BACKGROUND
all reactions performed by rna polymerase  are catalysed by a single active centre and are proposed to proceed through a general two mg2+ ion mechanism  <cit> .
structural studies revealed that conformational changes in the active centre might be required for efficient catalysis and biochemical studies are consistent with such a model  <cit> .
the major conformational change involves folding of the trigger loop  that brings the rnap active centre from 'open' to 'closed' conformation .
though crystal structures of rnaps with folded tl were obtained only in the presence of cognate ntp  bound in the i+ <dig> site of the active centre, tl folding is also thought to be required for efficient catalysis of pyrophosphorolysis and phosphodiester bond hydrolysis  <cit> .
although, in the absence of tl, the rate of phosphodiester bond formation is decreased by four orders of magnitude, the actual mechanism by which tl participates in rnap activity remains unknown.
crystallographic studies revealed that folded tl tightly interacts with cntp base and triphosphate moieties  <cit>  .
this observation was used to build three distinct models explaining the role of tl folding in phosphodiester bond formation.
in the first model, based on a crystal structure of yeast rnap ii, folded tl was proposed to directly participate in catalysis through an invariant histidine residue  that withdraws electron density from the β-phosphate of the incoming ntp thus activating nucleophilic attack on the α-phosphorus  <cit> .
according to the second model h <dig> participates in acid catalysis as a proton donor for the leaving pyrophosphate  <cit> .
this model is based on studies of single-subunit rnaps that are evolutionary unrelated to multi-subunit rnaps and may, therefore, use a different mechanism for catalysis.
in the third model, based on a crystal structure of t. thermophilus rnap, folding of tl was proposed in order to stabilize, via r <dig> and h <dig> residues, the triphosphate moiety of the i+ <dig> site-bound ntp in the active  conformation   <cit> .
a recent study of escherichia coli rnap revealed that mutation of h <dig> had only minor effect on catalysis, indicating that acid/base catalysis is not its primary role  <cit> .
however, the relatively mild effects of substitutions of amino acids that, according to crystallographic data, interact with the triphosphate moiety of incoming ntp observed in this work  are also inconsistent with the transition state stabilisation hypothesis  <cit> .
the small effects of single substitutions, compared to dramatic catalytic defect caused by deletion of the entire tl, suggest that tl may have an allosteric effect on other amino acids that are also required for catalysis .
mutations in tl and surrounding amino acids were found to alter discrimination against non-complementary ntp  and complementary 2'- and 3'-deoxy ntps   <cit> .
these observations led to suggestions that tl could participate in substrate discrimination by either:  selectively facilitating incorporation of correct substrates; or  by providing sufficient time for accurate ntp selection prior to the act of catalysis.
by analogy with dnaps and ssrnaps, the existence of a conformational change in the active centre of multi-subunit rnaps  that brings the active centre to a catalytically active conformation suggests that substrate discrimination may take place via differential efficiency of conformational change occurrence upon the binding of cntps versus ncntps.
in support of this idea, inhibition of tl folding of yeast rnap ii by α-amanitin resulted in a substantial decrease in the accuracy of ntp addition  <cit> .
in contrast, the recent study on e. coli rnap suggested that the open state of the active centre  may be a major checkpoint for discrimination against ncntps and c2'dntps  <cit> .
here, by using structure-based mutagenesis and fast kinetic analysis of phosphodiester bond formation by wild-type  and mutant t. aquaticus rnaps we uncover a stepwise mechanism of transcription fidelity, and dissect the role of tl in this process.
our results also reveal the role played by tl in the catalysis of phosphodiester bond formation.
RESULTS
experimental set up and kinetic analysis
in order to establish the roles of the open and closed states of the active centre in catalysis and fidelity, we have constructed rnap lacking tl   <cit>  and several rnaps bearing substitutions of tl amino acids that directly face/contact the cntp bound in the i+ <dig> site when tl is folded  <cit> .
these amino acids included: q <dig>  m <dig>  r <dig>  f <dig>  h <dig> and t <dig> .
compared to wt, f1241a and t1243a substitutions caused only minor, three- to fourfold defects in all reactions catalysed by the rnap active centre .
we considered these effects non-specific and did not investigate them further.
for the remaining mutants, we determined kpol  and kd  of single nucleotide incorporation and misincorporation reactions using artificially assembled elongation complexes  <cit>  .
the kinetic analysis of the data was performed as described in materials and methods and additional file 1: supplementary methods.
given that the rates of the reactions catalysed by rnap can be influenced by the translocation state of the elongation complex , we used elongation complexes in which the translocation state equilibriums were not significantly affected by rnap mutations .
we have previously shown that misincorporated ncnmps are efficiently removed from nascent rna through transcript-assisted hydrolysis of the second  phosphodiester bond of the transcript  <cit> .
therefore, in order to measure the rate of misincorporation we needed to monitor both the extension of the nascent rna with ncnmp and the subsequent transcript cleavage.
in order to achieve this goal, initial transcripts were labelled at the 3' end allowing us to simultaneously observe single-nucleotide addition upon misincorporation and the generation of dinucleotide through transcript-assisted proofreading .
note, for example, that, as seen from figure 2b, it would have been virtually impossible to compare misincorporation by r1239a and h1242a rnaps if only the rna extension is monitored.
although they have similar rates of misincorporation, these rnaps have very different rates of proofreading: the r1239a substitution does not influence intrinsic cleavage, while h1242a strongly diminishes it.
this result also suggests that tl plays an important role in intrinsic rna hydrolysis, and this is being investigated in a separate study.
active centre of Δtl rnap is stabilized in the open conformation
in order to investigate the role of tl in the active centre function, we constructed an rnap mutant that lacked the entire tl , Δtl rnap  <cit> .
in accordance with previous results  <cit> , Δtl rnap incorporated cntp four orders of magnitude slower than wt rnap , while the values of kd for cntp remained largely unchanged .
the same defect in cntp incorporation was observed for wt rnap inhibited by the antibiotic streptolydigin   <cit> , which locks the active centre in an open state by blocking the folding of tl  <cit> .
stl binds to but does not inhibit Δtl rnap, indicating that blocking of tl folding is the main pathway of stl action  <cit> .
taken together, these data indicate that the active centre of Δtl rnap permanently remains in an open state, where the enzyme can accept incoming ntps but cannot efficiently incorporate them into rna.
comparison of the properties of wt and Δtl rnaps gave us an opportunity to distinguish between tl-dependent and tl-independent functions and to reveal the role played by tl in catalysis and fidelity.
incorporation of cgtp was studied in ecg <dig>  misincorporation of ncgtp, c2'datp and c3'datp - in eca.
kd and kpol  for incorporation by wt rnap were obtained by fitting the kinetics data into a simple kinetic model as described in additional file 1:supplementary methods.
the rest of the data were fitted into the michaelis-menten equation as described in materials and methods.
errors are the standard error of data fitting.
the values were rounded to two significant digits.
incorporation of cgtp was studied in ecg <dig>  misincorporation of ncgtp - in eca.
kd and kpol  for incorporation by r1239a and h1242a rnaps were obtained by fitting the kinetics data into a simple kinetic model as described in additional file 1: supplementary methods.
kd and kpol for incorporation by other rnaps and misincorporation by all rnaps was obtained by fitting into the michaelis-menten equation as described in materials and methods.
errors are the standard error of data fitting.
the values were rounded to two significant digits.
fidelity of rnap active centre in the open state
in order to establish the role played by tl in discrimination against non-complementary substrates we analysed kinetic discrimination for all possible misincorporation events by Δtl and wt rnaps .
most misincorporation reactions by Δtl rnap proceeded extremely slowly even in the presence of  <dig> mm ncntp substrates, indicating that high  levels of discrimination are achieved against these misincorporation events in the open active centre.
although further improved by tl, discrimination against these events predominantly takes place in the open active centre .
for simplicity we refer to these misincorporation events as unntps .
some misincorporation events, however, were poorly discriminated against by Δtl rnap .
for simplicity, we refer to these misincorporation events as ncntps .
the presence of tl significantly  improved discrimination against ncntps.
the most pronounced tl-dependency was observed for discrimination against ncgtp misincorporation in position coding for catp .
we therefore focused on the examination of this misincorporation event as an example of tl-dependent discrimination, and refer to it as ncntp throughout the text.
