpause duration more than threefold, without mucheffect on efficiency (#5 and #6, Figure 2). This resultdiffers from that observed for bacterial RNAP,where homopolymer and alternating copolymertracts reduced pause duration.8 However, it is con-sistent with the finding that bending in T-tractsincreases transcriptional arrest by RNAPII invitro.11,27

To define the positions at which sequencechanges affect pausing, we varied both the lengthand location of T-tracts (#7–#17, Figure 2). T-tractsbetween þ2 and þ8 all gave elevated pause half-lives (#7–#10). The strongest effect (3.6-fold, #10)was observed with a T-tract of only 4 nt located atpositions þ5 to þ8. A region of lesser effect wasobserved at positions þ8 to þ12 (#12 and #14).Other homopolymers or random sequences(#19–#24, Figure 2) placed at þ5 to þ8 had littleeffect on pausing. Thus, the effect of the T-tractwas principally attributable to four Ts between þ5and þ8 (#10).

At least three downstream regions (12–4,15–8, and 110–13) affect pausing

We next investigated the role of flankingsequences and of the length, position, and orien-tation of T-tracts (#25–#52; Figure 3A). Thesestudies yielded templates that increased pauseduration as much as 20-fold and clear evidence forat least three different regions of downstreamDNA that influence pausing. These regions likely

reflect different RNAPII–DNA interactions; how-ever, their boundaries are arbitrary. They aredefined here to facilitate analysis of the resultsand are unlikely to reflect precise physical entities.

The first region is þ2 to þ4 (red boxes, Figure3A). The effect of region 1 is best illustrated bycomparing template #25 to wt and template #26 to#27. When the wild-type TT at þ3,4 (wt and #26)was replaced by CG (#25 and #27), pause durationincreased three- to fivefold. This effect was sup-pressed when a G was present at þ2 (compare#25 to #30 and #27 to #29), showing thatsequences in the þ2–4 region affect pausinginterdependently.

The second region is þ5 to þ8 (green boxes,Figure 3A). The effect of region 2 is defined by theT-tract described above and best illustrated bycomparing template #10 to #18, template #25 to#31, and template #27 to #33–#40. The þ5 to þ8T-tract increased pause duration , threefold,and the þ5 to þ8 sequence could either increase(#31–#36, #39, #40) or decrease (#37, #38) pauseduration relative to the wild-type AAGC.

The third region is þ10 to þ13 (blue boxes,Figure 3A). Region 3 corresponds to the weakeffect of sequences at þ8–12 described above. Itseffect is best illustrated by comparison of template#49 to #50. The effect of the þ10 nt also is evidentcomparing template #25 to #27 and #31 to #33,where a G increased pause duration , twofold.

In several cases, the effects of sequence changesin different regions were approximately additive(black arrows, Figure 3A, left). For instance, combi-nation of templates #8 and #25 to give #31 yieldedan 11.2-fold increase in pause half-life, versus 7.8-fold predicted for exactly additive effects; combi-nation of templates #25 and #26 to give #27 yieldeda sixfold increase versus 3.6-fold predicted; andcombination of templates #8 and #27 to give #33yielded a 16-fold increase versus 16.8-fold pre-dicted. These approximately additive effectssuggest that independent RNAPII–DNA inter-actions affect a single step in the pathway ofpause escape (effects on multiple unimolecularsteps cannot be additive because only one stepcan be rate-limiting). This additivity could resultfrom multiple RNAPII–DNA interactions, fromcumulative effects on DNA conformation, such asDNA bending, or from a combination of theseeffects.

In contrast, examples of non-additivity also wereobserved (e.g., combination of templates #25 and#28 to give #30 yielded a 1.3-fold increase versus3.1-fold predicted). Such non-additive effects mayoccur if the individual sequence changes affect thesame or interdependent interactions.

Downstream sequence effects on pausing donot result from difficulty melting DNA

As was also seen for bacterial RNAP,8 the effectof downstream DNA on pausing was not attri-butable to melting the duplex DNA. For RNAPII,


GC-rich sequences, which would be expected toresist melting and thus favor backtracking and dis-favor forward translocation, slowed RNAP lessthan AT-rich sequences (e.g., compare templates#1 and #2 to templates #5 and #6, Figure 2).Although CG at þ3,4 increased pause duration,

GC at the same positions did not (compare #25to #41, Figure 3A). Further, increasing the GCcontent of region 1 to GCG (#30) eliminated thepause-enhancement of CG. Finally, a templatethat increased the AT-content of the immediatelydownstream DNA significantly prolonged pause

