From the Waksman Institute and Department of
Genetics, State University of New Jersey, Rutgers, Piscataway, New
Jersey 08854, the § Public Health Research Institute, New
York, New York 10016, and ¶ The Rockefeller University, New York,
New York 10021
Received for publication, December 7, 2000, and in revised form, January 16, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Using a modification of a highly selective
affinity labeling protocol, we demonstrated that the
DNA-dependent RNA polymerase
(RNAP)1 is a multisubunit,
multifunctional molecular machine. RNAP from Escherichia
coli (subunit composition Because the biochemical functions of the enzyme are lost upon the
separation of RNAP subunits (3), the assignment of partial functions to
a particular subunit or subunit segment is done indirectly by a
combination of genetic and biochemical approaches. To directly establish the roles of different subunits in transcription, new approaches need to be developed. In this report, we used a modification of the highly selective affinity labeling technique (4) to study the
ability of RNAP subassemblies to interact with the transcription initiation substrate and the transcription initiation inhibitor Rif. We
present evidence that the purine-specific initiation site of RNAP (5,
6) is essentially complete in the physiological Preparation of RNA Polymerase Subunits and RNAP
Reconstitution--
RNAP subunits were overexpressed and purified as
described elsewhere (9). RNAP reconstitution was performed essentially as described by Borukhov and Goldfarb (10). Inclusion bodies containing
overexpressed Coupled Affinity Labeling and RNA Polymerase
Reconstitution--
The synthesis of the affinity labeling reagents,
AMP- and ATP-based aldehydes and derivatized Rif-A, was as
described (4, 11). 10 µl of individually renatured RNAP subunits or
subunit combinations were incubated for 15-min at 30 °C in
renaturation buffer (10) in the presence of varying concentrations of
cross-linking reagent followed by the addition of 10 mM
borohydride and an additional 15 min incubation at 37 °C. Missing
RNAP subunits were then added, and reactions (total volume 25 µl)
were incubated for an additional 30 min at 30 °C. Reactions were
supplemented with 0.5 µg of recombinant In Vitro Transcription--
Coupled RNAP reconstitution
and affinity labeling were performed as above. Reactions were
supplemented with NTPs (500 µM ATP, 50 µM
CTP and GTP, 2.5 µM CNBr Cleavage--
Coupled RNAP reconstitution and
affinity labeling reactions and product separation were performed as
above, and affinity labeled Rationale--
Available cross-linking and mutational data
indicate that the RNAP
The original selective affinity labeling protocol consists of two
steps. In the first step, the open RNAP-promoter complex is formed and
then RNAP is cross-linked to a derivatized, cross-linkable initiating
nucleotide as specified by the +1 position of the template. In the
second step, the cross-linked nucleotide is extended ("developed") in a template-dependent manner with radiolabeled nucleoside
triphosphate specified by the +2 position of the template. As a result,
a radioactive dinucleotide is covalently attached to the RNAP
subunit(s), which can then be visualized by autoradiography after
SDS-PAGE.
The purine-specific initiation site (i-site) is present even
in the free RNAP core (5, 6), so it should be possible to carry out the
first step of the affinity labeling protocol in the absence of promoter
DNA, followed by promoter complex formation and the
template-dependent extension of cross-linked nucleotide. One can extend this approach even further and perform the cross-linking step in the absence of one or two RNAP subunits. To visualize the
cross-link, one would then have to add the missing RNAP subunit(s) to
reconstitute the active enzyme. The standard RNAP reconstitution protocol involves mixing the RNAP subunits under denaturing conditions followed by dialysis into reconstitution buffer. However, active RNAP
can also be assembled from individually renatured subunits (3, 13).
Therefore, after the addition of RNAP
Because the assembly of active RNAP is required for
Additional control experiments are presented in Fig. 1c.
When the initiating AMP analogue alone was reacted with 10 mM sodium borohydride for 15 min, followed by the
addition of RNAP core subunits, no labeling was observed (Fig.