Δtl
wt
~1200
~3000
~350
~750
kinetic discrimination  was quantified as a ratio of the rate of incorporation of  <dig> mm catp, cctp, cgtp and cutp ) to the rate of misincorporation of these  <dig> mm nucleotide triphosphates  in elongation complexes identified in the left column .
the data for Δtl rnap  are approximate due to very low efficiency of some of the misincorporation events by Δtl rnap.
the data for wt rnap  are approximate because incorporation was fitted using single exponent equation.
wt rnap incorporated ncntp three orders of magnitude slower than cntp.
the rate of misincorporation of ncntp by Δtl rnap was only slightly slower than incorporation of the cntp .
wt/stl rnap misincorporated ncntp at a rate similar to that of Δtl rnap .
given that the active centres of Δtl and wt/stl rnaps are stabilized in the open state, we conclude that the open state does not allow efficient discrimination based on the kinetics of incorporation of cntp versus ncntp misincorporation.
this important result was not influenced by the differences in the elongation complexes we used for incorporation and misincorporation analysis .
induced fit discrimination and active expulsion of ncntp from the active centre
as mentioned above, tl does not influence the affinity of the rnap active centre for cntp.
we measured kd  for wt and Δtl rnaps.
the affinity of Δtl  rnap for ncntp was ~ <dig> times lower than that for cntp .
the difference in kd  and kd  is probably due to improper hydrogen bonding with the template base in the case of ncntp and is in agreement with theoretical calculations of base pair formation between non-cognate nucleotide bases  <cit> .
unexpectedly, kd  for wt rnap was significantly higher than that for Δtl  or wt/stl  rnaps, indicating that ncntp bound in the i+ <dig> site and the folded tl compete with each other.
according to the structural data on t. thermophilus rnap and yeast rnap ii, cntp and folded tl are structurally complementary   <cit> .
the competition between folded tl and ncntp can, therefore, be explained by the absence of such complementarity, probably caused by an altered geometry of a non watson-crick  base pair and, as a result, by a steric collision between them.
given that the folded tl participates in phosphodiester bond formation , steric competition of ncntp bound in the i+ <dig> site with the folded tl leads to a diminished rate of phosphodiester bond synthesis - to a slow misincorporation.
no such competition takes place in the case of cntp bound in the active centre , which allows efficient tl folding and catalysis.
therefore, tl-dependent cntp incorporation proceeds much faster than tl-dependent ncntp misincorporation, leading to kinetic discrimination against ncntp.
the mainly kinetic  based selection of ncntps by rnap suggests that discrimination takes place via an induced fit mechanism  <cit> .
from our results it follows that:  tl is required for catalysis ; and  tl is sterically conflicting with the ncntp bound in the active centre .
this suggests that the conformational change  in the active centre required for incorporation of cntps is obstructed in the presence of ncntps, thus supporting the induced fit discrimination hypothesis.
tl-dependent induced-fit kinetic discrimination, therefore, is another step assuring fidelity of transcription.
m <dig> governs tl folding and expulsion of ncntp from the active centre
in order to further investigate the competition between folded tl and ncntp bound in the active centre, we analysed kd  for rnaps bearing single substitutions in tl.
from the mutant enzymes tested  only m1238a rnap had kd  that was significantly lower than the wt rnap value and close to that of Δtl rnap .
this suggests that m <dig> is required for ncntp expulsion from the active centre.
in the folded tl, m <dig> stacks on the base of cntp in the i+ <dig> site .
we hypothesized that m <dig> is required for stabilization of the tl in the folded conformation via interaction with the incoming substrate base and, thus, is responsible for the observed competition between folded tl and ncntp.
to test this notion, we analysed rnaps with substitutions of m <dig> to hydrophobic amino acids with larger than alanine side chains, m1238v and m1238l .
in support of our hypothesis, m1238v rnap had kd  =  <dig> ±  <dig> μm  while m1238l rnap had the same kd  =  <dig> ±  <dig> μm as wt rnap .
importantly, the ability to expel ncntp from the active centre by m1238a, m1238v, and m1238l rnaps correlated with catalytic activities of these enzymes: in cntp addition reaction, m1238v rnap was slightly faster than m1238a, while m1238l was as fast as wt rnap .
the correlation between catalytic activity and efficiency of competition of folded tl with ncntp bound in the active centre suggests that 'tl folding required for catalysis' and 'tl folding required for ncntp expulsion from the active centre' are one and the same process.
interestingly, in eukaryotic rnaps ii and iii, the residue equivalent to m <dig> of bacterial rnap is leucine.
taken together, our data suggest that m <dig> is required for stabilization of the folded state of the tl in the presence of correct substrate in the i+ <dig> site.
in the case of ncntp, we propose that this stabilization leads to a steric competition between bound ncntp and the folded tl and, as a result, to the removal of incorrect substrate from the active centre.
the removal of erroneous ntp from the active centre upon tl folding contributes substantially to overall transcription fidelity , thus being another step assuring accurate transcription.
discrimination,
kinetic discrimination was quantified as a ratio of the rate of incorporation of  <dig> mm cntp to the rate of misincorporation of  <dig> mm of a corresponding c2'dntp or c3'dntp in elongation complexes identified.
role of tl in catalysis
we further investigated the basis for the tl-dependent kinetic step of transcription fidelity.
in the crystal structure, r <dig> and h <dig> of the folded tl contact phosphates of the triphosphate moiety of cntp bound in the i+ <dig> site  <cit>  .
alanine substitutions of either r <dig> or h <dig> reduce the rate of cntp incorporation 50-100-fold .
the effects of these substitutions were cumulative: the double alanine substitution  led to a further decrease in the rate of cntp incorporation , which was approximately the multiplicative product of individual defects observed for r1239a and h1242a mutants.
this indicates that r <dig> and h <dig> act at the same kinetic step of catalysis.
furthermore, the rate of pyrophosphorolysis was also affected similarly by r1239a and h1242a substitutions , suggesting that r <dig> and h <dig> play functionally analogous roles during catalysis of phosphotransfer reactions.
in order to test if the decreased rate of cntp incorporation by mutant rnaps is caused by distortion of tl folding, we analysed the ability of r1239a/h1242a tl to displace ncntp from the active centre.
as can be seen from table  <dig>  r1239a/h1242a rnap has kd  similar to that of wt .
furthermore, r1239a substitution had no effect on intrinsic rna hydrolytic activity , which also requires tl folding  <cit> .
these data indicate that tl with r1239a/h1242a double substitution has retained its ability to fold and that the defect caused by this mutation is caused by deficiency in catalysis per se.
the magnitude of r1239a/h1242a substitution effect on phosphodiester bond formation indicates that these two amino acids are the main determinants of tl function in catalysis.
the similarity of the effects caused by individual r1239a and h1242a substitutions on phosphotransfer reactions disfavours the exclusive role of h <dig> in catalysis as a brønsted-lowry or lewis acid  <cit> .