Figure 3. DNA bending alone cannot explain T-tract-enhancement of pause duration. A, Results collected and pre-sented as described in the legend to Figure 2. Colored boxes represent the three distinct regions in the downstreamDNA that affect pause half-life (see the text). Colored lines adjacent to the boxes indicate pair-wise comparisons.Black lines and arrows next to the template names show template combinations compared for additive effects. The his-togram and errors were generated as described in the legend to Figure 2, except that pause half-lives longer than 100seconds are shown on a logarithmic scale. B, RL value versus chain length for selected downstream sequences and con-trols C129 (GCAAAAAACG) and C232 (GCGAATTCGC). RL was calculated from the relative electrophoretic mobilitiesof control and experimental ladders (Materials and Methods), as illustrated for #43. C, Comparison of measured bendangles and to pause half-lives for selected templates (see Materials and Methods). Open circles, template strand T-tract.Filled circles, other sequences. The dotted line indicates the lower limit for detection of DNA bending (,38) by thismethod.29


duration (#7, Figure 2). Thus, downstream DNAeffects on pausing must be mediated by RNAPII–DNA interaction or by features of downstreamDNA structure other than the ease of DNAmelting.

Effect of DNA bending in region 2 is context-dependent

To investigate whether DNA bending explainsthe effects of region 2, we examined the correlationof effects on pause duration with known sequenceeffects on DNA bending. These correlationswere tantalizing, but imperfect, suggesting thatthe effect of DNA bending depends on its sequencecontext. T-tracts 4 nt or longer can cause intrinsicDNA bending, which may be localized near thejunctions of the T-tract and enhanced when theT-tract is flanked by GC-rich sequences.28 – 32 Sev-eral variants that shortened the T-tract below the4-nt threshold still increased pause duration rela-tive to #27 (#35, #36, and #39; Figure 3A), but to alesser extent than the 4-nt T-tract (#33). Increasedpause duration was sometimes evident when aT-tract known to bend DNA was in the non-tem-plate strand (#4430) but usually not when theT-tract was in the template strand (#43,30 #4531 and#46;32 Figure 3A). However, certain cases contra-dicted each of these observations. The template-strand T-tract #51 increased pausing, whereas thenon-template-strand T-tract #49 had a minimaleffect. Some of this variation could reflect effectsof flanking sequences on bending. GC-rich flankingsequences enhanced the effect of T-tracts (#31–#33;Figure 3A); however, the GC-rich flankingsequences alone increased the pause duration , -sixfold even when the T-tract was absent (#25, #26and #27; Figure 3A). In most cases, the presence ofGCG at positions 11–13 appeared to dampen theeffect of T-tracts on pause duration.

To account for possible context effects, we exam-ined the correlation of pause duration with DNAbend angle measured directly by EMSA of ligatedduplex oligonucleotides28 (Figure 3B and C;Materials and Methods). Previously measuredsequences gave the expected bend angles (#43(same as #44), #46, C1, and C2; Figure 3B). Ifthe template-strand T-tracts were discounted(#43, #51, and #52), bend angle and pauseduration correlated well except for #44 and #49(Figure 3C). Template #44 contained sequences inregions 1 and 3 that decreased pause duration(compare templates #27 and #42, Figure 3A); thus,the effect of DNA bending in region 2 of template#44 may be suppressed by interactions in regions1 and 3. We conclude that DNA bending by non-template-strand T-tracts in region 2 may increasepause duration, but that this effect is context-dependent. Further, template-strand T-tracts inregion 2 may not affect RNAPII equivalently tonon-template-strand T-tracts.

The effect of region 2 is not exclusivelythrough interaction of RNAPII with the DNAminor groove

A feature of T-tract DNA other than bending thatcould explain its effects on pausing is its character-istic minor groove. Some DNA-binding proteinsrecognize the minor groove of AT-rich DNA bythe narrow spacing of the phosphodiester back-bone, contacts to bases in the minor groove, orboth (e.g., a-CTD of bacterial RNAP33,34 andTATA-binding protein35). In such cases, recognitioncan be preserved when cytosine and inosinereplace thymine and adenine, respectively, becauseboth the narrow minor groove and the pattern ofhydrogen bond donors and acceptors in the minorgroove are maintained.32,35 Thus, to ask if exclu-sively minor-groove interactions can explain theeffects of region-2 T-tracts on pausing, we replacedthem with a C-tract paired with a template-strandI-tract (Figure 4 and Materials and Methods). Wefound that replacing T-A bp with C-I bp in region2 eliminated its ability to slow pause escape. There-fore, interactions of RNAPII with only the minorgroove of downstream DNA are unlikely toexplain the effect of downstream DNA T-tracts onpausing.

