From the Departments of Biology and ¶ Chemistry,
Indiana University, Bloomington, Indiana 47405
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ABSTRACT |
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There is a kinetic limitation to Rho function at
the first intragenic terminator in the lacZ gene (tiZ1)
which can be overcome by NusG: Rho can terminate transcription with
slowly moving, but not rapidly moving, RNA polymerase unless NusG is
also present. Here we report further studies with two other
Rho-dependent terminators that are not kinetically limited
(tiZ2 and Termination of RNA transcription in Escherichia coli is
mediated by two distinct mechanisms. Intrinsic termination requires only RNA polymerase and specific signals in the DNA template. Factor-dependent termination is mediated by the action of
Rho protein (1) with the assistance of NusG (2). Although Rho can act
by itself to terminate transcription efficiently in vitro at
a number of terminators, an in vivo requirement for NusG has been shown to exist for several terminators. The nature of this intracellular requirement for NusG and the mechanism by which NusG acts
to enhance Rho function must be understood to present a clear picture
of factor-dependent termination in E. coli.
The action of NusG has been examined for several
Rho-dependent terminators in vitro (3-5). When
reactions are carried out under conditions that are optimized for Rho
function, NusG does relatively little. It can enhance the efficiency of
termination somewhat and also activate promoter proximal termination
stop points that are not normally used by Rho. However, a requirement for NusG in Rho function was identified for the first intragenic terminator in lacZ (tiZ1) when transcription reactions were
performed using in vivo concentrations of NTPs which allow
RNA polymerase to elongate RNA chains at the in vivo rate
(3). In this case, NusG is required to overcome a kinetic limitation to
Rho function which is likely to exist in the intracellular environment.
However, under these same conditions a second terminator downstream
from tiZ1 on that same template, tiZ2, functioned as well in the
absence of NusG as in its presence, suggesting that either it is a
NusG-independent terminator or its position with respect to the start
point of transcription can influence the relative requirement for NusG as a cofactor.
Several individual activities of Rho factor are required to achieve
termination of RNA transcription (6). Rho must first bind to the RNA.
This interaction then activates an ATPase activity that allows the Rho
hexamer to track along the RNA in the 5' to 3' direction (7-9) and to
dissociate the RNA from the transcription complex at a pause site (10,
11). Each of these steps has a kinetic component that could be affected
by NusG. The effects of NusG on many of these steps of Rho action have
been measured. NusG does not change the Kd of the
Rho-RNA binding interaction, the Km for RNA to
activate ATPase, the Vmax of ATP hydrolysis, the
concentration of Rho necessary for half-maximal termination, or the
rate with which it can dissociate a single-stranded DNA base pairing to
the 3'-end of the transcript (the helicase activity) (4, 5, 12). It has
been shown to slow the dissociation rate of Rho from nascent RNA from 2 to 5 min (13). However, because RNA polymerase passes through the tiZ1
terminator in about 5 s, it is not clear how such a stabilization
of the Rho-RNA interaction might enable termination of rapidly
elongating polymerases at tiZ1.
The final component activity is the dissociation of RNA from the
transcription complex. This has been examined for transcription complexes randomly stopped in the trp t' terminator region
(5). NusG was shown to increase the rate of dissociation of RNA from complexes that were stopped at the promoter proximal termination points, sites at which Rho-dependent termination occurs
very slowly, if at all, in the absence of NusG. Thus, this result did
not distinguish whether NusG enhances the rate of Rho-mediated RNA
release or was simply allowing Rho to act at positions where it
normally does not.
In this report we investigate further the generality of the requirement
for NusG to allow Rho to cause termination of rapidly moving RNA
polymerase and the effects of NusG on the rate of transcript release.