nevertheless, we measured ph dependences of catalytic rates of wt, r1239a, h1242a and r1239a/h1242a rnaps.
we used  <dig> mm cntp to avoid effects of ph on kd .
the shapes of curves for all four rnaps were the same .
the descending limb that corresponds to protonation of the leaving group was not observed in either of the plots.
inflection at ph = ~ <dig> - <dig>  can be assigned to pka of deprotonation of rna 3' hydroxyl - that is, of a general base.
note that the substitution of either r <dig> or h <dig> shifts this pka towards a more basic value, though by less than one ph unit .
therefore, h <dig> and r <dig> participate in general base catalysis , but are likely do so as a part of a network of other groups of the active centre.
this view is supported by the fact that the slopes of log-log curves were less than  <dig> indicating that the inflection point does not reflect a single pka that is being titrated.
furthermore, substitution of r <dig> to asparagine  only slightly affected catalysis , although the side chains of these amino acids are of different chemical nature.
therefore, in agreement with crystallographic studies of t. thermophilus rnap elongation complex  <cit> , our results suggest that r <dig> and h <dig> coordinate the triphosphate moiety of cntp in the i+ <dig> site to allow efficient transition state formation , although they are also indirectly participating in general base catalysis.
we therefore propose that the kinetic induced-fit discrimination against ncntp is based on different efficiencies of transition state stabilization and/or reactants orientation and, in part, of base catalysis during cntp incorporation versus ncntp misincorporation.
discrimination against 2'-deoxyntp
incorporation of 2'-deoxyntps in rna may be detrimental to cells as 2' hydroxyls are frequently involved in rna ternary structure and function and may influence mrna translation.
we therefore investigated the mechanisms assuring discrimination against complementary 2'-deoxyntps  during transcription.
as can be seen from table  <dig>  Δtl rnap only poorly distinguished between c2'dntp and cntp, while discrimination by wt rnap was strong .
the observed poor kinetic discrimination by Δtl rnap was not due to differences in the elongation complexes used for cntp and c2'dntp incorporation .
low kinetic discrimination by Δtl rnap was observed for all c2'dntps .
the highest discrimination was achieved against cttp, which was incorporated only  <dig> times slower than cutp .
the result indicates that in the open state of the active centre, discrimination against c2'dntp is ineffective; in other words, discrimination against c2'dntp mainly depends on the presence of tl.
the kinetic discrimination against c2'dntps implies that the induced fit mechanism is involved  <cit> .
we tested if tl folding, that apparently determines induced fit discrimination, would result in expulsion of c2'dntp from the active centre - that is, if kd  of wt rnap would be higher than that of Δtl rnap.
as can be seen from table  <dig>  wt rnap indeed had ~10-fold higher kd  than Δtl rnap, indicating that tl competes with c2'dntp in the i+ <dig> site.
the competition between c2'dntp and tl leads to the inhibition of tl folding and, thus, of productive catalysis compared to cntp which does not compete with tl.
the result supports the idea that discrimination against c2'dntp proceeds via induced fit mechanism.
the result also suggests that, as in the case of ncntps, tl participates in the active removal of c2'dntp from the active centre.
it is likely that the absence of an interaction of the 2'oh with some determinant in the active centre makes c2'dntp adopt a conformation that results in competition with the folded tl and the expulsion of c2'dntp.
we tested two amino acids, β' n <dig> and q <dig>  that were earlier suggested to participate in the discrimination against c2'dntp based on crystallographic and biochemical data with bacterial rnaps and yeast rnap ii  <cit> .
the substitution n737a had a small effect on c2'dntp discrimination .
in contrast, the substitution q1235a led to a ~25-fold decrease in discrimination against c2'dntp .
the result suggests that q <dig> participates in recognition of the 2'oh of cntps, but does it along with some other determinants.
q <dig> may be partnered by β'r <dig> which, in the crystal structures of t. thermophilus rnap and yeast rnap ii elongation complexes, is close to the 2'oh of the substrate  <cit> .
the contributions of different steps in discrimination against c2'dntp are summarized in table  <dig> and are schematically shown in figure  <dig>
values were quantified using data of table 1: 'open active centre' values were generated from the row identifier ratios using the Δtl data; 'total' values were generated from the row identifier ratios using the wild-type  data; 'tl folding' values were generated from the row identifier ratios using the ratio of the wt ratio over the Δtl ratio.
discrimination against 3'-deoxyntp
incorporation of ntps bearing modifications of the 3' hydroxyl or lacking this group abolishes further transcription.
we therefore analysed the mechanism used by rnap to discriminate against complementary 3'-deoxyntps .
wt rnap incorporated c3'dntp ~ <dig> times slower than cntp .
in contrast, Δtl rnap practically failed to discriminate against c3'dntp , suggesting that discrimination strictly depends on the presence of tl.
the phenomenon was not due to the differences in elongation complexes or the identity of c3'dntp .
next, we measured kd  for wt and Δtl rnaps.
surprisingly, wt rnap had the same kd  as Δtl rnap , indicating that c3'dntp does not compete with, and is therefore not expelled by, the folded tl.
therefore, only kinetic tl-dependent discrimination is utilized against c3'dntps.
we investigated the mechanism of tl-dependent kinetic discrimination against c3'dntp.
based on crystallographic studies of yeast rnap ii elongation complexes, it was suggested that q <dig> of tl and β'n <dig> may act in concert to check for the presence of the 3' oh  <cit> .
we tested rnap bearing q1235a substitution for its ability to discriminate against c3'dntp.
as can be seen from figure 4a, q1235a rnap showed only ~fivefold discrimination against c3'dntp, compared to ~700-fold discrimination exhibited by wt rnap.
therefore, q <dig> of tl is the primary determinant of kinetic discrimination against c3'dntp.
in line with this idea, n737a rnap had a slight  decrease in c3'dntp discrimination which was, however, tl-dependent, because in the absence of tl, n737a substitution had no effect  .
we investigated the mechanism of q1235-dependent kinetic discrimination.
q1235a substitution slowed down cntp incorporation ~ <dig> fold as compared to wt .
however, this effect was not due to altered tl folding as inferred from kd  .
furthermore, the q1235a substitution did not significantly influence intrinsic transcript hydrolysis , which also requires tl folding  <cit> .
in order to test if q <dig> participates, along with r <dig> and h <dig>  in catalysis, we analysed q1235a/r1239a/h1242a triple mutant rnap.
curiously, while the single q1235a substitution had a significant effect on the rate of reaction, the triple mutant did not have any further defect in catalysis than the double r1239/h <dig> mutant .
the result suggests that q <dig> does not directly participate in catalysis along with r <dig> and h <dig> but is somehow linked to the function of these amino acids, possibly acting at the step preceding transition state stabilization by r1239/h <dig>  note also that q1235a and r1239a substitutions have similar effects on catalysis and, in the crystal structure of yeast rnap ii elongation complex, q <dig>  interacts with both n <dig>  and the 3'oh of the substrate  <cit> .
these data suggest that q <dig> may participate in orientation of r <dig> into a catalytically active conformation.
when the incoming ntp lacks the 3' oh, q <dig> fails to perform this role, leading to inefficient catalysis and inefficient kinetic discrimination against c3'dntps.
the contributions of different steps in discrimination against c3'dntp are summarized in table  <dig> and are also shown schematically in figure  <dig>
discussion
taken together, our results suggest that the fidelity of transcription is achieved via a stepwise process and that each step contributes differently to discrimination against non-complementary, 2'-deoxy, and 3'-deoxy erroneous substrates.