1c, compare lanes 5 and 6). This
order-of-addition experiment establishes that the cross-linking reaction was complete by the time the missing subunits were added at
the second stage of the reaction. Further, no labeling was observed
when the initiating AMP analogue was incubated with individually renatured
We used several controls to establish that the observed labeling of
To map the initiating nucleotide cross-link sites, purified
radiolabeled subunits were cleaved at Met residues under
"single-hit" conditions to yield families of nested, easily
identifiable fragments (Fig. 2b). The pattern of radioactive
CNBr-generated fragments was the same whether
The apparent Km values for initiating ATP analogue
binding to The The principal result of this work is the demonstration
that the purine-specific RNAP initiation i-site is
essentially complete in the In contrast, our results reveal an absolute requirement of Recently, an atomic structure of the RNAP core enzyme
from the Eubacterium T. aquaticus was determined (8). The
structure revealed a molecule with a "crab-claw" shape. In the view
presented in Fig. 4, the upper jaw of the
claw is made mostly of 2
subassembly of Escherichia coli
RNA polymerase efficiently and specifically interacts with the
initiating purine nucleotide. Isolated
is also active in this
reaction. In contrast, neither
nor
2
is able to
interact with a chimeric molecule composed of rifampicin attached to an initiation substrate. Based on these results, we conclude that the
RNA polymerase initiation site, specific for purine nucleotides, which ultimately become the 5'-end of the transcript, is essentially complete in the absence of the largest subunit,
'. However, the rifampicin binding center is formed only in the
2
'
core enzyme. We interpret our results in light of the high resolution
structure of core RNA polymerase from Thermus
aquaticus.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2
'
) is the most
studied enzyme of its class. Apart from its ability to catalyze
phosphodiester bond formation, the partial biochemical functions of
E. coli RNAP include the binding of single- and
double-stranded DNA, limited melting of double-stranded DNA, binding of
single- and double-stranded RNA, binding of nucleoside triphosphates, and binding of transcription inhibitors rifampicin and
streptolydigin. Most of the functions common to all RNAPs are carried
out by the two largest subunits,
' and
. The two largest subunits
are the most evolutionary conserved and constitute >70% of the enzyme
mass. Together,
' and
contain 17 segments conserved from
bacteria to man (1, 2). It is likely that the conserved segments
participate in the formation of the enzyme functional sites as well as
in intersubunit interactions stabilizing the core assembly.
2
subassembly (7) and is also present in the isolated
subunit. In
contrast, we found that the Rif-binding site is present only in the
complete RNAP core, suggesting that
' plays an unexpectedly critical
role in the assembly of this site. We interpret our data in light of
the high resolution three-dimensional structure of the RNAP core from
Thermus aquaticus (8).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
or
' subunit were solubilized in denaturing buffer
(10) to a final concentration of 0.25 mg/ml and dialyzed against two
changes of a 30-fold excess of reconstitution buffer (10). The
supernatant after dialysis was aliquoted and stored at
80 °C until
further use. RNAP was reconstituted in 25-µl reactions containing 2.5 µg of
, 2.5 µg of
', and 0.6 µg of hexahistidine-tagged
prepared as described (9). RNAP was assembled by incubating the
reactions for 30 min at 30 °C.
70 (9), 50 ng
of a DNA fragment containing the T7 A1 promoter (12), and 0.3 µM
-[32P]UTP (3000 Ci/mmol). Reactions
proceeded for 3 h at 25 °C and then were terminated by
the addition of an equal volume of Laemmli loading buffer. Proteins
were resolved on polyacrylamide gel electrophoresis on 8% SDS-gels.
Labeled subunits were visualized by autoradiography and quantified by phosphorimagery.
-[32P]UTP (300 Ci/mmol)), and transcription was allowed to proceed for 15 min at
37 °C. Reactions were terminated by the addition of
formamide-containing loading buffer. Products were analyzed by
urea-PAGE (7 M urea, 20% polyacrylamide) followed by
autoradiography and PhosphorImager analysis.
subunits were extracted from the SDS
gels by crushing the gel chips and soaking them in 0.2% SDS overnight.
Extracted subunits were dried in vacuo and dissolved in a
small volume of water, and HCl was added to 50 mM. Cleavage
was initiated by the addition of CNBr to a final concentration of 50 mM and allowed to proceed for 5 min at room temperature.