Specifically, we test the effects of NusG on two terminators, lac tiZ2 and Enzymes and Reagents--
Wild-type Rho, purified as described
previously (14), was provided by Lislott Richardson. NusG was from
Barbara Stitt (Temple University) (15), and NusA was from Richard
Burgess (University of Wisconsin) (16). EcoRI-Gln111 mutant
endonuclease was from Paul Modrich (Duke University) (17). F62S Rho
(18) was provided by Chon Martinez. The variant form of
Micrococcus luteus Rho, Mlu des(60-300) Rho lacks most of
the 256-residue RNA-binding domain insertion segment that is a
characteristic of M. luteus Rho (19) and also contains a
hexahistidine amino-terminal extension. Mlu des(60-300) Rho was
expressed in E. coli and purified using standard procedures
(14) plus chromatography on a Ni2+-nitrilotriacetic acid
agarose (20). E. coli RNA polymerase was purchased from
Epicentre Technologies (Madison, WI). Enzymes used for DNA
manipulations were from New England Biolabs. E. coli MRE600
tRNA was from Boehringer Mannheim. DNA oligonucleotides were from the
Indiana Institute for Molecular Biology or Life Technologies, Inc.
Gelman BioTrace NT filters were from Baxter Scientific Products
Division. Other enzymes and reagents were from Sigma and J. T. Baker.
Plasmid Constructions--
pCBZ6, an
8,318-bp1 plasmid, was
constructed from pTL61T (21) and pCBZ4 (3). pTL61T was digested with
Bsu36I, the ends blunted with Klenow DNA polymerase, and
then digested with EcoRI. The 8,073-bp fragment of pTL61T is
missing the lac DNA sequences from
pCBC1, a 4,086-bp plasmid, was constructed by insertion of the 751-bp
Sau3A fragment of pDE13 (22), which contains a C to G change
at position 6 of the cro gene, into pIF2 (23) cut with BglII. This generates a complete cro gene driven
by its natural pR promoter. The C6G change allows preparation of stable
ternary transcription complexes (A24 complexes) by transcription in the absence of CTP.
pCBC2, a 3,979-bp plasmid, was constructed from pCBC1 by digestion with
BglII and AvaI, filling in the ends with Klenow
DNA polymerase and recircularization with DNA ligase. It contains a
107-bp deletion in the early region of the cro gene from
position +86 to +192.
Transcription Templates--
The integrity of the DNA sequence
of each plasmid in the transcription template region was confirmed by
sequencing the DNA (24). The DNA templates used for in vitro
transcription from pCBZ4 and pCBZ6 were prepared by amplification of a
segment of the lacZ DNA from (
The transcription templates derived from the plasmids pCBC1 and pCBC2
were prepared by digestion of the plasmids with PvuI. This
enzyme cuts the DNA downstream from the cro gene (at
position 649) and allows examination of all stop points in tR1. Both
templates contain the C6G mutation necessary for A24 complex formation.
The DNA templates used for the release assays were prepared by
amplification of pCBZ4 or pCBC2 with Vent DNA polymerase. The pCBZ4
template is from position RNA Transcription--
In vitro transcription
reactions were carried out using ternary transcription complexes that
were stopped by omission of CTP. A16 complexes on lacZ
templates from pCBZ4 and pCBZ6 were prepared as described (3). A24
complex formation on cro templates from pCBC1 and pCBC2 was
carried out by a modification of this procedure. 3 pmol of RNA
polymerase was mixed with approximately equal molar amounts of DNA
template in transcription buffer (150 mM potassium glutamate, pH 7.8, 40 mM Tris-HOAc, pH 7.8, 4 mM Mg(OAc)2, 1 mM dithiothreitol,
0.02% Nonidet P-40, 0.002% acetylated bovine serum albumin, 1%
glycerol) and preincubated for 5 min at 37 °C. GTP, UTP, and
[
Termination reactions (20 µl) were done using approximately 5 fmol of
isolated complex in transcription buffer. Transcription factors were
mixed with transcription complexes on ice and preincubated for 3 min at
37 °C. NTP mixtures were then added to either low (0.2 mM GTP, 0.2 mM ATP, 0.02 mM UTP,
0.2 mM CTP, 4 mM Mg(OAc)2) or high
(1.1 mM GTP, 2.7 mM ATP, 1.4 mM
UTP, 0.7 mM CTP, 10 mM Mg(OAc)2)
concentrations and the reactions incubated for 3 min at 37 °C. The
reactions were stopped and analyzed by 6% polyacrylamide and 7 M urea gel electrophoresis as described (3). Quantitation of autoradiograms was done using a Molecular Dynamics scanning densitometer.