The effect of downstream DNA on pause half-life and efficiency are uncorrelated

Although less common than effects on pauseduration, some downstream DNA sequences

Figure 4. Exclusively minor groove-interactions do notmediate downstream DNA effects on pausing. Emax andt1/2 were determined for the templates shown (changesin bold) as described in the legend to Figure 1. I, inosine.Results shown are an average of three independentmeasurements that varied by less than 15%.


significantly affected pause efficiency (e.g., #7, #23,#28, #49 and #52; Figures 2 and 3A). To ask ifthese effects correlated with the more prominenteffects on pause duration, we compared pauseduration and efficiency directly for all 53 templatestested (Figure 5A). The two parameters were com-pletely uncorrelated (R ¼ 0.03, P ¼ 0.81). Further,the lack of correlation was not attributable toexperimental error. Variation between templateswas far greater than error estimates (Figure 5B) orthe standard deviation among multiple measure-ments (e.g., see errors for templates #27, #28, and#33, Figure 3A). These different effects of down-stream DNA on efficiency and duration reinforceour conclusion from truncation experiments(Figure 1E) that downstream-DNA does not inhibitnucleotide addition equivalently in paused and

active TECs (because that would yield correlatedincreases in efficiency and pause duration).

Downstream T-tracts affect nucleotide additionfrom the unactivated intermediate

As described above, one or more paused state ofRNAPII responds to downstream DNA differentlythan does the active state. To ask if the two knownHIV-1 paused states differ in their response, weplaced a downstream T-tract after a mutant pausesite at which the unactivated intermediate and thebacktracked TEC can be distinguished kinetically.The following results establish that the selectiveeffect of downstream DNA occurs primarily in theunactivated intermediate by slowing steps leadingback to the active TEC.

The mutant pause site we used encodes a morestable hybrid that dramatically slows formation ofthe backtracked TEC (called here the stabilizedhybrid template; same as template #5 of Palangatand Landick24). When GTP substrate is present,RNAPII occupies only the active and unactivatedstates on the stabilized hybrid template, and theunactivated intermediate can be detected as aslow population at #100 mM GTP, but not at1 mM GTP (#25% of total U62 TEC).24 When thedownstream T-tract (#10, Figure 2) was added tothe stabilized hybrid to yield the combined mutanttemplate (Figure 6A), the unactivated intermediatebecame detectable even at 1 mM GTP (Figure 6Aand B, left panel). When GTP was withheld fromTECs positioned at U62 on the stabilized hybridand combined mutant templates, the backtrackedTEC formed slowly and was readily detectableafter two minutes (Figure 6B, right panels). Thatthis slow forming species is the backtrackedTEC was established previously by examiningTFIIS-mediated cleavage.24

The key result establishing that the effect of thedownstream T-tract occurred selectively in theunactivated intermediate was the near identicalbehavior of the backtracked TEC on the two tem-plates when the time of GTP addition was varied(Figure 6C and D). At intermediate delay times(15–60 seconds), pause escape on the combinedmutant template was biphasic. The unactivatedintermediate was evident as a faster species(green, kobs

e ¼ 55 £ 1023 s21) that converted tothe slower, backtracked species (blue,kobs

e ¼ 6 £ 1023 s21) with increasing delay time.However, the rates at which the backtrackedspecies formed and at which it escaped to thenucleotide addition pathway were indistinguish-able on the two templates (Figure 6D). Thus,the downstream T-tract had little effect on thebacktracked state. It also had little effect on forma-tion of the unactivated intermediate, since the 15–25% of U62 that formed unactivated intermediateon the combination template (green lines, Figure6C) was about equal to the ,25% previouslyestimated to form unactivated intermediate on thestabilized hybrid template at #100 mM GTP (see

Figure 5. There is no correlation between pause half-life and efficiency. A, Scatter plot of Emax versus t1/2 forall templates in Figures 2 and 3. B, Semi-log plot ofpause RNA concentration as a function of time forselected templates, quantified as described in the legendto Figure 1.


Figure 6C described earlier24). Thus, the 3.6-foldeffect of the downstream T-tract on pause durationmust occur primarily at steps by which theunactivated intermediate re-enters the nucleotideaddition pathway (green lines, Figure 6D). Itremains possible that some downstream sequenceeffects on efficiency occur in the active TEC confor-mation (see Discussion); however, our resultsstrongly suggest that the major effects of down-stream DNA on pause longevity occur in theunactivated intermediate.