Reactions were terminated by the addition of a double volume of Laemmli
loading buffer. The reaction products were resolved by 12.5% SDS-PAGE
and visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit makes extensive contacts with the
initiating purine nucleotide, which will ultimately become the 5'-end
of the transcript (4, 14, 15). Thus,
appears to be principally responsible for the formation of the 5'-face of the catalytic center.
We were interested to know whether the purine-specific initiation site
might be present in the
2
RNAP subassembly or in the
subunit alone. Accordingly, we developed an assay to monitor the
interaction of isolated RNAP with derivatized initiating nucleotide
analogues. Our assay combines two powerful methods of RNAP study,
highly selective affinity labeling with initiation substrate analogues
(4) and in vitro RNAP reconstitution (3, 10).
and template DNA, the original
cross-link can be developed with radioactive nucleoside
triphosphate specified by position +2 of the promoter.
and the
2
Subassembly Can Be
Affinity Labeled with an Initiation Substrate Analogue Specific for
Lys1065--
We used the modified affinity
labeling protocol described above to ask whether the RNAP subassembly
2
, and even
alone, can be affinity labeled with
initiating AMP derivatized with a cross-linkable aldehyde group (4).
In this experiment, as shown in Fig.
1, derivatized AMP was added to the
indicated renatured RNAP subunits or subunit combinations
(Crosslinking to), and the cross-linking reaction was
induced by the addition of 10 mM sodium borohydride for 15 min at 37 °C. The RNAP core was then assembled by the addition of
individually renatured subunits omitted in the first step
(Addition of). The cross-link was then developed by the
addition of
, the T7 A1 promoter-containing DNA fragment, and
-[32P]UTP specified by the +2 position of the
template. As expected, robust labeling of
was observed in the
complete reaction, which contained all the RNAP core subunits at the
initial cross-linking step (lane 10). Approximately 5% of
was radioactively labeled in the complete reaction, which
represented ~40% of the labeling achieved in the standard affinity
labeling protocol when RNAP open complex was used at the cross-linking
stage (data not shown). Surprisingly, a high level of
labeling was
observed when the mixture of
and
was modified in the absence of
' (lane 9). The labeling efficiency dropped to ~20% of
that seen in the complete reaction when
alone was used at the
cross-linking step (lane 7). Similar results were obtained
with initiating ATP derivatized with a cross-linkable aldehyde group
positioned at the
-phosphate (data not shown). Importantly, no
labeling occurred in reactions when no extra core subunits were added
in the development step, indicating that the preparations of renatured
and
were free of contaminating RNAP or
', which would have
revealed themselves by affinity labeling of
in Fig. 1a
(lanes 6 and 8).
View larger version (40K):
[in a new window]
Fig. 1.
RNAP and the
2
subassembly
bind initiating nucleotide analogue. a, the indicated
RNAP subunits were individually renatured and combined in the presence
of 500 µM initiating AMP analogue derivatized with an
aldehyde group (AMP(1065), Ref. 4), and cross-linking was induced by
the addition of borohydride (Crosslinking to). After
the cross-linking reaction was complete, additional RNAP subunits were
added as indicated (Addition of). Reactions were then
provided with the recombinant
subunit, the T7 A1
promoter-containing DNA fragment, and
-[32P]UTP
specified by the second position of the promoter. Reactions were
incubated for 10 min at room temperature and terminated by the addition
of SDS-containing loading buffer. Reaction products were resolved on an
8% SDS-gel, stained, and revealed by autoradiography. b,
aliquots of reactions shown in panel a, lanes 2,
4, and 5, were supplemented with 25 µM NTPs and incubated for 15 min at 37 °C. Reaction
products were resolved by 20% denaturing urea-PAGE and revealed by
autoradiography. c, affinity labeling reactions were
performed as in a with individual RNAP subunits or subunit
mixtures in the order indicated. Reaction products were resolved by 8%
SDS-PAGE. An autoradiograph is presented.
labeling
to occur, the amount of radioactive labeling depends not only on the
binding of the initiating nucleotide analogue by the i-site at the cross-linking step but also on the amount of active RNAP present
at the final stages of the reaction. To determine the efficiency of the
RNAP assembly, aliquots of reactions from lanes 7,
9, and 10 of Fig. 1a were supplemented
with NTPs and in a steady-state transcription assay (Fig.