the scheme summarizing our findings is presented in figure  <dig>  these steps are based on two conformational states of the rnap active centre  and on a structural rearrangement of the active centre during transition between these two states.
the open active centre can efficiently discriminate only against unntps  but is error-prone in the case of ncntps, c2'dntp and c3'dntps.
similar conclusions were made during analysis of yeast rnap ii  <cit> .
that a 'motionless' active centre  is error-prone appears to be a general concept, as illustrated, for example, by primases, which have error rates similar to that of Δtl rnap  <cit> .
the poor fidelity achieved in the open state of the rnap active centre does not have a strong influence on the accuracy of transcription because this state is catalytically very inefficient.
folding of tl that makes the active centre catalytically efficient therefore determines the following steps that contribute to transcription fidelity.
our results indicate that the catalytic role of tl is primarily determined by r <dig> and h <dig>  which stabilize reaction transition state  and also indirectly contribute to general base catalysis.
thus, folding of tl, which is governed by m <dig>  is mainly required in order to bring r <dig> and h <dig> to the position from which they can accomplish this function.
the proposed steric competition between folded tl and ncntp or c2'dntp bound in i+ <dig> site makes catalysis with these incorrect nucleotides inefficient.
there is no such competition between folded tl and cntp.
therefore, though the rate of misincorporation is increased during tl folding , the increase of cntp incorporation rate is much higher, leading to kinetic discrimination against ncntps and c2'dntps.
interestingly, in the case of c3'dntp, tl is proposed to fold but fails to properly orient its catalytic residues in the absence of the functional relationship between q <dig> and the 3'oh.
the tl-controlled fidelity checkpoint is an example of an induced fit mechanism of enzymatic accuracy.
dnaps and single-subunit rnaps also use an induced fit mechanism to ensure fidelity.
in these enzymes, the binding of correct substrate induces a large conformational change converting the complex to a catalytically active, closed, conformation  <cit>  .
the principal finding of our study is that the apparent competition of the folded tl with ncntp and c2'dntp bound in the i+ <dig> site results in the erroneous substrates' displacement from the active centre, further improving the fidelity by another order of magnitude .
the contribution of this step to transcription fidelity may be more important for c2'dntps, because these nucleotides bind in the active site with the same affinity as cntps and, therefore, should compete with correct substrates much more efficiently than ncntps.
however, it should be noted that this step has no effect on the discrimination against c3'dntps.
misincorporation rates by t. aquaticus wt rnap measured in our study  were qualitatively similar to those observed for yeast wt rnap ii  <cit> , with two exceptions.
t. aquaticus wt rnap misincorporated ncgtp in place of catp and ncutp in place of cctp much faster  than did yeast wt rnap ii.
this suggests that discrimination against some non-cognate ntps may depend on the sequence of elongation complex or may vary between bacterial and eukaryotic rnaps.
curiously, results from a recent study on e. coli rnap contradict some of our conclusions  <cit> .
by studying e. coli Δtl rnap, the authors concluded that the open state of the active centre is able to efficiently discriminate against ncntp.
although our conclusions agree on some misincorporation events , there is a discrepancy in the case of ncgtp/catp misincorporation.
zhang et al.
<cit>  argue that they failed to observe meaningful misincorporation at  <dig> mm ncgtp, while t. aquaticus rnap misincorporated only six times slower than cntp .
the elongation complexes used in both studies were of the same sequence.
this factor can, therefore, be excluded when analysing the possible source of this discrepancy.
we have constructed an e. coli Δtl rnap with a tl deletion identical to the one used by zhang et al.
<cit>  and tested it for ncgtp misincorporation .
we observed efficient misincorporation even in  <dig> mm ncgtp by the mutant enzyme, which was ~ <dig> times slower than incorporation rate in the presence of  <dig> mm cgtp.
taking into account the higher kd  value, ~25-fold kinetic discrimination is expected in the open active centre, which is slightly higher than that observed for t. aquaticus Δtl rnap for the same misincorporation event.
we also observed higher kinetic discrimination against c2'dntp and c3'dntp by e. coli Δtl rnap compared to t. aquaticus Δtl rnap in the same elongation complexes .
better discrimination by open-state active centre of e. coli rnap is achieved due to the relatively faster tl-independent incorporation of cntp by e. coli Δtl rnap compared to t. aquaticus Δtl rnap .
this suggests that some amino acids of the e. coli rnap active centre that lie outside tl may support catalysis and, thus, contribute to kinetic discrimination against erroneous ntps in the open state of the active centre.
this idea is supported by the fact that alanine substitutions of e. coli homologues of r <dig> and h <dig> have a far less drastic effect on catalysis  than the corresponding t. aquaticus rnap substitutions .
therefore, e. coli possesses some additional catalytic mechanism that is lacking or diminished in t. aquaticus rnap.
our results indicate that, in addition to tl-dependent transition state stabilization, the active centre performs general base catalysis which only partly depends on tl.
it is therefore possible that e. coli rnap relies on tl-independent base catalysis more than t. aquaticus rnap and that this determines better kinetic discrimination in the open active centre.
the drastic catalytic effect of the tl deletion in e. coli rnap, however, indicates that this additional catalytic mechanism is still dependent on the tl, possibly through allosteric action of tl on amino acids of the active centre.
overall, our results suggest that the low accuracy of the open state of the active centre  is a general feature of multi-subunit rnaps, though some species-specific variations are possible.
according to our results, the total fidelity of transcription that can be achieved through steps dissected in our study is ~10- <dig> for ncntps, ~10- <dig> for c2'dntps and ~10- <dig> for c3'dntps .
at high ntp concentrations present in cells, accuracy is expected to drop by two orders of magnitude in the case of ncntps and one order of magnitude for c2'dntps, but to remain the same for c3'dntps.
however, the fidelity of transcription can be further improved by a factor of 10-1-10- <dig> during the proofreading of transcription via transcript-assisted and cleavage factor-assisted  removal of erroneous nucleotides  <cit> .
CONCLUSIONS
transcription is characterized by high fidelity of copying of genetic information into rna.
discrimination against non-cognate and 2'- and 3'-deoxy ntps entering the rnap active centre proceeds via a stepwise mechanism involving not only discrimination based on watson-crick pairing but also active recognition of substrates by the catalytic domain of the rnap active centre, the tl.
each step contributes differently to the overall fidelity against various erroneous substrates.
a unique property of the multi-subunit rnap active centre, compared to single-subunit rnaps and dnaps active centres, is the ability of the former to actively expel wrong substrates upon tl folding.
this property may be particularly important in the case of 2'-deoxy ntps, which are not discriminated during initial binding and, therefore, compete with the cognate rntps.
induced fit discrimination based on kinetics of incorporation versus misincorporation is determined by the direct involvement of tl in catalysis along with the two mg2+ ions of the rnap active centre.
interestingly, such combinatorial way of catalysis  indicates that the regulation of the activity of the enzyme can be accomplished through controlling tl folding, rather than the orientation of the catalytic mg2+ ions.
these effects were observed during analysis of rnaps with mutations in non-catalytic amino acids surrounding the active site  <cit> .
regulation of catalysis via tl may also be accomplished by external factors  <cit> .
'combinatorial' catalysis also suggests that rnaps of different organisms can adapt to various conditions by changing the amino acid composition of tl and surrounding amino acids, while leaving the two mg2+ binding sites virtually intact  <cit> .
for example, changes in the f-loop, a domain close to tl, determine the temperature optima of catalysis by closely related rnaps from thermophilic thermus aquaticus and mesophilic deinococcus radiodurans  <cit> .