Discussion

By examining the relationship of downstreamDNA sequence and structure to pausing byhuman RNAPII, we have characterized the natureof RNAPII–downstream DNA contacts and theireffects on the rate of nucleotide addition. We findthat downstream DNA affects pause durationby selectively affecting a subpopulation of TECs ina transiently paused state called the unactivated

intermediate. Downstream DNA in at least threedifferent regions may exert the selective effects.Different sequences in these three regions alsoaffect the efficiency with which actively elongatingTECs form the unactivated intermediate; these lesscommon effects may occur in the active TEC orthe transition state between the active and unacti-vated conformations. The capacity for DNA bend-ing in region 2 (þ5 to þ8) partially correlates withincreased pause duration. However, these effectscannot be explained by interactions of static DNAbends with RNAPII or by effects of duplex stabilityon translocation of RNAPII. We propose insteadthat RNAPII’s DNA-entry channel may havegreater mobility in the unactivated intermediateand that movements of parts of the channel com-bine with the sequence-dependent malleability ofDNA to generate strong effects of downstreamDNA on transcriptional pausing.

Downstream DNA–RNAPII interaction

To consider the nature of downstream

Figure 6. RNAPII–downstream T-tract interaction only affects the unactivated intermediate. A, Emax and t1/2 forwild-type, downstream mutant template (#10, Figure 2), stabilized hybrid template, and the combination mutanttemplate (determined as described in the legend to Figure 1). B, Delay experiment. TECs were advanced to A60 bystepwise transcription on either the stabilized hybrid template (top) or on the combination mutant template (bottom)and then incubated with CTP, GTP and UTP (1 mM each, left) or incubated with CTP and UTP (1 mM each, right)for 120 seconds followed by addition of GTP to 1 mM (Materials and Methods). Samples in the lanes designated “C”were chased as described in the legend to Figure 1. C, Fraction U62 RNA versus time after GTP addition for differentdelay times prior to GTP addition. Data from experiments similar to those shown in B, but with different delaytimes, were plotted versus time after GTP addition. The data shown are representative of three independent measure-ments that varied by less than 15%. Blue lines and points represent RNAs assigned to the long-lived, backtrackedTECs. Green lines and points represent RNAs assigned to the unactivated intermediate. D, Pause mechanism withintermediates colored as in C and steps affected by downstream DNA in green. Observed rates were estimated bynon-linear regression of the quantities of paused species versus time; the lower limit for escape from the unactivatedintermediate on the stabilized hybrid template ($200 £ 1023 s21) was estimated from the results in C.29


DNA–RNAPII interaction, we examined a modelbased on a TEC crystal structure of Saccharomycescerevisiae RNAPII16 (Figure 7). The location andorientation of downstream DNA in this model isbased on weak density in the crystal structure andis consistent with the crosslinking data fromboth bacterial and eukaryotic transcriptioncomplexes.26,36,37 The disordered trigger loop inRPB1 was modeled based on its location in theScRNAPII–TFIIS co-crystal structure,38 where itsinteractions with TFIIS may fix its position relativeto the partially resolved location in the ScRNAPTEC16 and the location in T. thermophilis RNAP.39

We positioned the bent DNA in the model usingthe crystal structure of DNA30 (Figure 7A; non-tem-plate strand T-tract as in #44, yellow; templatestrand T-tract as in #43, light green). The precisestructure of this bent DNA probably is dictated bycrystal packing forces,31 and the trajectory of DNArelative to RNAPII depends on the location ofthe T-tract within the downstream duplex (,208bending toward the major groove arises frombends at the junctions of the T-tract with flankingDNA, mostly at the 50 junction). Nonetheless, themodel illustrates the range of contacts possiblebetween downstream DNA and RNAPII even ifthe bend structure is inaccurate in detail. For tem-plate #44, the model predicts a bend toward theRPB1 clamp and RPB5, whereas for template #43it predicts a bend away from the clamp and RPB5and toward the RPB1 jaw. Other T-tract locations(not shown) produce other bend angles andpotential RNAPII interactions.

We used the model to identify parts of RNAPIInear the downstream DNA. Multiple segments ofRPB1 or RPB2 contact or approach the downstreamDNA in each of the three regions that affected theHIV-1 pause (segments 1–7 in region 1, segments7–13 in region 2, segments 13–14 in region 3;colored numbers in Figure 7B; described in Table1). A fourth region of potential contacts of down-stream nucleotides þ14 to þ18 to the RNAPII jawdomain (parts of RPB1 and RPB5) was alsoevident, as described.15,16,19 In all four regions,most potential contacts involve the phosphatebackbone of downstream DNA (Table 1). In a fewinstances, non-polar side-chains located over themajor or minor grooves could make van der Waalsinteractions within the grooves19 (e.g., RPB2 P231in segment 11 and RPB5 P86 and P118 in segments15 and 16; Figure 7B, Table 1), but no side-chains