RNA Release Assays--
Ternary transcription complexes were
prepared with lacZ (A16 complexes) or cro (A24
complexes) DNA templates containing EcoRI sites. After
complex formation, but prior to isolation, EcoRI-Gln111 mutant endonuclease (17) was added to 400 nM and the
reactions incubated for 3 min at 37 °C. NTPs were then added to the
low level with 10 µg/ml rifampicin and the reactions incubated an additional 3 min at 37 °C. The EcoRI-stopped complexes
were then isolated by gel filtration chromatography.
EcoRI-Gln111 acts as a roadblock to stop RNA polymerase 14 bp upstream from the EcoRI binding site (25). The position
of RNA polymerase stopped on the lacZ template was +175, and
on the cro template it was +205 (which corresponds to
position +312 of wild-type cro). These positions were
verified by gel electrophoretic analysis of the RNA from the complex.
Release assays were done in transcription buffer at 37 °C.
Transcription factors were mixed with the transcription complexes on
ice, preincubated for 3 min at 37 °C, and the reactions started by
addition of 1 mM ATP. The 50-µl reactions were stopped after different times by the addition of 500 µl of 0.5 M
KCl, 10 µg/ml poly(C), and 10 µg/ml heparin. The reactions were
then incubated on ice for 5 min and filtered through BioTrace NT
filters. The filters were washed three times with 2 volumes of stop
solution. The amount of RNA that remained associated with the
transcription complexes was determined by measuring radioactivity with
a liquid scintillation spectrometer.
Role of Terminator Position on NusG Dependence--
To determine
the importance of terminator position within a transcription unit on
the ability of Rho to act on rapidly moving RNA polymerase, we prepared
a derivative of the lacZ DNA template with the segment from
bp 76 to bp 276 deleted (Fig. 1). The
deleted segment contains the termination stop point region for tiZ1 (bp 140 to bp 229) plus its likely rut sequences. In this
derivative, called tiZ2d (in the plasmid pCBZ6), the stop point region
of the tiZ2, which normally runs between bp 360 and bp 480, is now between bp 160 and 280 or nearly in the same position as the stop point
region of tiZ1 in the normal template (pCBZ4). This was verified by
examination of the size distributions of the RNA molecules produced
from transcription of the two templates with low NTPs in the presence
of Rho (shown in Fig. 2A). As
expected, the addition of NusG under these conditions caused some RNA
polymerase molecules to terminate transcription at earlier stop points
in the tiZ1 region of pCBZ4 and in similar places in the tiZ2d region
of pCBZ6. The overall efficiencies of termination at each terminator,
determined by scanning densitometry of the autoradiogram shown in Fig.
2 and others like it, are presented in Fig.
3A. The results demonstrate that tiZ2 functions in the absence of tiZ1 and thus confirms that tiZ1
and tiZ2 are distinct terminators.
The critical test is whether tiZ2 can still function in this new
position when transcription is done with high NTPs in the absence of
NusG. The results (Figs. 2A and 3B) show that
virtually no termination occurred at tiZ2d in the pCBZ6 template in the absence of NusG and that only low levels of termination occurred with NusG present. As was found previously, however, Rho by itself terminated transcription in tiZ2 when it was in its normal position (pCBZ4; see Figs. 2A and 3B). Hence, deletion of
202 bp upstream of tiZ2 introduced a kinetic limitation to Rho function
which was similar to that observed for tiZ1 in the normal template. This suggests that the amount of upstream RNA is an important determinant of the ability of Rho to act alone with rapidly moving RNA polymerase.