 all reactions performed by rna polymerase are catalysed by a single active centre and are proposed to proceed through a general two mg2+ ion mechanism. structural studies revealed that conformational changes in the active centre might be required for efficient catalysis and biochemical studies are consistent with such a model. the major conformational change involves folding of the trigger loop that brings the rnap active centre from'open' to'closed' conformation. though crystal structures of rnaps with folded tl were obtained only in the presence of cognate ntp bound in the i+ site of the active centre, tl folding is also thought to be required for efficient catalysis of pyrophosphorolysis and phosphodiester bond hydrolysis. although, in the absence of tl, the rate of phosphodiester bond formation is decreased by four orders of magnitude, the actual mechanism by which tl participates in rnap activity remains unknown. crystallographic studies revealed that folded tl tightly interacts with cntp base and triphosphate moieties. this observation was used to build three distinct models explaining the role of tl folding in phosphodiester bond formation. in the first model, based on a crystal structure of yeast rnap ii, folded tl was proposed to directly participate in catalysis through an invariant histidine residue that withdraws electron density from the β-phosphate of the incoming ntp thus activating nucleophilic attack on the α-phosphorus. according to the second model h participates in acid catalysis as a proton donor for the leaving pyrophosphate. this model is based on studies of single-subunit rnaps that are evolutionary unrelated to multi-subunit rnaps and may, therefore, use a different mechanism for catalysis. in the third model, based on a crystal structure of t. thermophilus rnap, folding of tl was proposed in order to stabilize, via r and h residues, the triphosphate moiety of the i+ site-bound ntp in the active conformation. a recent study of escherichia coli rnap revealed that mutation of h had only minor effect on catalysis, indicating that acid/base catalysis is not its primary role. however, the relatively mild effects of substitutions of amino acids that, according to crystallographic data, interact with the triphosphate moiety of incoming ntp observed in this work are also inconsistent with the transition state stabilisation hypothesis. the small effects of single substitutions, compared to dramatic catalytic defect caused by deletion of the entire tl, suggest that tl may have an allosteric effect on other amino acids that are also required for catalysis. mutations in tl and surrounding amino acids were found to alter discrimination against non-complementary ntp and complementary 2'- and 3'-deoxy ntps. these observations led to suggestions that tl could participate in substrate discrimination by either: selectively facilitating incorporation of correct substrates; or by providing sufficient time for accurate ntp selection prior to the act of catalysis. by analogy with dnaps and ssrnaps, the existence of a conformational change in the active centre of multi-subunit rnaps that brings the active centre to a catalytically active conformation suggests that substrate discrimination may take place via differential efficiency of conformational change occurrence upon the binding of cntps versus ncntps. in support of this idea, inhibition of tl folding of yeast rnap ii by α-amanitin resulted in a substantial decrease in the accuracy of ntp addition. in contrast, the recent study on e. coli rnap suggested that the open state of the active centre may be a major checkpoint for discrimination against ncntps and c2'dntps. here, by using structure-based mutagenesis and fast kinetic analysis of phosphodiester bond formation by wild-type and mutant t. aquaticus rnaps we uncover a stepwise mechanism of transcription fidelity, and dissect the role of tl in this process. our results also reveal the role played by tl in the catalysis of phosphodiester bond formation.  experimental set up and kinetic analysis in order to establish the roles of the open and closed states of the active centre in catalysis and fidelity, we have constructed rnap lacking tl and several rnaps bearing substitutions of tl amino acids that directly face/contact the cntp bound in the i+ site when tl is folded. these amino acids included: q m r f h and t. compared to wt, f1241a and t1243a substitutions caused only minor, three- to fourfold defects in all reactions catalysed by the rnap active centre. we considered these effects non-specific and did not investigate them further. for the remaining mutants, we determined kpol and kd of single nucleotide incorporation and misincorporation reactions using artificially assembled elongation complexes. the kinetic analysis of the data was performed as described in materials and methods and additional file 1: supplementary methods. given that the rates of the reactions catalysed by rnap can be influenced by the translocation state of the elongation complex, we used elongation complexes in which the translocation state equilibriums were not significantly affected by rnap mutations. we have previously shown that misincorporated ncnmps are efficiently removed from nascent rna through transcript-assisted hydrolysis of the second phosphodiester bond of the transcript. therefore, in order to measure the rate of misincorporation we needed to monitor both the extension of the nascent rna with ncnmp and the subsequent transcript cleavage. in order to achieve this goal, initial transcripts were labelled at the 3' end allowing us to simultaneously observe single-nucleotide addition upon misincorporation and the generation of dinucleotide through transcript-assisted proofreading. note, for example, that, as seen from figure 2b, it would have been virtually impossible to compare misincorporation by r1239a and h1242a rnaps if only the rna extension is monitored. although they have similar rates of misincorporation, these rnaps have very different rates of proofreading: the r1239a substitution does not influence intrinsic cleavage, while h1242a strongly diminishes it. this result also suggests that tl plays an important role in intrinsic rna hydrolysis, and this is being investigated in a separate study. active centre of Δtl rnap is stabilized in the open conformation in order to investigate the role of tl in the active centre function, we constructed an rnap mutant that lacked the entire tl, Δtl rnap. in accordance with previous results, Δtl rnap incorporated cntp four orders of magnitude slower than wt rnap, while the values of kd for cntp remained largely unchanged. the same defect in cntp incorporation was observed for wt rnap inhibited by the antibiotic streptolydigin, which locks the active centre in an open state by blocking the folding of tl. stl binds to but does not inhibit Δtl rnap, indicating that blocking of tl folding is the main pathway of stl action. taken together, these data indicate that the active centre of Δtl rnap permanently remains in an open state, where the enzyme can accept incoming ntps but cannot efficiently incorporate them into rna. comparison of the properties of wt and Δtl rnaps gave us an opportunity to distinguish between tl-dependent and tl-independent functions and to reveal the role played by tl in catalysis and fidelity. incorporation of cgtp was studied in ecg misincorporation of ncgtp, c2'datp and c3'datp - in eca. kd and kpol for incorporation by wt rnap were obtained by fitting the kinetics data into a simple kinetic model as described in additional file 1:supplementary methods. the rest of the data were fitted into the michaelis-menten equation as described in materials and methods. errors are the standard error of data fitting. the values were rounded to two significant digits. incorporation of cgtp was studied in ecg misincorporation of ncgtp - in eca. kd and kpol for incorporation by r1239a and h1242a rnaps were obtained by fitting the kinetics data into a simple kinetic model as described in additional file 1: supplementary methods. kd and kpol for incorporation by other rnaps and misincorporation by all rnaps was obtained by fitting into the michaelis-menten equation as described in materials and methods. errors are the standard error of data fitting. the values were rounded to two significant digits. fidelity of rnap active centre in the open state in order to establish the role played by tl in discrimination against non-complementary substrates we analysed kinetic discrimination for all possible misincorporation events by Δtl and wt rnaps. most misincorporation reactions by Δtl rnap proceeded extremely slowly even in the presence of mm ncntp substrates, indicating that high levels of discrimination are achieved against these misincorporation events in the open active centre. although further improved by tl, discrimination against these events predominantly takes place in the open active centre. for simplicity we refer to these misincorporation events as unntps. some misincorporation events, however, were poorly discriminated against by Δtl rnap. for simplicity, we refer to these misincorporation events as ncntps. the presence of tl significantly improved discrimination against ncntps. the most pronounced tl-dependency was observed