1b). RNAP activity in this assay correlated with the
affinity labeling results; the amount of full-sized transcript produced
was the highest when the
/
mixture was used for cross-linking, followed by the addition of
' (lane 2), or when
individually renatured
,
, and
' were combined simultaneously
(lane 3). Only ~20% of RNAP activity could be
recovered when
alone was cross-linked to the initiating substrate
analogue followed by the addition of
and
' (lane 1).
Thus, the relative cross-linking efficiency of the initiating AMP
analogue to
in reactions containing the isolated subunit or in the
/
mixture is comparable with that achieved in the complete RNAP
labeling reaction.
' followed by the reaction with 10 mM sodium
borohydride for 15 min and the addition of the missing
and
(Fig. 3c, lane 3). Thus, the reagent used is
strictly specific for the
subunit, as expected from previous work
(4).
resulted from the specific binding of the purine nucleotide analogue to
the RNAP i-site. The presence of unlabeled ATP during the
cross-linking step abolished labeling (Fig.
2a). This suggests that the
affinity labeling of
reflects the interaction of the derivatized
AMP with a specific nucleotide-binding site rather than a nonspecific
interaction of the cross-linker with the lysines of the protein.
Interestingly, the addition of UTP did not interfere with the labeling,
suggesting as expected that the pyrimidine nucleotide poorly competes
for the purine-specific i-site (data not shown).
View larger version (28K):
[in a new window]
Fig. 2.
The interaction of initiating nucleotide with
RNAP and the
2
subassembly
is specific. a, affinity labeling was performed as
described in the legend for Fig. 1. In lanes 2,
4, and 6, cross-linking was performed in the
presence of 5 mM ATP competitor. In lanes 1,
3, and 5, 5 mM ATP was added together
with
,
-[32P]UTP, and DNA. Reaction products were
resolved on an 8% SDS-gel. An autoradiograph is presented.
b, affinity modification reactions were performed as
described in the legend for Fig. 1 with either AMP-based reagent
(lanes 4-6) or ATP-based reagent (lanes 1-3 and
7-9). Radiolabeled
subunit was extracted from the gel
and treated with 50 mM CNBr for 5 min, and reaction
products were resolved on a 12.5% gel and revealed by autoradiography.
Lanes 1-3 are control lanes without CNBr treatment.
alone, the
/
mixture, or the complete reaction containing
,
, and
' was
used at the cross-linking step (compare lanes 4-6 and
7-9). Previous work demonstrated that standard affinity
labeling with the AMP-based reagent resulted in exclusive cross-linking
to universally conserved Lys1065 in the
conserved
segment H (14); the ATP-based reagent cross-linked to
Lys1242 in the universally conserved segment
I.2 The experiment of Fig.
2b reveals that the ATP-based reagent also cross-links to
Lys1242 in our protocol because the smallest radioactive
fragment visible on the gel corresponded to cleavage at
Met1243 whether
, the
/
mixture, or the
complete mixture of RNAP core subunits was modified. The smallest
radiolabeled CNBr fragment observed with the AMP-based reagent
corresponded to cleavage at
Met1085, not to
Met951 as has been previously reported for this reagent
(14). Thus, with our labeling protocol at least a fraction of the
cross-links occurred to a lysine between
Met1066 and
Met1085, leaving Lys1073 and/or
Lys1078 as possible candidates. Lys1073 is the
most likely target because structural analysis of the T. aquaticus RNAP core (8) reveals that the T. aquaticus
Lys846 (E. coli Lys1073) is located
within 5 Å of Lys838 (E. coli
Lys1065), which is the site of AMP reagent cross-linking at
standard affinity labeling conditions (4, 14). In contrast, the
T. aquaticus Lys851 (E. coli
Lys1078) is more than 17 Å away from Lys838.
The shift in the cross-link site can be explained by conformational changes, which occur during the holoenzyme and/or promoter complex formation. We also note that affinity labeling of the E. coli RNAP carrying the K1065A mutation results in
Lys1073 labeling even when the standard affinity labeling
protocol is used.3
,
/
, or
/
/
' were calculated from the
extent of
affinity labeling as a function of the analogue
concentration (data not shown). The calculated values were similar (650 µM (
), 500 µM (
/
), and 575 µM (
/
/
')) and close to the published Km value for the RNAP i-site determined
by independent methods (5, 6). We conclude that the observed affinity
labeling is the result of the specific interaction of the initiating
nucleotide with the
2
subassembly or isolated
and
that this interaction occurs in the i-site.