The results also show that the weak intrinsic terminator located within
tiZ2 (26) is partially affected by the deletion and by the level of
NTPs. This confirms similar observations on the importance of the
overall sequence context (27) and the elongation rate (28, 29) in the
Rho-independent termination process.
The
The ability of Rho to act was not greatly affected by this deletion.
The distribution of stop points closely resembled those of the natural
tR1 terminator but shifted closer to the promoter by the expected 107 nucleotides (Fig. 2B). With the low NTP level, termination
at tR1 and tR1d was indistinguishable with and without NusG (Fig. 3).
With the high NTP level, termination at tR1d was reduced slightly
compared with tR1 and was not affected by NusG (Fig. 3). Thus, although
position within a transcription unit with its consequent effect on the
size of the nascent transcript can influence whether Rho action depends
on NusG, other aspects of the terminator also contribute to this requirement.
Response of Variant Rho Factors to Elongation Kinetics--
We
have shown previously that NusG can partially compensate for a
defective Rho factor. F62S Rho is partially defective in vivo but almost completely defective in vitro at tR1,
giving only 8% termination with low NTPs. NusG was able to restore
in vitro termination to 65%, nearly the in vivo
efficiency (18). We test here the ability of F62S Rho to terminate
transcription at the lacZ intragenic terminators (Figs.
4 and 5).
The results with the low NTP level closely parallel those for the tR1
terminator in that F62S Rho caused very little termination on its own,
but it was able to terminate transcription with moderate efficiency when NusG was also present (Figs. 4 and 5).
F62S Rho did not cause significant termination of transcription at tiZ1
with the high NTP level, even when NusG was present (Figs. 4 and 5).
The inability of F62S Rho to terminate at tiZ1 with the high NTP level
suggests that the kinetic limitation that exists for wild-type Rho
factor is more substantial for F62S Rho because NusG can no longer
overcome this limitation. Further, F62S Rho was unable to cause any
increase of termination above the intrinsic levels at tiZ2 (Figs. 4 and
5) or any termination at all at tR1 (not shown) with the high NTP
level, even with NusG present. This was not expected as wild-type Rho
was able to act at these terminators as efficiently with high NTPs as
with low NTPs. This suggests that the rate of chain elongation imposes limitations on Rho action at all terminators but that wild-type Rho can
normally overcome these limitations on its own for many terminators.
The Rho factor from M. luteus is unusual. It terminates with
higher efficiency and at more promoter proximal positions than E. coli Rho at tR1 (19). We have examined the ability of this Rho and
a deletion variant that behaves more like E. coli Rho (20)
to terminate at the lacZ intragenic terminators. The
wild-type M. luteus Rho terminated transcription at tiZ1
with near 100% efficiency at both the low and high NTP levels and was
only slightly affected by NusG (not shown). The deletion variant
M. luteus des(60-300) Rho factor behaved very similarly to
the E. coli Rho protein during transcription of the
lacZ template with E. coli RNA polymerase. The
positions of the stop points were similar to those caused by E. coli Rho, although some earlier points were also observed (Fig.
4). Using the low NTP level, this Rho factor terminated transcription
with very high efficiency (>90%) at both tiZ1 and tiZ2 (Fig. 5). With
the high NTP level, the efficiency of termination was somewhat lower at
both terminators. Termination efficiency at tiZ2 was similar to that
with E. coli Rho (about 60%). However, the efficiency of
termination at tiZ1 was very different from that caused by E. coli Rho (Fig. 5). At this terminator, the deletion variant of
M. luteus Rho terminated transcription on its own with high
efficiency (about 50%). This efficiency was enhanced by NusG to about
70%. These results suggest that the kinetic limitation of E. coli Rho to cause termination at tiZ1 does not exist for M. luteus des(60-300) Rho and also that E. coli NusG can
activate the termination activity of a heterologous Rho factor.
