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Molecular Cell
Article
The RNA Polymerase II Trigger LoopFunctions in Substrate Selectionand Is Directly Targeted by
a
-Amanitin
Craig D. Kaplan,
1,
*Karl-Magnus Larsson,
and Roger D. Kornberg
1
Department of Structural Biology, Stanford University, Stanford, CA 94305, USA *Correspondence:cdkaplan@stanford.eduDOI 10.1016/j.molcel.2008.04.023
SUMMARY
Structural, biochemical, and genetic studies have ledto proposals that a mobile element of multisubunitRNA polymerases,theTriggerLoop(TL),playsacriti-cal role in catalysis and can be targeted by antibioticinhibitors.Herewepresentevidencethatthe
Saccha- romyces cerevisiae
RNA Polymerase II (Pol II) TLparticipatesinsubstrateselection.Aminoacidsubsti-tutionswithinthePolIITLpreferentiallyaltersubstrateusage and enzyme fidelity, as does inhibition of transcription by
a
-amanitin. Finally, substitution of His1085 in the TL specifically renders Pol II highly re-sistant to
a
-amanitin, indicating a functional interac-tion between His1085 and
a
-amanitin that is sup-ported by rerefinement of an
a
-amanitin-Pol II crystalstructure. We propose that
a
-amanitin-inhibited PolIIelongation,whichisslowandexhibitsreducedsub-strate selectivity, results from direct
a
-amanitin inter-ference with the TL.
INTRODUCTION
Recent structural studies complement a large body of work thathasstrivedtoilluminatethemechanismoftranscriptionbymulti-subunit RNA polymerases. Three-dimensional X-ray structuresof eukaryotic Pol II and prokaryotic RNA polymerase (RNAP)highlight the structural conservation of these enzymes, particu-larly within their active sites ( Cramer et al., 2000, 2001; Gnattet al., 2001; Hirata et al., 2008; Murakami et al., 2002a, 2002b;Vassylyev et al., 2002; Vassylyev et al., 2007a, 2007b; Wangetal.,2006;Westoveretal.,2004;Zhang etal.,1999 ).RNApoly-merases must balance speed of transcription with fidelity, effi-ciently selecting correct NTP substrates specified by the DNA template. Additionally, RNA polymerases must select correctNTP substrates over 2
0
-dNTP substrates. Multisubunit RNA polymerases are observed to make contacts with the 2
0
-OHand 3
0
-OH groups of incoming NTPs ( Vassylyev et al., 2007b;Wang et al., 2006; Westover et al., 2004 ), but these contactscannot fully explain the robust selection of NTPs over 2
0
-dNTPs.Several conserved sequence blocks have been identified ineukaryotic Pol I, II, and III and their prokaryotic counterpart,RNAP ( Jokerst et al., 1989 ). One of these, homology block G,includes a highly conserved Trigger Loop (TL), named due toits mobile nature and possible relationship to the translocationmechanism ( Vassylyev et al., 2002 ).In the presence of template-specified ‘‘matched’’ NTP sub-strates,theTLsofbothPolIIandRNAParestabilizedinaconfor-mation that directly interacts with base-paired substrates ( Vas-sylyev et al., 2007b; Wang et al., 2006 ). This orientation of theTL is hypothesized to promote catalysis of phosphodiesterbond formation ( Vassylyev et al., 2007b; Wang et al., 2006 ).The observed contacts between NTPs and TLs of Pol II andRNAP are similar, but not identical. In both systems, an abso-lutely conserved histidine (His1085 in
S. cerevisiae
Rpo21, re-ferred to as Rpb1 hereafter, His1242 in
Thermus thermophilus
b
0
, and His936 in
E. coli
b
0
) within the TL appears to interactwith NTP substrates and has been proposed to be critical forTL function based on the recent structures ( Wang et al., 2006 ).In
E.coli
RNAP, thisresidueisessential forviability( Weilbaecheret al., 1994 ).TheexactroleoftheTLintranscriptionisunclear.TheTLisnotrequired for matched NTP base pairing with the template DNA,but rather may function bystabilizing the reaction product or en-abling a conformational change in the substrate that promotescatalysis. We have previously proposed that TL contacts withbase-paired NTP substrates may be specifically tailored tosuch matched substrates, and thus, the TL may function in tran-scriptional fidelity ( Wang et al., 2006 ). The TL is not absolutelyrequired for bond formation, as deletion or severe mutation of the TL in either