Figure 7. Downstream DNA interactions of RNAPII.RPB1 (pink), RPB2 (cyan), and RPB5 (white) are shownas worms, with the segments of the subunits that poten-tially interact with downstream DNA highlighted inmagenta, blue, purple, or orange (for RPB1, RPB2, thetrigger loop, or RPB5 segments, respectively). RPB3,RPB6, RPB8, RPB9-12 are shown as white CPK surfaces.The RNA transcript is red. The template DNA strand inthe hybrid and the downstream DNA are green. Thebridge helix is black. Four regions of potential down-stream DNA interaction referred to in the text areshown as numbered bars above the structure. The down-stream DNA trajectories predicted for bent DNA in tem-plates #43 (light green) or #44 (yellow) are renderedsemi-transparent. A, View of entire RNAPII structure.The box demarcates the view in B. B, Close-up view ofdownstream DNA interactions. Segments of RPB1,RPB2, and RPB5 that potentially interact with down-stream DNA are 1, RPB1 329–333 (clamp); 2, RPB2 500–510 (fork loop 2); 3, RPB1 307–311 (rudder); 4, RPB11401–1406 (switch 1); 5, RPB1 835–837 (bridge); 6, RPB11082–1088 (trigger loop); 7, RPB1 169–178 (clamp); 8,RPB1 1383–1389 (switch 1); 9, RPB1 1105–1109 (cleft);10, RPB1 133–144 (clamp); 11, RPB2 226–237 (lobe); 12,RPB2 259–267 (lobe); 13, RPB1 1310–1315, 1274–1279,1331–1341 (bacterial jaw); 14, RPB1 1219–1224, 1243–1254,1254–1270 (jaw); 15, RPB5 114–120 (jaw); 16, RPB5

86–90 (jaw) (names and numbering for S. cerevisiae RNA-PII are as described;15 see Table 1 for more information).The trigger loop (purple) is depicted in the conformationobserved in the co-crystal with TFIIS.38 Possible alternatepositions of the trigger loop are depicted as a semi-trans-parent worm (conformation in T. thermophilis RNAP39) oras purple spheres (modeled route near active site basedon trajectory of trigger loop segments in ScRNAPIITEC16).


make base-specific interactions within the major orminor grooves, even though the crystal structuredid contain a T-tract from þ1 to þ6 (similar to tem-plate #7, Figure 2). Nonetheless, it is possible thatpause-enhancing downstream sequences causesequence-specific interactions not evident in themodel. However, we think it is much more likelythat most effects of downstream DNA on RNAPIIreflect indirect readout, which is the recognition ofDNA structure by its sequence-dependent confor-mation, including the ability to be deformed uponprotein binding.40 – 43

The effects of the region-2 T-tracts offer insightinto how indirect readout might operate withinthe RNAPII DNA-entry channel. Strong effects of

flanking sequences must explain why some butnot all demonstrably bent sequences gave strongeffects on pausing (Figure 3C). These flanking-sequence effects on pausing were much greaterthan their reported effects on DNA bending,28

making it highly unlikely that the simpleencounter of a static bend by RNAPII explains ourresults.

Instead, we suggest that the effects of down-stream DNA T-tracts on pausing arise from a com-plex interplay between the anisotropic flexibilityof T-tract DNA and the effect of RNAPII side-chains on DNA conformation.44 – 46 The outcome ofthis interplay may control both downstream DNAtrajectory and the conformation of parts of RNAPII

Table 1. Potential contacts of RNAPII to downstream DNA

Contact Name Regiona SegmentbClosest

residue(s)cDNA

contactd H. sapiens E. coli T. aquaticus

1 Clamp 1 RPB1 329–333e G331 ,T1P2 343–347 331–335 607–6102 Fork loop 2 1 RPB2 500–510 NT1-3 527–537 532–546 412–3263 Rudder 1 RPB1 307–311 I308f T2P3 321–325 310–314 585–589

A309f T3P44 Switch 1 1 RPB1 1401–1406 E1403 T2P3 1439–1444 1325–1330 1439–14445 Bridge 1 RPB1 835–837 Y836 ,T1-2 858–860 794–796 1091–10936 Trigger loopg 1 RPB1 1082–1088 H1085g NT3P4 1105–1111 933–939 1239–12457 Clamp 2 RPB1 169–178 R175 ,NT8P9 186–195 131–142 119–130h