NusG Increases the Rate of Rho-mediated RNA Release--
Because
NusG allows Rho to function within a kinetic window that is too short
for the action of Rho alone, NusG likely affects the rate of Rho action
to allow termination of rapidly elongating polymerase. To determine if
NusG changes the rate of Rho-mediated dissociation of RNA from
transcription complexes, RNA release assays were done using
transcription complexes in which RNA polymerase had been elongated to
position 175 on the lacZ DNA where it was stopped by an
EcoRI-Gln111 roadblock (17, 25). RNA polymerase stopped in
this way is a substrate for Rho action (25). The site at position 175 was chosen because it is the first position in tiZ1 at which Rho acts
to cause termination in the absence of NusG. Reactions were then
started by the addition of ATP and the amount of RNA released by Rho
determined by separating the released RNA away from transcription
complexes using a nitrocellulose filter binding method (31). The
complexes were stable over the time course of these experiments, as
less than 5% of the complexes dissociated in the absence of Rho
factor. NusG did not change this Rho-independent release of RNA (not shown).
Fig. 6A shows that the
addition of NusG increased the rate of dissociation of RNA from the
ternary complexes. The data points for each of the release reactions
fit well to a curve for the equation for the progress of a first-order
reaction. The values of the first-order rate constant (k)
for the reactions with Rho alone and with Rho plus NusG were 0.010 s
To determine if this correlation between the rate of RNA release and
the ability to terminate rapidly moving RNA polymerase could be
extended, additional Rho factors were examined. The M. luteus des(60-300) Rho, which caused efficient termination at tiZ1 during transcription with high NTPs even in the absence of NusG
(Fig. 4), catalyzed release with a k value of 0.031 s
The effect of NusA was also examined as it acts in an opposing manner
to NusG both on Rho function and on RNA polymerase elongation (32). The
rate constant for release of RNA by wild-type E. coli Rho in
the presence of NusA was 0.005 s
To determine if this rate of release correlation could be extended
still further to include other terminators, we examined the rate of
transcript release at the NusG assists Rho protein in mediating the termination of
transcription by overcoming kinetic limitations of Rho to act by itself. We showed here that with stable, isolated transcription complexes it increased by a factor of 2 or more the rate with which Rho
dissociated the RNA transcript. We also found that the extent of the
kinetic limitation of Rho to act by itself depends on the specific
terminator, the Rho factor, and the position of the terminator in its
transcription unit.
Nehrke et al. (5) investigated the effects of NusG on the
release of transcripts from isolated ternary transcription complexes in
which RNA polymerase had been arrested by deprivation of NTPs at a
number of sites preceding, through, and beyond the trp t' region. They showed that NusG acted to assist Rho in releasing transcripts from complexes arrested at upstream sites, sites where Rho
did not cause termination by itself but did cause termination with NusG
present. They thus concluded that a function of NusG in termination is
to enhance an otherwise negligible rate of Rho-dependent RNA polymerase release at early sites (5). Their results (Fig. 7B in Ref. 5) also showed that NusG increased the rate of
transcript dissociation from the sites where Rho could cause
termination by itself. However, the extent of that rate enhancement was
not as dramatic as for the upstream sites and was not specifically noted. In our studies we focused on measuring rates for the relatively rapid dissociation of ternary complexes stopped at single points where
Rho could also function by itself, and we were able to obtain quantitative differences that could account for the function of NusG in
overcoming the kinetic limitations of Rho to function by itself at some terminators.
To cause termination, Rho must act in the limited time RNA polymerase
takes to traverse the termination region. The faster RNA polymerase
moves the less time Rho has to bind and to act. Even though NusG
accelerates the rate of elongation by ~25% (32), it can still
increase the efficiency of termination because it increases the rate of
RNA release by an even greater extent.
In the presence of NusG and with in vivo levels of NTPs RNA
polymerase elongates chains in lacZ at a rate of 56 nucleotides/s (32). At this rate, the interval between the time when
RNA is large enough for Rho to bind (~100 nucleotides) and the time
when RNA polymerase has fully cleared the tiZ1 region (at bp 240) is less than 5 s. Within this time interval NusG and Rho can act together to terminate transcription with an efficiency of 20% (Fig.