E. coli
or
T. thermophilus
RNAP does not inacti-vate the enzyme but renders elongation very slow ( Temiakovet al., 2005; Toulokhonov et al., 2007; Vassylyev et al., 2007b ).TLmutantsalterelongationpropertiesofRNAPandPolII,bothnegatively and positively, and affect the ability of RNAP to re-spond to elongation factors ( Bar-Nahum et al., 2005; Malagonetal.,2006;Svetlovetal.,2007;Temiakovetal.,2005;Toulokho-nov et al., 2007; Vassylyev et al., 2007b; Weilbaecher et al.,1994 ). The TL of RNAP has been identified as essential forNTP-dependent aspects of the transcription reaction observedin kinetic experiments, which are proposed to relate to a stepother than chemistry ( Toulokhonov et al., 2007 ) and have beenattributed to translocation or NTP binding to an allosteric site( Bar-Nahum et al., 2005; Foster et al., 2001; Nedialkov et al.,2003; Toulokhonov et al., 2007 ). The RNAP TL has also beenimplicated in active site rearrangements during transcriptionalpausing, where the TL interacts with the 3
0
Molecular Cell
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, 547–556, June 6, 2008
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2008 Elsevier Inc.
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In bacterial RNAP, the TL is required for inhibition of transcrip-tion by Streptolydigin (Stl) ( Temiakov et al., 2005; Tuske et al.,2005 ). Stl slows RNAP elongation, and a crystal structure of the
Tth
RNAP EC with Stl shows the TL positioned away fromamatched NTPsubstrate base pairedwithDNAintheRNAPac-tivesite,suggestingStlblocksTLinteractionwithsubstrate( Vas-sylyev et al., 2007b ). In the eukaryotic enzyme, the mushroomtoxin
a
-amanitin, a specific Pol II inhibitor, binds near the regiontraversedbyTLmovement,raisingthepossibilitythat
a
-amanitininhibits Pol II by preventing or altering TL movement ( Bushnellet al., 2002; Wang et al., 2006 ).Genetic data concerning
a
-amanitin resistant Pol II mutantsfrom several species and the crystal structure of
a
-amanitinbound to
S. cerevisiae
Pol II are in agreement that
a
-amanitinfunctionally bindsthePolIIactivesitejustbelowthe‘‘BridgeHe-lix’’ domain ( Bushnell et al., 2002and references therein).
a
-am-anitin appears not to alter substrate association with Pol II, butinsteadslows elongationconsiderably, therebyfavoringpausingand backtracking events ( Chafin et al., 1995; Rudd and Luse,1996 ).
a
-amanitininhibitionofhumanPolIIinkineticexperimentshasbeeninterpretedasresultingfromablocktoPolIItransloca-tion, possibly through restraint of Bridge Helix conformationalchanges ( Gong et al., 2004 ), as had been previously proposedbyBushnell et al., 2002. In the presence of bound
a
-amanitin,PolIIisable toadd severalnucleotidesbefore encounteringnor-malpausesitesthatare,inturn,exacerbatedby
a
-amanitin,sug-gestingthatanytranslocationblock isnotabsolute( Chafinetal.,1995; Rudd and Luse, 1996 ).Inthiswork,wepresentevidence thatthePolIITLfunctions insubstrate selectivity, promoting the fast addition of template-specified NTP substrates, but not of mismatched NTPs ormatched 2
0
-dNTPs. A loss-of-function substitution within theTL strongly compromises matched NTP usage but has moremodest effects on 2
0
-dNTP incorporation or NTP misincorpora-tion, indicating that slow addition of inappropriate substratesmay represent a distinct, TL-independent mode of synthesisby Pol II. Other substitutions in the TL increase incorporationrate for either all substrates or for inappropriate substrates, illus-trating the close relationship between the TL and correct sub-strate usage. We show that
a
-amanitin specifically restrainsthe Pol II TL through a functional interaction with TL residueHis1085 and that the consequence of
a
-amanitin inhibition of transcription is conversion of Pol II from a fast, accurate, TL-de-pendent mode to a slow, inaccurate, TL-independent mode.