1 K176 ,T3P48 Switch 1 1 RPB1 1383–1389 R1386 T2P3 1413–1419 1308–1313 1422–1427

2 H1387 NT5P69 Cleft 2 RPB1 1105–1109 K1109 ,NT5P6 1128–1132 1145–1149 1262–1266

2 N1110 ,NT4P510 Clamp 2 RPB1 133–144 K143 NT7P8/mj. grv. 139–150 210–222 485–497h

11 Lobe 2 RPB2 226–237 P231/P233 ,T6P7/mj. grv. 208–218 158–170 149–15812 Lobe 2 RPB2 259–267 Contact only

if movesR261 , T6P7 240–254 189–192 177–180

13 Bacterialjawi

2–3 RPB1 1310–1315 Possible homologueof bacterial

RNAP jaw, contactonly if moves

,T8-11 1340–1345 1152–1158 1269–1275h

1274–1279 1299–1304 1170–1174 1287–12901331–1341 1181–1191 1298–1308h

1361–1371 1211–1226 1328–134014 Jawj 3 RPB1 1254–1270 Contact only

if moves,NT12-14 1274–1295 k k

4 1243–1254l Contact onlyif moves

,T17-19 1263–1274 k k

4 1219–1224 Contact onlyif moves

,T17-19 1238–1243 k k

15 Jaw 4 RPB5 114–120 T117 NT15P16 106–112 k k

S119 NT15P1616 Jaw 4 RPB5 86–90 P86 mj. grv. 78–82 k k

K91 ,NT15P16,16P17

a Downstream DNA contact regions as shown in Figures 3 and 7.b Protein segments responsible for the contact. Numbering is that of S. cerevisiae RNAPII.15

c Amino acid in RNAPII that approaches downstream DNA most closely.d Part of downstream DNA that approaches RNAPII most closely. T, template DNA strand. N, non-template DNA strand. P, phos-

phate between the indicated nt numbered as in Figures 3 and 7. mj. grv., major groove. , , potential contact within ,5 A of RNAPII,but contact does not occur in RNAPII TEC model.

e Also contacts P-2 and P-3 of DNA in RNA:DNA hybrid (K332).f The conformation of the rudder differs in different RNAP structures. I308 and A309 correspond to Arg residues in bacteria, which

appear to contact þ2 and þ3 phosphates of template DNA.g Contacts mapped for location of the trigger loop in T. thermophilis RNAP39 because TFIIS constrains the trigger loop location in

TFIIS–RNAPII,38 as depicted in Figure 7. H1085 is not near downstream DNA in the TFIIS–RNAPII co-crystal structure.h These segments of bacterial RNAP occupy locations to those of the RPB1 segments, but form different structures.i This segment of RPB1 is closest in structure to the bacterial RNAP jaw.18

j No contact to downstream DNA in TEC crystal structure. Crystal packing and absence of DNA after þ21 prevent interaction. Thejaw appears readily able to move to contact downstream DNA if more DNA were present.15,16

k No bacterial homologue exists for this RNAPII segment.l This portion of RPB1 is disordered in available crystal structures.


near the downstream DNA. The trajectory of aDNA duplex is thought to be controlled by thebalance of the counteracting forces of base-stackingand electrostatic repulsion.45 T-tracts may perturbone or both of these forces, resulting in anisotropicflexibility of the DNA (a tendency to flex in a par-ticular direction), rather than a static bend.44 Theinteractions of protein side-chains also mayalter DNA trajectory by perturbing the symmetricbalance of phosphate repulsion.46,47 Thus, theinherent pliancy of T-tract DNA and other DNA-sequence effects on backbone structure may com-bine with effects of RNAPII side-chains to dictatethe conformation of downstream DNA, which forsome sequences alters RNAPII activity. Becausethe number of combinations of such effects in theDNA-entry channel is large, no simple set of rulesfor effects of DNA sequence on RNAPII activitymight be expected. However, inherently deform-able sequences, such as T-tracts, might increasethe chances for such effects. Indeed, these are thetypes of results we observe. We may be no moreable to predict effects of particular downstreamsequences on RNAPII activity than we can predictthe sequence-specificity of any other protein–nucleic acid interaction without empirical informa-tion. Suitably designed in vitro selection experi-ments, however, could define downstream DNAsequences that maximally favor or disfavorpausing.