3B). The rate of release of RNA from the complexes with RNA
polymerase blocked at bp 175 in lacZ was close to being
sufficient to account for the efficiency of termination at tiZ1; 13%
of the releasable RNA was released by 5 s (Fig. 6). In contrast,
when transcription was done with the low NTP level, RNA polymerase moved at only about 7 nucleotides/s resulting in a kinetic window of
susceptibility to termination of about 20 s. Rho was able to release RNA sufficiently rapidly to achieve high efficiency termination on its own in this case, releasing ~21% of the releasable RNA by
20 s (Fig. 6).
Much more rapid release was found for the complexes arrested at the
site in The finding that Rho action at tiZ2 was NusG-independent when tiZ2 was
in its normal position (360-480 bp from the transcription start point)
but was NusG-dependent when it was moved upstream by 200 bp
can be explained in terms of the kinetics. When tiZ2 was at the normal
downstream position, 4-5 s more were available for Rho to bind to the
nascent RNA and start interactions that could lead to release than when
tiZ2 was at the upstream position. This extra time might be sufficient
to allow Rho to release RNA without the assistance of NusG. However,
because the change of position also changed the sequence and the
conformation of the RNA, the difference in the dependence of tiZ2 on
NusG could also be a consequence of changes in the interactions of Rho
with the RNA.
We have shown previously that NusG does not affect the affinity of Rho
for lacZ RNA or enhance ATPase activity of Rho with lacZ RNA as a cofactor (12). Nehrke et al. (5)
also could not detect an effect of NusG on the activation of Rho-ATPase
with trp t' RNA, nor could they detect any effect of NusG on
the stability of binary Rho·trp t' RNA complexes or on the
RNA·DNA helicase activity of Rho. Further, we have also observed
that NusG does not alter the oligomerization state of Rho factor in the
presence or absence of its RNA or ATP substrates (12). Although NusG can form separate physical interactions with Rho factor (4) and RNA
polymerase (33), the lack of functional effects on NusG of reactions
involving Rho alone has prompted us and others (15) to conclude that
NusG is exerting its effects on transcription termination from its
interactions with RNA· polymerase. More specifically, we contend that
the interaction of NusG with RNA polymerase is favoring the formation
of a conformation or a state of RNA polymerase which is more
susceptible to Rho actions that mediate release.
Sullivan and Gottesman (2) showed that termination of transcription at
tR1) which show that the requirement for NusG depends on
the properties of the terminator and its location in the transcription
unit. NusG is also shown to increase the rate of Rho-mediated
dissociation of transcription complexes arrested at a specific
termination stop point in the tiZ1 region and the rates of dissociation
with three different Rho factors and two different terminators
correlated with their sensitivity to RNA polymerase elongation
kinetics. These results suggest a model of NusG function which involves
an alteration in the susceptibility of the transcription complex to Rho
action which allows termination to occur within the short kinetic
window when RNA polymerase is traversing the termination region.
INTRODUCTION
Top
Abstract
Introduction
References
tR1, placed at two different positions in
transcription units. Our results indicate that both the location of the
terminator sequence and the nature of the terminator itself influence
greatly the kinetic restraints on Rho function and the requirement for NusG to overcome these restraints. To examine the effects of NusG on
the rate of RNA release caused by Rho factor we made use of homogeneously stopped transcription complexes that were positioned at
termination stop points within tiZ1 and
tR1 which were normally used by Rho alone. The results from these assays reveal that Rho can
mediate release of RNA from the complex at tR1 much more rapidly than
from that at tiZ1 and that NusG enhances the rate of release for both.
This action of NusG suggests that its effects on release are what
allows it to overcome the kinetic limitations of Rho to function alone
at certain terminators.
EXPERIMENTAL PROCEDURES
171 to + 277. The
region from
171 to +75 was prepared by amplification of pCBZ4 from
178 to +75 with Vent DNA polymerase followed by cleavage with
EcoRI, which reduces this segment to
171 to +75. The two
fragments were then joined with DNA ligase. pCBZ6 has a deletion of 202 bp in the lacZ DNA from position +76 to +277.