for discrimination against ncgtp misincorporation in position coding for catp. we therefore focused on the examination of this misincorporation event as an example of tl-dependent discrimination, and refer to it as ncntp throughout the text. Δtl wt ~1200 ~3000 ~350 ~750 kinetic discrimination was quantified as a ratio of the rate of incorporation of mm catp, cctp, cgtp and cutp ) to the rate of misincorporation of these mm nucleotide triphosphates in elongation complexes identified in the left column. the data for Δtl rnap are approximate due to very low efficiency of some of the misincorporation events by Δtl rnap. the data for wt rnap are approximate because incorporation was fitted using single exponent equation. wt rnap incorporated ncntp three orders of magnitude slower than cntp. the rate of misincorporation of ncntp by Δtl rnap was only slightly slower than incorporation of the cntp. wt/stl rnap misincorporated ncntp at a rate similar to that of Δtl rnap. given that the active centres of Δtl and wt/stl rnaps are stabilized in the open state, we conclude that the open state does not allow efficient discrimination based on the kinetics of incorporation of cntp versus ncntp misincorporation. this important result was not influenced by the differences in the elongation complexes we used for incorporation and misincorporation analysis. induced fit discrimination and active expulsion of ncntp from the active centre as mentioned above, tl does not influence the affinity of the rnap active centre for cntp. we measured kd for wt and Δtl rnaps. the affinity of Δtl rnap for ncntp was ~ times lower than that for cntp. the difference in kd and kd is probably due to improper hydrogen bonding with the template base in the case of ncntp and is in agreement with theoretical calculations of base pair formation between non-cognate nucleotide bases. unexpectedly, kd for wt rnap was significantly higher than that for Δtl or wt/stl rnaps, indicating that ncntp bound in the i+ site and the folded tl compete with each other. according to the structural data on t. thermophilus rnap and yeast rnap ii, cntp and folded tl are structurally complementary. the competition between folded tl and ncntp can, therefore, be explained by the absence of such complementarity, probably caused by an altered geometry of a non watson-crick base pair and, as a result, by a steric collision between them. given that the folded tl participates in phosphodiester bond formation, steric competition of ncntp bound in the i+ site with the folded tl leads to a diminished rate of phosphodiester bond synthesis - to a slow misincorporation. no such competition takes place in the case of cntp bound in the active centre, which allows efficient tl folding and catalysis. therefore, tl-dependent cntp incorporation proceeds much faster than tl-dependent ncntp misincorporation, leading to kinetic discrimination against ncntp. the mainly kinetic based selection of ncntps by rnap suggests that discrimination takes place via an induced fit mechanism. from our results it follows that: tl is required for catalysis; and tl is sterically conflicting with the ncntp bound in the active centre. this suggests that the conformational change in the active centre required for incorporation of cntps is obstructed in the presence of ncntps, thus supporting the induced fit discrimination hypothesis. tl-dependent induced-fit kinetic discrimination, therefore, is another step assuring fidelity of transcription. m governs tl folding and expulsion of ncntp from the active centre in order to further investigate the competition between folded tl and ncntp bound in the active centre, we analysed kd for rnaps bearing single substitutions in tl. from the mutant enzymes tested only m1238a rnap had kd that was significantly lower than the wt rnap value and close to that of Δtl rnap. this suggests that m is required for ncntp expulsion from the active centre. in the folded tl, m stacks on the base of cntp in the i+ site. we hypothesized that m is required for stabilization of the tl in the folded conformation via interaction with the incoming substrate base and, thus, is responsible for the observed competition between folded tl and ncntp. to test this notion, we analysed rnaps with substitutions of m to hydrophobic amino acids with larger than alanine side chains, m1238v and m1238l. in support of our hypothesis, m1238v rnap had kd = ± μm while m1238l rnap had the same kd = ± μm as wt rnap. importantly, the ability to expel ncntp from the active centre by m1238a, m1238v, and m1238l rnaps correlated with catalytic activities of these enzymes: in cntp addition reaction, m1238v rnap was slightly faster than m1238a, while m1238l was as fast as wt rnap. the correlation between catalytic activity and efficiency of competition of folded tl with ncntp bound in the active centre suggests that'tl folding required for catalysis' and'tl folding required for ncntp expulsion from the active centre' are one and the same process. interestingly, in eukaryotic rnaps ii and iii, the residue equivalent to m of bacterial rnap is leucine. taken together, our data suggest that m is required for stabilization of the folded state of the tl in the presence of correct substrate in the i+ site. in the case of ncntp, we propose that this stabilization leads to a steric competition between bound ncntp and the folded tl and, as a result, to the removal of incorrect substrate from the active centre. the removal of erroneous ntp from the active centre upon tl folding contributes substantially to overall transcription fidelity, thus being another step assuring accurate transcription. discrimination, kinetic discrimination was quantified as a ratio of the rate of incorporation of mm cntp to the rate of misincorporation of mm of a corresponding c2'dntp or c3'dntp in elongation complexes identified. role of tl in catalysis we further investigated the basis for the tl-dependent kinetic step of transcription fidelity. in the crystal structure, r and h of the folded tl contact phosphates of the triphosphate moiety of cntp bound in the i+ site. alanine substitutions of either r or h reduce the rate of cntp incorporation 50-100-fold. the effects of these substitutions were cumulative: the double alanine substitution led to a further decrease in the rate of cntp incorporation, which was approximately the multiplicative product of individual defects observed for r1239a and h1242a mutants. this indicates that r and h act at the same kinetic step of catalysis. furthermore, the rate of pyrophosphorolysis was also affected similarly by r1239a and h1242a substitutions, suggesting that r and h play functionally analogous roles during catalysis of phosphotransfer reactions. in order to test if the decreased rate of cntp incorporation by mutant rnaps is caused by distortion of tl folding, we analysed the ability of r1239a/h1242a tl to displace ncntp from the active centre. as can be seen from table r1239a/h1242a rnap has kd similar to that of wt. furthermore, r1239a substitution had no effect on intrinsic rna hydrolytic activity, which also requires tl folding. these data indicate that tl with r1239a/h1242a double substitution has retained its ability to fold and that the defect caused by this mutation is caused by deficiency in catalysis per se. the magnitude of r1239a/h1242a substitution effect on phosphodiester bond formation indicates that these two amino acids are the main determinants of tl function in catalysis. the similarity of the effects caused by individual r1239a and h1242a substitutions on phosphotransfer reactions disfavours the exclusive role of h in catalysis as a brønsted-lowry or lewis acid. nevertheless, we measured ph dependences of catalytic rates of wt, r1239a, h1242a and r1239a/h1242a rnaps. we used mm cntp to avoid effects of ph on kd. the shapes of curves for all four rnaps were the same. the descending limb that corresponds to protonation of the leaving group was not observed in either of the plots. inflection at ph = ~ - can be assigned to pka of deprotonation of rna 3' hydroxyl - that is, of a general base. note that the substitution of either r or h shifts this pka towards a more basic value, though by less than one ph unit. therefore, h and r participate in general base catalysis, but are likely do so as a part of a network of other groups of the active centre. this view is supported by the fact that the slopes of log-log curves were less than indicating that the inflection point does not reflect a single pka that is being titrated. furthermore, substitution of r to asparagine only slightly affected catalysis, although the side chains of these amino acids are of different chemical nature. therefore, in agreement with crystallographic studies of t. thermophilus rnap elongation complex, our results suggest that r and h coordinate the triphosphate moiety of cntp in the i+ site to allow efficient transition state formation, although they are also indirectly participating in general base catalysis. we therefore propose that the kinetic induced-fit discrimination against ncntp is based on different efficiencies of transition state stabilization and/or reactants orientation and, in part, of base catalysis during cntp incorporation versus ncntp misincorporation. discrimination against 