Role of downstream DNA in pausing

The most important aspect of our findings is thatthe effects of downstream DNA on RNAPII activityare strongly manifest only in an off-line, unacti-vated conformation of the TEC. This suggests thatthe nature of RNAPII–downstream DNA inter-action differs in the unactivated intermediate,most likely that parts of RNAPII in the down-stream DNA channel exhibit a greater range ofmotion in the off-line state and thus more readilyengage or favor altered conformations of thedownstream DNA. In this way, sequences thathave little effect on the active TEC may generateinteractions that are specific to the unactivatedTEC. The idea that RNAP exhibits greater confor-mational flexibility in the unactivated state is ingeneral agreement with its other properties,including the rearrangement of catalytic or reactivegroups in the active site, the ability to backtrack,and the ability to be stabilized by nascent RNAhairpins in the case of bacterial RNAP.2,14,24,48 – 50 Amore rigid conformation of the active TEC mayfacilitate rapid transcription by optimizing translo-cation, NTP binding, and catalysis and minimizingthe effects of nucleic acid sequence on the enzyme.Differences in the conformations of the activeand unactivated TECs also may explain whyeffects of downstream DNA on pause efficiencyexhibit a different sequence specificity (Figure 5).These effects could be manifest either in the active

TEC or in the transition state between the activeand unactivated states.

How downstream DNA interaction alters theactivity of the unactivated intermediate remainsan open question. We favor the idea that theseinteractions indirectly affect nucleotide additionby affecting movement of a flexible loop, calledthe trigger loop, that lies within the secondarychannel just downstream of the active site. Move-ments of the trigger loop have been variously pro-posed to affect translocation, NTP-binding, andthe conformation of the active site,16,39,51 and arestrongly indicated by detection of different trig-ger-loop locations in different RNAP crystalstructures (Figure 7).16,38,39 Interestingly, at a bac-terial pause site that does not permit backtracking,the trigger loop crosslinks to the RNA 30 nucleotide(I. Toulokhonov, unpublished results). Thus, thetrigger loop likely moves toward the active site inthe unactivated intermediate, consistent with themodeled location based on the ScRNAPII TEC(Figure 7B). Although we do not know whichmechanistic step is inhibited by downstream DNA(translocation, NTP-binding, an active-site confor-mational change, or catalysis), alternating inter-action of the trigger loop with downstream DNAin the active TEC and with active-site proximalresidues in the unactivated intermediate, coupledwith the interplay of anisotropic DNA flexibilityand RNAPII–DNA interactions in the unactivatedintermediate, can explain the selective effect ofdownstream DNA sequence on the unactivatedintermediate.

Alternative mechanisms are possible, but lessattractive. First, RNAPII–downstream DNA inter-actions, mediated by indirect readout and DNAdeformation, could directly inhibit translocation.However, effects solely on translocation are incon-sistent with some of our results. Translocationshould melt 1 bp of downstream DNA betweenþ2 and þ4.16,36 However, changing þ2 to an ATbp did not decrease pause duration (template #7,Figure 2), and changing þ3 and þ4 to GC base-pair did not increase pause duration (templates#30 and #41, Figure 3A), as would be predictedfrom a direct translocation-effect model. If down-stream DNA–RNAPII interaction affects pausingby affecting translocation, then translocationmust contribute significantly to pausing and thesesubstitutions should have predictable effects onpause duration by altering the energetics of trans-location. Additionally, there was no effect of thedownstream DNA T-tract on reverse translocationof a paused TEC (i.e., backtracking), even whenthe same sequence strongly affected pause dura-tion (Figure 6).

Another possibility, recently proposed by twogroups, is that NTPs bind to the downstreamDNA by Watson–Crick pairing and that thebound NTPs affect RNAPII activity. In one versionof this model, NTPs are fed into the active sitebound to the template DNA strand;13 in the other,a single bound NTP acts as an allosteric effector of


the active-site conformation.14 Thus, changes indownstream DNA–RNAPII interactions couldaffect these putative NTP-binding sites. However,these models are based on the complex kinetics ofnucleotide addition, for which equally parsimo-nious models involving NTP-binding through thesecondary channel and active-site conformationalchanges can be hypothesized. In the absence ofdirect biochemical evidence for NTP-binding inthe downstream DNA channel, we find a trigger-loop mediated effect of RNAP–downstream DNAinteraction on active-site conformation to be amore attractive explanation. Further, some down-stream DNA effects involve sequence changes inregions of the downstream DNA that all availabledata suggest are double-stranded in the TEC andthus not accessible to NTP-binding (e.g., at þ8 toþ15 in templates #14 and #16, Figure 2).19,26

In conclusion, we note that the large effects ofdownstream DNA on RNAPII activity suggest onepossible mechanism for the plethora of eukaryoticregulatory factors known to affect transcriptelongation.3,4 The downstream DNA is by nomeans the only possible target for such factors.1

However, the fact that DNA regions that affectRNAPII activity are partially exposed in the TECmakes it easy to envisage how a regulatory proteincould interact with DNA, RNAPII, or both tostabilize or destabilize certain conformations, asproposed for the bacteriophage Nun protein,52 andthereby positively or negatively affect transcriptelongation.