' Subunit Is Required for the Formation of the Rif-binding
Site--
Rifampicin forms a tight complex with RNAP and prevents
promoter escape (3, 16). Mutations in
abolish or weaken Rif binding
to RNAP, suggesting that
forms at least a part of the Rif binding
center (17, 18). To test this idea directly and to study the binding of
Rif to RNAP subassemblies, we used a chimeric compound comprising Rif
covalently attached to ATP (Rif-A, as reviewed in Ref. 11). The
chimeric molecule interacts with RNAP core with high affinity because
of the strong Rif-RNAP interaction. The molecule also serves as a
primer for the transcription reaction because the linker between Rif
and the nucleotide moiety allows Rif-A to interact with RNAP in a
bifunctional manner such that each ligand occupies its natural binding
site (11). Rif-A was derivatized with the cross-linkable aldehyde group
positioned next to the
-phosphate of the nucleotide and specific for
Lys1065 (11). RNAP
,
2
, or a
mixture of
,
, and
' was preincubated with varying
concentrations of derivatized Rif-A, and cross-linking was induced by
the addition of borohydride. After the cross-linking reaction was
complete, the missing RNAP subunits were added, followed by the
template-dependent development of the cross-link using radioactive UTP (Fig. 3a). As
a control, affinity labeling reactions were also performed with various
concentrations of the AMP-based reagent (Fig. 3b). In
reactions containing all RNAP subunits, comparable levels of labeling
were achieved with both Rif-A and AMP. However, the concentration
dependence of labeling was dramatically different, as expected.
Reactions containing Rif-A saturated at low concentrations of the
reagent because of the very high affinity of Rif for its RNAP-binding
site. In contrast,
labeling with the AMP-based reagent increased
linearly over the whole concentration range tested because of the lower
affinity of AMP for the i-site. Strikingly, no labeling with
the Rif-A reagent was observed when cross-linking was performed with
isolated
or in the mixture of the
and
subunits (Fig.
3a, lanes 1-6 and 1'-6'). We
conclude that the
' subunit is required for the formation of the
high affinity Rif-binding site. The complete lack of labeling of RNAP subassemblies by Rif-A at 50 µM concentrations contrasts
with observable levels of modification with 50 mM
derivatized AMP (compare lanes 4 and 4' in Fig.
3, a and b). Thus, Rif binding may inhibit AMP
binding in the i-site, consistent with earlier kinetic data (16).
View larger version (32K):
[in a new window]
Fig. 3.
Modification of RNAP subassemblies with Rif-A
chimeric molecule. Cross-linking was performed using the indicated
concentrations of AMP(1065) or Rif-A(1065), followed by the addition of
the missing subunits, RNAP assembly, and the development of the
cross-link in the presence of ,
-[32P]UTP, and DNA.
Autoradiographs of 8% SDS-gels are presented.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2
subassembly and is even
present in the isolated
subunit. In contrast, the Rif-binding site
requires
' for its formation. The critical role of
in the
formation of the i-site is supported by affinity labeling
data, which demonstrate that the initiating substrate makes extensive
contacts with several regions of the
subunit (14, 15). Here, we
demonstrated that
residues in conserved segments H and I can
specifically interact with the initiating nucleotide even in the
absence of other RNAP subunits. Preliminary experiments demonstrate
that the assembly-competent C-terminal module of
(amino acids
643-1342), which contains conserved segments F, G, H, and I, can also
be affinity-labeled by the derivatized initiating nucleotides in
our assay, albeit with low
efficiency.4
' for the
formation of the Rif-binding site as we were unable to detect an
interaction between the
2
subassembly and
Rif-A. Extensive genetic data indicate that mutations that cause Rif resistance and decrease Rif binding to RNAP are found exclusively in
the rpoB gene, coding for
. Early biochemical experiments demonstrated that, in agreement with our data, rifampicin derivatives cross-link to
nonspecifically (20). On the other hand, it has been
reported that
2
weakly binds Rif as judged by
spectral, gel filtration-based, and proteolytic assays (21,
22). However, in these later studies the authors did not determine whether their
2
preparation was free of traces of the
RNAP core and whether the observed interaction was specific because no
controls using
2
containing the
subunit carrying
a Rif resistance mutation were used. In addition, there is a
formal possibility that the difference between our results and those
reported by previous researchers is attributable to differences between
Rif and Rif-A interactions with RNAP. However, the absence of Rif-A
binding and cross-linking to RNAP harboring known Rif resistance
mutations makes this possibility unlikely (11).