RESULTSRpb1 TL Residue His1085 Is Important for Pol IIFunction In Vivo
Recent crystal structures of both Pol II and prokaryotic RNAPhave revealed a previously uncharacterized conformation of the TL, showing a direct interaction between the TL and amatched NTP positioned at the 3
0
-end of the nascent RNA ( Fig-ure 1 A). In both enzymes, a conserved histidine residue withinthe TL is observed to directly interact with the NTP substrate.In order to probe the requirement for this histidine in TL functionand transcription in general, we engineered different amino acidsubstitutions in place of
S. cerevisiae
Rpb1 His1085 ( Figure 1B).Substitution of alanine or phenylalanine at position 1085 resultsin inviability, while substitution of tyrosine for histidine (H1085Y)supports viability but confers a severe growth defect.
Substitution of Rpb1 His1085 Causes Defects in Pol IIElongation and Reduces Selection of NTP Substratesover 2
0
-dNTP and Mismatched NTP Substrates
In order to understand the molecular defects underlying theseveregrowthdefectcausedbytheH1085Ysubstitution,wepu-rified this enzyme from yeast and examined its activity with avariety of substrates ( Figure 2;Figure S1available online). Wild-
type (WT) and H1085Y enzymes were assembled onto nucleicacid scaffolds similar to those used for structural studies of PolII, and their ability to utilize various substrates was examined.H1085Yexhibitedastrongelongationdefectforrun-offtranscrip-tion at saturating NTP concentration ( Figure 2 A;Figure S1 A).
Compared to its defect with NTPs, H1085Y was less impairedfor the addition of 2
0
-dNTPs ( Figure 2B;Figure S1B). Similarly,
H1085Y exhibited a milder defect for misincorporation of GTPatapositionwhereATPwasspecifiedthanitdidforNTPaddition
Figure 1. Substitution of Rpb1 His1085 Confers Severe GrowthDefects or Lethality In Vivo
(A) Structural model of TL interaction with matched GTP in Pol II active site(PDB 2E2H) ( Wang et al., 2006 ). TL residues Rpb1 1070–1105 (magenta) areshown together with a GTP substrate (orange), template DNA (blue), down-stream nontemplate DNA (green), and RNA (red). Side chains for some resi-dues in the TL (Rpb1 1081–1082 and 1084–1086) are also shown. His1085 ispositioned to interact with the
b
-phosphate of the GTP.(B) Phenotypes of Rpb1 His1085 substitutions. In the absence of functionalRpb1, cells cannot lose an
RPB1
plasmid marked with
URA3
.
rpb1-H1085A
and
rpb1-H1085F
plasmids fail to confer viability to an
rpb1
D
strain, renderingcellssensitivetothedrug5-FOAbecausetheyhavebeenforcedtomaintainan
RPB1-URA3
plasmid for Rpb1 function (bottom row).
rpb1-H1085Y
canprovide the sole source of Rpb1 function but exhibits a severe growth defect.
Molecular Cell
Pol II Substrate Selection and
a
-Amanitin
548
Molecular Cell
30
, 547–556, June 6, 2008
ª
2008 Elsevier Inc.
( Figure2C;FigureS1D).WTandH1085Ywerealsocharacterized
inamodifiedsinglenucleotideadditionassay( Wangetal.,2006 ).In this assay, substrate concentration is lowered significantly toallow a single incorporation to be observed. Strong H1085Yeffects on ATP or GTP incorporation were observed using thesame templates where H1085Y had only mild defects in2
0
-dATP or 2
0
-dGTP incorporation or GTP misincorporation ( Fig-ureS1E).ThedefectinH1085Yisprimarilyoncatalysis,asK
M
cat
/K
M
0
-dGTP and a 5000-fold selection of NTPs over 2
0
-dATP.H1085Y shows only a 60-fold selection for NTPs over 2
0
-dGTPand a 600-fold selection for NTPs over 2
0
-dATP. These resultssuggest that His1085 promotes selection of correct NTP sub-strates over incorrect NTPs and matched 2
0
-dNTPs.