2'-deoxyntp incorporation of 2'-deoxyntps in rna may be detrimental to cells as 2' hydroxyls are frequently involved in rna ternary structure and function and may influence mrna translation. we therefore investigated the mechanisms assuring discrimination against complementary 2'-deoxyntps during transcription. as can be seen from table Δtl rnap only poorly distinguished between c2'dntp and cntp, while discrimination by wt rnap was strong. the observed poor kinetic discrimination by Δtl rnap was not due to differences in the elongation complexes used for cntp and c2'dntp incorporation. low kinetic discrimination by Δtl rnap was observed for all c2'dntps. the highest discrimination was achieved against cttp, which was incorporated only times slower than cutp. the result indicates that in the open state of the active centre, discrimination against c2'dntp is ineffective; in other words, discrimination against c2'dntp mainly depends on the presence of tl. the kinetic discrimination against c2'dntps implies that the induced fit mechanism is involved. we tested if tl folding, that apparently determines induced fit discrimination, would result in expulsion of c2'dntp from the active centre - that is, if kd of wt rnap would be higher than that of Δtl rnap. as can be seen from table wt rnap indeed had ~10-fold higher kd than Δtl rnap, indicating that tl competes with c2'dntp in the i+ site. the competition between c2'dntp and tl leads to the inhibition of tl folding and, thus, of productive catalysis compared to cntp which does not compete with tl. the result supports the idea that discrimination against c2'dntp proceeds via induced fit mechanism. the result also suggests that, as in the case of ncntps, tl participates in the active removal of c2'dntp from the active centre. it is likely that the absence of an interaction of the 2'oh with some determinant in the active centre makes c2'dntp adopt a conformation that results in competition with the folded tl and the expulsion of c2'dntp. we tested two amino acids, β' n and q that were earlier suggested to participate in the discrimination against c2'dntp based on crystallographic and biochemical data with bacterial rnaps and yeast rnap ii. the substitution n737a had a small effect on c2'dntp discrimination. in contrast, the substitution q1235a led to a ~25-fold decrease in discrimination against c2'dntp. the result suggests that q participates in recognition of the 2'oh of cntps, but does it along with some other determinants. q may be partnered by β'r which, in the crystal structures of t. thermophilus rnap and yeast rnap ii elongation complexes, is close to the 2'oh of the substrate. the contributions of different steps in discrimination against c2'dntp are summarized in table and are schematically shown in figure values were quantified using data of table 1:'open active centre' values were generated from the row identifier ratios using the Δtl data;'total' values were generated from the row identifier ratios using the wild-type data;'tl folding' values were generated from the row identifier ratios using the ratio of the wt ratio over the Δtl ratio. discrimination against 3'-deoxyntp incorporation of ntps bearing modifications of the 3' hydroxyl or lacking this group abolishes further transcription. we therefore analysed the mechanism used by rnap to discriminate against complementary 3'-deoxyntps. wt rnap incorporated c3'dntp ~ times slower than cntp. in contrast, Δtl rnap practically failed to discriminate against c3'dntp, suggesting that discrimination strictly depends on the presence of tl. the phenomenon was not due to the differences in elongation complexes or the identity of c3'dntp. next, we measured kd for wt and Δtl rnaps. surprisingly, wt rnap had the same kd as Δtl rnap, indicating that c3'dntp does not compete with, and is therefore not expelled by, the folded tl. therefore, only kinetic tl-dependent discrimination is utilized against c3'dntps. we investigated the mechanism of tl-dependent kinetic discrimination against c3'dntp. based on crystallographic studies of yeast rnap ii elongation complexes, it was suggested that q of tl and β'n may act in concert to check for the presence of the 3' oh. we tested rnap bearing q1235a substitution for its ability to discriminate against c3'dntp. as can be seen from figure 4a, q1235a rnap showed only ~fivefold discrimination against c3'dntp, compared to ~700-fold discrimination exhibited by wt rnap. therefore, q of tl is the primary determinant of kinetic discrimination against c3'dntp. in line with this idea, n737a rnap had a slight decrease in c3'dntp discrimination which was, however, tl-dependent, because in the absence of tl, n737a substitution had no effect. we investigated the mechanism of q1235-dependent kinetic discrimination. q1235a substitution slowed down cntp incorporation ~ fold as compared to wt. however, this effect was not due to altered tl folding as inferred from kd. furthermore, the q1235a substitution did not significantly influence intrinsic transcript hydrolysis, which also requires tl folding. in order to test if q participates, along with r and h in catalysis, we analysed q1235a/r1239a/h1242a triple mutant rnap. curiously, while the single q1235a substitution had a significant effect on the rate of reaction, the triple mutant did not have any further defect in catalysis than the double r1239/h mutant. the result suggests that q does not directly participate in catalysis along with r and h but is somehow linked to the function of these amino acids, possibly acting at the step preceding transition state stabilization by r1239/h note also that q1235a and r1239a substitutions have similar effects on catalysis and, in the crystal structure of yeast rnap ii elongation complex, q interacts with both n and the 3'oh of the substrate. these data suggest that q may participate in orientation of r into a catalytically active conformation. when the incoming ntp lacks the 3' oh, q fails to perform this role, leading to inefficient catalysis and inefficient kinetic discrimination against c3'dntps. the contributions of different steps in discrimination against c3'dntp are summarized in table and are also shown schematically in figure discussion taken together, our results suggest that the fidelity of transcription is achieved via a stepwise process and that each step contributes differently to discrimination against non-complementary, 2'-deoxy, and 3'-deoxy erroneous substrates. the scheme summarizing our findings is presented in figure these steps are based on two conformational states of the rnap active centre and on a structural rearrangement of the active centre during transition between these two states. the open active centre can efficiently discriminate only against unntps but is error-prone in the case of ncntps, c2'dntp and c3'dntps. similar conclusions were made during analysis of yeast rnap ii. that a'motionless' active centre is error-prone appears to be a general concept, as illustrated, for example, by primases, which have error rates similar to that of Δtl rnap. the poor fidelity achieved in the open state of the rnap active centre does not have a strong influence on the accuracy of transcription because this state is catalytically very inefficient. folding of tl that makes the active centre catalytically efficient therefore determines the following steps that contribute to transcription fidelity. our results indicate that the catalytic role of tl is primarily determined by r and h which stabilize reaction transition state and also indirectly contribute to general base catalysis. thus, folding of tl, which is governed by m is mainly required in order to bring r and h to the position from which they can accomplish this function. the proposed steric competition between folded tl and ncntp or c2'dntp bound in i+ site makes catalysis with these incorrect nucleotides inefficient. there is no such competition between folded tl and cntp. therefore, though the rate of misincorporation is increased during tl folding, the increase of cntp incorporation rate is much higher, leading to kinetic discrimination against ncntps and c2'dntps. interestingly, in the case of c3'dntp, tl is proposed to fold but fails to properly orient its catalytic residues in the absence of the functional relationship between q and the 3'oh. the tl-controlled fidelity checkpoint is an example of an induced fit mechanism of enzymatic accuracy. dnaps and single-subunit rnaps also use an induced fit mechanism to ensure fidelity. in these enzymes, the binding of correct substrate induces a large conformational change converting the complex to a catalytically active, closed, conformation. the principal finding of our study is that the apparent competition of the folded tl with ncntp and c2'dntp bound in the i+ site results in the erroneous substrates' displacement from the active centre, further improving the fidelity by another order of magnitude. the contribution of this step to transcription fidelity may be more important for c2'dntps, because these nucleotides bind in the active site with the same affinity as cntps and, therefore, should compete with correct substrates much more efficiently than ncntps. however, it should be noted that this step has no effect on the discrimination against