167 to +839) with Vent DNA
polymerase using primers designed to add EcoRI sites to both
ends of the product DNA for cloning purposes. Both templates contain
the C10T mutation necessary for A16 complex formation as well as the
UV5 and L8 promoter mutations.
167 to +189 of the lacZ DNA, and
the pCBC2 template is from position
188 to +219. In both templates,
primer-directed EcoRI sites were added immediately downstream from the gene segments as is required for this experiment.
-32P]ATP (350 nCi/pmol) were then added to 4 µM each and the reactions incubated at 16 °C for 10 min. The transcription complexes were isolated by gel filtration
chromatography on Sephacryl S-300HR columns as described (3). All
enzymes used for transcription studies were diluted in transcription
buffer containing 0.012% acetylated bovine serum albumin and 0.12%
Nonidet P-40.
RESULTS
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Fig. 1.
Diagram of transcription templates. The
templates used for in vitro transcription studies are
diagrammed to scale. They are aligned from the start points of
transcription which are indicated by the arrows. The
positions of tiZ1, tiZ2, and tR1 are depicted by the boxes.
The solid areas represent the rut site areas for
each terminator. The open, stippled, and
shaded portions of the boxes represent the
termination stop point regions.
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Fig. 2.
Rho-dependent termination on
wild-type and deletion templates. RNA transcription reactions were
carried out with 50 nM Rho (hexamers) with and without 25 nM NusG, using both the low and high NTP levels as
indicated in the figure. The lanes designated U
and C were generated by RNA chain terminating sequence
reactions using 3'-dUTP or 3'-dCTP. The sizes of the transcripts were
determined from the mobility of RNAs in these sequence lanes.
Panel A, transcription of the lacZ DNA templates.
pCBZ4 is the wild type, and pCBZ6 is a deletion variant that
repositions tiZ2 202 bp closer to the promoter. These reactions were
analyzed on a single polyacrylamide gel, but some lanes have been
deleted from this figure. Panel B, transcription of the
cro DNA templates. pCBC1 is the wild type, and pCBC2 is a
deletion variant that repositions tR1 107 bp closer to the
promoter.
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Fig. 3.
Efficiencies of termination with different
terminator constructs. Transcription reactions were done without
any addition (open bars), with 50 nM Rho
(stippled bars), or with 50 nM Rho plus 25 nM NusG (black bars). RNAs were separated by
polyacrylamide gel electrophoresis and quantitated by scanning
densitometry. The efficiency of termination is the percent of RNA
polymerase molecules that encounter a terminator which terminate at
that terminator. tiZ1 and tiZ2 were measured on the pCBZ4 template and
tiZ2d from the deletion construct pCBZ6. tR1 was measured on a template
from pCBC1 and tR1d from the deletion construct in pCBC2. Panel
A, termination with the low NTP condition. Panel B,
termination with the high NTP condition.
tR1 terminator is the only terminator to be studied in
vitro which has been shown to be NusG-dependent
in vivo (2). It has been examined at the low NTP level and
also at an intermediate NTP level (200 µM each NTP). In
neither case was Rho function greatly dependent on NusG (4, 15, 18,
19). We examined Rho function at tR1 during transcription with the low
and high concentrations of NTPs which revealed the kinetic limitation
to Rho function at tiZ1. With both NTP levels, Rho functioned very efficiently on its own, and the addition of NusG did not significantly improve the termination efficiency (Fig. 2B, pCBC1 template,
and Fig. 3). Like tiZ2, tR1 has a longer upstream sequence than tiZ1 does (Fig. 1). To determine if this extra upstream DNA was responsible for the ability of Rho to act alone at tR1 with the high NTP level, a
deletion variant (referred to as tR1d) lacking residues +86 to +192 was
made in which the tR1 stop points are positioned at a distance from the
promoter similar to those in tiZ1, while leaving the rut
region (residues 224-380; (30)) intact (Fig. 1).