Materials and Methods

Template DNA

DNA templates were prepared by PCR-amplificationof plasmid pLL283 or pPM130 (stabilized hybrid mutant)using a biotinated upstream primer, as described,24 and adownstream primer that included 36 bp of DNA down-stream of the pause site. To generate truncated tem-plates, a StuI site was introduced at appropriatepositions in the reverse primer and the resulting PCRproducts were digested with StuI. The sequences of allPCR primers used are available on request. Transcriptiontemplates were immobilized on streptavidin-agarosebeads (Sigma, St. Louis, MO) through the streptavidin–biotin linkage.

To put inosine in the downstream DNA (Figure 5), anoligonucleotide specifying þ40 to þ98 of the templatestrand (relative to the transcription start site) withinosine at positions þ67 to þ70 was annealed to a comp-lementary strand containing cytosine bases to pair withthe inosine bases. The annealed duplex was digestedwith NheI and ligated to an NheI-digested PCR frag-ment generated from pLL283. The ligated templatefragment was agarose-gel purified and immobilized onstreptavidin-agarose beads.

Proteins and substrates

Nuclear extracts were prepared53 from HeLa cells pro-cured from Cell Culture Center (Connrapids, MN). FPLC

purified NTPs and 20-dNTPs were obtained from Phar-macia (Piscataway, NJ). HPLC purified oligonucleotideswere from Operon Technologies (Alameda, CA).

Preparation of halted TECs andstepwise transcription

U14 TECs were prepared as described.24 Briefly, pre-initiation complexes were formed by incubating HeLanuclear extracts with immobilized templates in tran-scription buffer (10 mM Hepes–Kþ, pH 7.9, 33 mM KCl,8 mM MgCl2, 1 mM DTT, 0.1 mM Na2EDTA, 0.1 mMdATP, 6% (v/v) glycerol) and 20 units Prime RNaseInhibitor for 20 minutes at 30 8C (100 ml). Transcriptionwas initiated by the addition of CTP, GTP (100 mMeach), and [a-32P]-UTP (0.5 mM; 3000 Ci/mmol), andhalted U14 TECs formed for one minute at 30 8C. Thehalted TECs were sequentially washed with ten volumeseach of (a) wash buffer (20 mM Tris–HCl, pH 8.0, 10 mMb-mercaptoethanol, 0.2 mM Na2EDTA, and 20% (v/v)glycerol) containing 1% Sarkosyl and 1 M KCl; (b) washbuffer containing 0.1 M KCl; and (c) transcription buffercontaining acetylated BSA (200 mg/ml) and finally sus-pended in transcription buffer. To measure Emax and t1/

2, U14 TECs were allowed to elongate through the pausesite in the presence of all four NTPs (1 mM each) at30 8C (Figure 1). For the experiment shown in Figure 6,U14 TECs were walked to position A60, and allowed toelongate to position U66 in the presence of CTP, GTPand UTP (1 mM each) at 30 8C, or in the presence ofCTP and UTP with subsequent addition of GTP.24

Samples were removed from the reactions, processed,electrophoresed through denaturing gels, and quantifiedas described.24

DNA bending measurements

Deoxyoligonucleotides (10mers; Operon, Alameda,CA) were designed to produce staggered ends uponannealing that ligate unidirectionally. The 10mers were50-phosphorylated with [g-32P]-ATP using T4 Polynucleo-tide kinase (New England Biolabs), annealed, andligated using T4 DNA Ligase (New England Biolabs) asdescribed.28 The ligated multimers were separated at7 V/cm, 23 8C on native 8% (w/v) polyacrylamide(acrylamide to bis-acrylamide, 29 : 1, w/w) gels in Tris–Borate (90 mM, pH 8.3) plus EDTA (2.5 mM). The ratioof apparent length to actual chain length (RL) was deter-mined by comparison to an MspI digest of pBR322and multimers of the sequence 50-CTTAAGCCTC. Bendangles (Figure 3C) were calculated as the product of 188(the curvature per helical turn of the standard bentsequence 50-GCAAAAAACG) and the relative curvatureof a given DNA sequence, as calculated from RL versusactual chain length as described29 (Figure 3B).

Acknowledgements

We thank members of our laboratory, R. Gourse,S. Rutherford, I. Artsimovitch, & W. Ross for criti-cal reading of the manuscript and for helpful sug-gestions, and R. Kornberg for coordinates ofdownstream DNA in the RNAPII TEC. This workwas supported by grants from the NIH (GM38660


to R.L.) and NSF (REU 9820163 for support toC.T.H.).

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Edited by J. Karn

(Received 17 February 2004; received in revised form 25 May 2004; accepted 3 June 2004)


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