and is bilobal; the lower jaw is made mostly
of
'. The deep cleft that separates the jaws harbors the catalytic
magnesium ion and binds the DNA template (8, 23). Analysis of the
T. aquaticus RNAP structure validates the biochemical data
obtained in this work with the E. coli enzyme. As can be
seen, the Rif-binding site (defined by sites that, when mutated,
prevent Rif binding and result in Rif resistance (green,
yellow, blue, and white spheres on
Fig. 4)) and the i-site (as defined by cross-link points in conserved segments H and I (red spheres in Fig. 4)) come
from clearly different "domains" of the structure.
5
View larger version (67K):
[in a new window]
Fig. 4.
Structural context of the Rif-binding
site and initiating nucleotide cross-link sites (see text for
details). A backbone representation of the T. aquaticus
RNAP core is shown. The subunit is in cyan,
' is in
pink,
and the poorly seen
dimer are in
white. The bottom panel represents the
entire RNAP. The boxed area is expanded at the top and is
shown in stereo. The active-center Mg2+ is in
magenta. Amino acids known to cross-link to initiating
nucleotide analogues used in this work are indicated as red
spheres. Sites of the known rifampicin resistance mutations are
indicated as white (N-terminal cluster), green
(cluster I), yellow (cluster II), and blue
(cluster III) spheres, correspondingly. The
' segment F
helix described in the text is indicated in red. Segment F
residues that make direct contacts with cluster II amino acids are
shown in magenta.
The i-site lies on the face of the sheet domain
made up from the
subunit conserved segments F, G, and H and a
portion of segment I. Proteolytic studies, in vitro
reconstitution, and two-hybrid analysis indicate that in E. coli, this domain is capable of independent folding and specific
binding to RNAP
subunit (24, 25) and that evolutionary conserved
residue Asp1084 in segment H is particularly important for
2
formation (25). Our results show that this domain
is also capable of interacting with the initiating nucleotide
-phosphate through
Lys1073. On the structure,
I
(white in Fig. 4) is immediately behind this domain,
consistent with the observed stimulatory effect of
on
labeling.
It should be noted that the i-site residues revealed by
affinity labeling cannot be essential for catalysis because detecting cross-linking at these residues in the affinity labeling protocol requires catalysis to attach the radioactive label (14, 26).
Unfortunately, the 5'-phosphate cross-links do not allow to
model the position of the initiating nucleotide on the structure with confidence. However, the highlighted residues of
must be very
close to the initiating nucleotide bound in the i-site to be
cross-linked to the derivatized nucleotide.
Mustaev et al. (27) used selective affinity
labeling of RNAP promoter complexes to study the extension of
initiating nucleotides cross-linked to the E. coli
Lys1065 and His1237. They
demonstrated that the initiating nucleotides cross-linked to
Lys1065 could be extended by only one nucleotide specified
by the second position of the template. In contrast, initiating
nucleotides cross-linked to His1237 could be extended by as
much as nine nucleotides. These results were taken as strongly
supporting the inchworming model of transcription. This model attempts
to explain the high processivity of transcription elongation by
postulating that the catalytic center of RNAP can move with respect to
the RNAP mainframe (28). In contrast, rigid body models postulate that
no extensive movements of the catalytic center occur and that the
addition of a single nucleotide to the nascent RNA 3'-terminus is
coupled to the translocation of the whole complex along the DNA.
Structural analysis indicates that the data of Mustaev et
al. (27) can be explained without invoking the inchworming model.