Resistance of TL Mutant H1085Y to
a
-Amanitin andSubstrate-Specific Pol II Inhibition by
a
-Amanitin
To probe His1085 and TL function, we assessed the effect of
a
-amanitin treatment on usage of matched and mismatchedNTP substrates and matched 2
0
-dNTP substrates by WT andH1085YformsofPolII.Ourresultsabovesuggested thatTLres-idue His1085 functions in efficient usage of matched NTP sub-strates but is less involved in utilization of inefficient substratessuch as 2
0
-dNTPs or mismatched NTPs. We reasoned that if
a
-amanitin inhibition targets TL function, then it might exhibitsubstrate-selective effects on elongation, similar to those ob-served for H1085Y ( Figure 3;Figure S2 ). Treatment of WT Pol II
with
a
-amanitinresultedinaseverereductioninelongationusingNTP substrates. The elongation activity of H1085Y, while re-duced compared to WT, was comparatively much less affectedby
a
a
-amanitin treatment was also much less effective for inhibi-tion of 2
0
-dNTP addition ( Figure 3B;Figure S2B) or for GTP mis-
incorporation ( Figure 3C;Figure S2C) by WT Pol II. In contrast to
WT,H1085Ywascompletelyresistanttoorslightlystimulatedby
a
-amanitin for these substrates. Our experiments with 2
0
-dNTPsubstrates or NTP misincorporation are limited to addition of asingle nucleotide due to an almost complete inability of Pol IIto incorporate more than one 2
0
-dNTP, even if matched,and the slowrate of multiple misincorporations. To more directlycompare
a
-amanitin inhibition of NTPs with suboptimal 2
0
-dNTP
Figure 2. Elongation Defects and AlteredSubstrate Selection by Rpb1 H1085Y Pol II
(A) H1085Y exhibits reduced elongation rate usingNTP substrates. Run-off transcription of an oligo-nucleotide scaffold template generates a 61 ntRNA product. Representative experiments forWT and H1085Y Pol II are shown in the left andright panels, respectively. Average elongationrates for each NTP concentration were measuredas the length of the transcribed region (51 nt) di-vided by the time of half-maximal accumulationofrun-offproduct(61nt).Averageelongationrateswere then plotted versus NTP concentration to in-fer maximum average elongation rate (seeExper-imental Proceduresfor details) (top right graph).Inferred maximum average elongation rates areshown in the bottom right graph with error barsrepresenting the 95% confidence interval (SeeExperimental Proceduresfor details).(B) H1085Y Pol II exhibits only modest defects for2
0
-dNTP incorporation. WT and H1085Y Pol II ECswere formed on oligonucleotide scaffolds con-taining 10-mer RNAs with templates specifyingaddition of different NTPs at position 11. Averageincorporation rates for different template-speci-fied2
0
-dNTPsweremeasuredas1/t
1/2
formaximalaccumulation of 11-mer RNA. Incorporation rateswere then plotted versus 2
0
-dNTP concentration,and maximum incorporation rate for either2
0
-dATP or 2
0
-dGTP was inferred (left panels).Maximum incorporation rates for WT Pol II andH1085Y are shown in the right panels with errorbars representing the 95% confidence interval(SeeExperimental Proceduresfor details).(C) H1085Y Pol II exhibits modest defects in GTPmisincorporation. WT and H1085Y ECs wereformed and labeled as in (B) with templates spec-ifying incorporation of ATP at the position beingmeasured, but were challenged with 1 mM GTP and misincorporation rate measured as the 1/t
1/2
for maximal incorporation. Mean misincorporation ratefrom at least three experiments is represented in the bar graph (error bars represent ± SD).
Molecular Cell
Pol II Substrate Selection and
a
-Amanitin
Molecular Cell
30
, 547–556, June 6, 2008
ª
2008 Elsevier Inc.
549
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