c3'dntps. misincorporation rates by t. aquaticus wt rnap measured in our study were qualitatively similar to those observed for yeast wt rnap ii, with two exceptions. t. aquaticus wt rnap misincorporated ncgtp in place of catp and ncutp in place of cctp much faster than did yeast wt rnap ii. this suggests that discrimination against some non-cognate ntps may depend on the sequence of elongation complex or may vary between bacterial and eukaryotic rnaps. curiously, results from a recent study on e. coli rnap contradict some of our conclusions. by studying e. coli Δtl rnap, the authors concluded that the open state of the active centre is able to efficiently discriminate against ncntp. although our conclusions agree on some misincorporation events, there is a discrepancy in the case of ncgtp/catp misincorporation. zhang et al. argue that they failed to observe meaningful misincorporation at mm ncgtp, while t. aquaticus rnap misincorporated only six times slower than cntp. the elongation complexes used in both studies were of the same sequence. this factor can, therefore, be excluded when analysing the possible source of this discrepancy. we have constructed an e. coli Δtl rnap with a tl deletion identical to the one used by zhang et al. and tested it for ncgtp misincorporation. we observed efficient misincorporation even in mm ncgtp by the mutant enzyme, which was ~ times slower than incorporation rate in the presence of mm cgtp. taking into account the higher kd value, ~25-fold kinetic discrimination is expected in the open active centre, which is slightly higher than that observed for t. aquaticus Δtl rnap for the same misincorporation event. we also observed higher kinetic discrimination against c2'dntp and c3'dntp by e. coli Δtl rnap compared to t. aquaticus Δtl rnap in the same elongation complexes. better discrimination by open-state active centre of e. coli rnap is achieved due to the relatively faster tl-independent incorporation of cntp by e. coli Δtl rnap compared to t. aquaticus Δtl rnap. this suggests that some amino acids of the e. coli rnap active centre that lie outside tl may support catalysis and, thus, contribute to kinetic discrimination against erroneous ntps in the open state of the active centre. this idea is supported by the fact that alanine substitutions of e. coli homologues of r and h have a far less drastic effect on catalysis than the corresponding t. aquaticus rnap substitutions. therefore, e. coli possesses some additional catalytic mechanism that is lacking or diminished in t. aquaticus rnap. our results indicate that, in addition to tl-dependent transition state stabilization, the active centre performs general base catalysis which only partly depends on tl. it is therefore possible that e. coli rnap relies on tl-independent base catalysis more than t. aquaticus rnap and that this determines better kinetic discrimination in the open active centre. the drastic catalytic effect of the tl deletion in e. coli rnap, however, indicates that this additional catalytic mechanism is still dependent on the tl, possibly through allosteric action of tl on amino acids of the active centre. overall, our results suggest that the low accuracy of the open state of the active centre is a general feature of multi-subunit rnaps, though some species-specific variations are possible. according to our results, the total fidelity of transcription that can be achieved through steps dissected in our study is ~10- for ncntps, ~10- for c2'dntps and ~10- for c3'dntps. at high ntp concentrations present in cells, accuracy is expected to drop by two orders of magnitude in the case of ncntps and one order of magnitude for c2'dntps, but to remain the same for c3'dntps. however, the fidelity of transcription can be further improved by a factor of 10-1-10- during the proofreading of transcription via transcript-assisted and cleavage factor-assisted removal of erroneous nucleotides.  transcription is characterized by high fidelity of copying of genetic information into rna. discrimination against non-cognate and 2'- and 3'-deoxy ntps entering the rnap active centre proceeds via a stepwise mechanism involving not only discrimination based on watson-crick pairing but also active recognition of substrates by the catalytic domain of the rnap active centre, the tl. each step contributes differently to the overall fidelity against various erroneous substrates. a unique property of the multi-subunit rnap active centre, compared to single-subunit rnaps and dnaps active centres, is the ability of the former to actively expel wrong substrates upon tl folding. this property may be particularly important in the case of 2'-deoxy ntps, which are not discriminated during initial binding and, therefore, compete with the cognate rntps. induced fit discrimination based on kinetics of incorporation versus misincorporation is determined by the direct involvement of tl in catalysis along with the two mg2+ ions of the rnap active centre. interestingly, such combinatorial way of catalysis indicates that the regulation of the activity of the enzyme can be accomplished through controlling tl folding, rather than the orientation of the catalytic mg2+ ions. these effects were observed during analysis of rnaps with mutations in non-catalytic amino acids surrounding the active site. regulation of catalysis via tl may also be accomplished by external factors.'combinatorial' catalysis also suggests that rnaps of different organisms can adapt to various conditions by changing the amino acid composition of tl and surrounding amino acids, while leaving the two mg2+ binding sites virtually intact. for example, changes in the f-loop, a domain close to tl, determine the temperature optima of catalysis by closely related rnaps from thermophilic thermus aquaticus and mesophilic deinococcus radiodurans. 
a flexible domain of the rna polymerase active centre, the trigger loop, was shown to play an important role in this process, but the mechanisms of this participation remained elusive. during transcription, the rna polymerase active centre discriminates against not just non-complementary ribo ntp substrates but also against complementary 2'- and 3'-deoxy ntps. during the second step, non-complementary and 2'-deoxy ntps are discriminated against based on differences in reaction transition state stabilization and partly in general base catalysis, for correct versus non-correct substrates. we demonstrate that fidelity of transcription by multi-subunit rna polymerases is achieved through a stepwise process. the initial binding in the active centre is the major discrimination step for some non-complementary substrates, although for the rest of misincorporation events discrimination at this step is very poor. in the following step, non-complementary and 2'-deoxy ntps are actively removed from the active centre through a rearrangement of the trigger loop. we show that individual steps contribute differently to discrimination against various erroneous substrates. here we show that transcription fidelity is achieved through a multi-step process. this step is determined by two residues of the trigger loop that participate in catalysis. the only step of discrimination against 3'-deoxy substrates, distinct from the ones above, is based on failure to orient the trigger loop catalytic residues in the absence of 3'oh. transcription is the first step of gene expression and is characterized by a high fidelity of rna synthesis. we define the mechanisms and contributions of each of these steps to the overall fidelity of transcription.
 transcription is the first step of gene expression and is characterized by a high fidelity of rna synthesis. during transcription, the rna polymerase active centre discriminates against not just non-complementary ribo ntp substrates but also against complementary 2'- and 3'-deoxy ntps. a flexible domain of the rna polymerase active centre, the trigger loop, was shown to play an important role in this process, but the mechanisms of this participation remained elusive.  here we show that transcription fidelity is achieved through a multi-step process. the initial binding in the active centre is the major discrimination step for some non-complementary substrates, although for the rest of misincorporation events discrimination at this step is very poor. during the second step, non-complementary and 2'-deoxy ntps are discriminated against based on differences in reaction transition state stabilization and partly in general base catalysis, for correct versus non-correct substrates. this step is determined by two residues of the trigger loop that participate in catalysis. in the following step, non-complementary and 2'-deoxy ntps are actively removed from the active centre through a rearrangement of the trigger loop. the only step of discrimination against 3'-deoxy substrates, distinct from the ones above, is based on failure to orient the trigger loop catalytic residues in the absence of 3'oh.  we demonstrate that fidelity of transcription by multi-subunit rna polymerases is achieved through a stepwise process. we show that individual steps contribute differently to discrimination against various erroneous substrates. we define the mechanisms and contributions of each of these steps to the overall fidelity of transcription. 