In the T. aquaticus RNAP structure Lys838, which
is homologous to the E. coli Lys1065, is located
in an inner strand of the
sheet domain and is firmly anchored on
the RNAP mainframe. In contrast, His999, which is
homologous to the E. coli His1237, is located on
an unstructured loop that traverses the main channel of the enzyme and
appears to be flexible. Thus, the extension of the cross-link at this
position may simply reflect the flexibility of the cross-link site
rather than the movement of the catalytic center.
The Rif pocket, as defined by Rif resistance mutations, is farther away
from the catalytic center, at the interface between several structural
elements of . Mutations toward Rif resistance occur at the base of
and in between the two lobes formed by the
jaw (Fig. 4). Residues
defining the N-terminal-most cluster of Rif mutations (E. coli position 146, T. aquaticus position 137 (white sphere)) and the C-terminal-most cluster (E. coli position 687, T. aquaticus position 566 (blue sphere)) occur at the place where the two lobes meet.
Cluster I mutations (E. coli positions 507-534, T. aquaticus positions 387-414 (green spheres)) affect both lobes. Cluster II mutations (E. coli positions
563-574, T. aquaticus
positions 443-454 (yellow
spheres)) affect the downstream lobe only. Cluster II amino acids
lie at the base of a hairpin-like structure (blue in Fig.
4). This hairpin is directly supported by the
' segment F helix
(running vertically on the left in Fig. 4 (red and
magenta)). In particular, evolutionary conserved T. aquaticus segment F residues 1085-1088, corresponding to E. coli
' positions 786-789, make van der Waals contacts
with cluster II amino acids 447-451, corresponding to the E. coli
amino acids 567-571. In addition, the T. aquaticus
'Arg1077 (E. coli
Arg780) is in direct contact with the T. aquaticus
Pro444. The corresponding residue in
E. coli,
Pro564, can be mutated toward Rif
resistance (18). Thus, it appears that cluster II would be unable to
fold properly in the absence of
', which may explain the requirement
for
' for the formation of the Rif site.
In E. coli, the segment F amino acid Ser793,
which is only three amino acids away from the ' amino acids involved
in the stabilization of the Rif-cluster II structure, could be mutated
toward streptolydigin resistance (19). The main cluster of
streptolydigin resistance mutations is in
, between Rif cluster I
and II. Our biochemical and structural analysis suggests that mutations
toward Rif resistance can also be obtained in the
' segment F. We
are currently performing targeted searches for such mutations. If
found, such mutants may explain the nature of infrequent Rif-resistant
bacterial isolates that do not contain any changes in the known Rif
resistance sites in
.
![]() |
FOOTNOTES |
---|
* This work was supported by The Burroughs Wellcome Fund for Biomedical Research Career Award and National Institutes of Health Grants RO1 59295 (to K. S.), GM53759 (to S. A. D), and GM37017 (to A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Waksman
Institute for Microbiology, 190 Frelinghuysen Rd., Piscataway, NJ
08854. Tel.: 732-445-6095; Fax: 732-445-5735; E-mail:
severik@waksman.rutgers.edu.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M011041200
2 M. Kozlov, I. Bass, K. Severinov, and A. Mustaev, unpublished observations.
3 A. Mustaev, V. Sagitov, and A. Goldfarb, unpublished observations.
4 K. Severinov, unpublished observations.
5 K. Severinov and A. Mustaev, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: RNAP, RNA polymerase; Rif, rifampicin; PAGE, polyacrylamide gel electrophoresis.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Allison, L. A., Moyle, M., Shales, M., and Ingles, C. J. (1985) Cell 42, 599-610[Medline] [Order article via Infotrieve] |
2. | Sweetser, D., Nonet, M., and Young, R. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1192-1196[Abstract] |
3. | Zillig, W., Palm, P., and Heil, A. (1976) in RNA Polymerase (Losick, R. , and Chamberlin, M., eds) , pp. 101-125, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York |
4. | Grachev, M. A., Kolocheva, T. I., Lukhtanov, E. A., and Mustaev, A. (1987) Eur. J. Biochem. 163, 113-121[Abstract] |
5. | Wu, C. W., and Goldthwait, D. A. (1969) Biochemistry 8, 4450-4458[Medline] [Order article via Infotrieve] |
6. | Wu, C. W., and Goldthwait, D. A. (1969) Biochemistry 8, 4458-4464[Medline] [Order article via Infotrieve] |
7. | Ishihama, A. (1981) Adv. Biophys. 14, 1-35[Medline] [Order article via Infotrieve] |
8. | Zhang, G., Campbell, L., Minakhin, L., Richter, C., Severinov, K., and Darst, S. A. (1999) Cell 98, 811-824[Medline] [Order article via Infotrieve] |
9. | Tang, H., Severinov, K., Goldfarb, A., and Ebright, R. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4902-4906[Abstract] |
10. | Borukhov, S., and Goldfarb, A. (1993) Protein Expression Purif. 4, 503-511[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Mustaev, A.,
Zaychikov, E.,
Severinov, K.,
Kashlev, M.,
Polyakov, A.,
Nikiforov, V.,
and Goldfarb, A.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
12036-12040 |
12. | Heumann, H., Lederer, H., Kammerer, W., Palm, P., Metzger, W., and Baer, G. (1987) Biochim. Biophys. Acta 909, 126-132[Medline] [Order article via Infotrieve] |
13. |
Markov, D.,
Naryshkina, T.,
Mustaev, A.,
and Severinov, K.
(1999)
Genes Dev.
13,
2439-2448 |
14. |
Mustaev, A.,
Kashlev, M.,
Lee, J. Y.,
Polyakov, A.,
Lebedev, A.,
Zalenskaya, K.,
Grachev, M.,
Goldfarb, A.,
and Nikiforov, V.
(1991)
J. Biol. Chem.
266,
23927-23931 |
15. |
Severinov, K.,
Mustaev, A.,
Severinova, E.,
Kozlov, M.,
Darst, S. A.,
and Goldfarb, A.
(1995)
J. Biol. Chem.
270,
29428-29432 |
16. | McClure, W. R., and Cech, C. L. (1978) J. Biol. Chem. 253, 8949-8956[Abstract] |
17. | Jin, D. J., and Gross, C. A. (1988) J. Mol. Biol. 202, 45-58[Medline] [Order article via Infotrieve] |
18. | Severinov, K., Soushko, M., Goldfarb, A., and Nikiforov, V. (1993) J. Biol. Chem. 268, 14280-14825 |
19. |
Severinov, K.,
Markov, D.,
Nikiforov, V.,
Severinova, E.,
Landick, R.,
Darst, S. A.,
and Goldfarb, A.
(1995)
J. Biol. Chem.
270,
23926-23929 |
20. | Lowder, J. F., and Johnson, R. S. (1987) Biochem. Biophys. Res. Commun. 147, 1129-1136[Medline] [Order article via Infotrieve] |
21. | Stender, W., Stutz, A. A., and Scheit, K. H. (1975) Eur. J. Biochem. 56, 129-136[Abstract] |
22. | Huaifeng, M., and Hartmann, G. R. (1983) Eur. J. Biochem. 131, 113-118[Abstract] |
23. |
Korzheva, N.,
Mustaev, A.,
Kozlov, M.,
Malhotra, A.,
Nikiforov, V.,
Goldfarb, A.,
and Darst, S. A.
(2000)
Science
289,
619-625 |
24. | Wang, Y., Severinov, K., Loizos, N., Fenyö, D., Heyduk, E., Heyduk, T., Chait, B. T., and Darst, S. A. (1997) J. Mol. Biol. 270, 648-662[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Naryshkina, T.,
Rogulja, D.,
Golub, L.,
and Severinov, K.
(2000)
J. Biol. Chem.
275,
31183-31190 |
26. | Kashlev, M., Lee, J., Zalenskaya, K., Nikiforov, V., and Goldfarb, A. (1990) Science 248, 1006-1009[Medline] [Order article via Infotrieve] |
27. |
Mustaev, A.,
Kashlev, M.,
Zaychikov, E.,
Grachev, M.,
and Goldfarb, A.
(1993)
J. Biol. Chem.
268,
19185-19187 |
28. | Uptain, S. M., Kane, C. M., and Chamberlin, M. J. (1997) Annu. Rev. Biochem. 66, 117-172[CrossRef][Medline] [Order article via Infotrieve] |