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Fig. 4.
Transcription termination by different Rho
factors at tiZ1 and tiZ2. RNA transcription reactions were carried
out using wild-type E. coli Rho (E.c.), the
deletion of M. luteus des(60-300) Rho (M.l.), or
F62S Rho at 50 nM (hexamers) each. Where indicated, 25 nM NusG was also included in the reactions. Experiments
were done using the low (L) or high (H) NTP
level. The sizes indicated on the left were determined from
the mobility of transcripts terminated by the incorporation of 3'-dNTPs
(not shown). The tiZ1 and tiZ2 regions are indicated on the
right.
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Fig. 5.
Efficiencies of termination by different Rho
factors at tiZ1 and tiZ2. The amounts of Rho and NusG used were as
described in Fig. 4. Efficiencies were determined as described in Fig.
3. Panel A, with low NTP level; panel B, with
high NTP level. Open bars, at tiZ1 with Rho alone;
gray bars, at tiZ1 with Rho and NusG; stippled
bars, at tiZ2 with Rho alone; solid bars, at tiZ2 with
Rho and NusG. The extents of termination in the absence of Rho were the
same as shown in Fig. 3. For tiZ2, which has a weak intrinsic
terminator, those values were the same as with the F62S Rho in the
absence of NusG.
1 and 0.023 s
1, respectively. The maximum
fraction released (Frmax) was 0.63 for each.
Thus, NusG increased the rate of release by more than a factor of
2.
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Fig. 6.
Kinetics of Rho-mediated RNA release.
Transcription complexes were stopped at position 175 of the
lacZ DNA or at position 205 in the tR1d DNA by using an
EcoRI-Gln111 roadblock and isolated by gel filtration
chromatography. Protein factors were then added and the reactions
started by the addition of 1 mM ATP. At increasing times,
reactions were stopped, and the amount of RNA that was dissociated from
the complexes was determined by scintillation counting. The data are
expressed as the fraction of the total RNA that was released. In the
absence of Rho factor, less than 5% of the RNA was released after 5 min. The curves are the best fits of the data to the equation
Fr = Frmax(1 e
kt), where Fr is the
fraction of RNA released, Frmax the extrapolated
value of Fr at infinite time, k the rate
constant, and t the time. Panel A, influence of
Nus factors on release kinetics. Reactions were done on the
lacZ template with 50 nM Rho without any other
factor (
) with 25 nM NusG (
), or with 250 nM NusA (×). Panel B, release kinetics of
different Rho factors. Reactions were done on the lacZ
template with 50 nM M. luteus des(60-300) Rho
in the absence (
) and presence of 25 nM NusG (
) or
with 50 nM F62S mutant E. coli Rho (
).
Panel C, RNA release kinetics at the tR1d terminator. The
release of RNA from complexes stopped on the tR1d DNA was examined in
the presence of 50 nM Rho alone (
) and with 25 nM NusG (
). The data for Rho alone on the
lacZ DNA shown in panel A are reproduced in
panels B and C for comparative purposes.
1 (Fig. 6B), which is substantially faster
than the rate with E. coli Rho. But even with this faster
acting Rho factor the rate was increased further by adding NusG to a
value of 0.054 s
1, which is an increase of 1.7-fold above
the rate without NusG. In contrast, F62S Rho that was
NusG-dependent even with the low NTP level was
substantially slower in catalyzing release. These results reveal a
strong correlation between the rate of RNA release and the ability to
terminate rapidly moving RNA polymerase.
1 Fig. 6A),
which is substantially lower than k for the reaction with
Rho alone. These results extend the release rate correlations and show
that NusA and NusG act in opposing manners on the same step in the
Rho-dependent termination reaction. Further, functional effects of NusA and NusG were observed when the proteins were added to
RNA polymerase that had already entered the elongation phase of
transcription, therefore indicating that they do not need to be present
from the onset to exert their effects.
tR1 terminator, which is not kinetically
limited. Transcription complexes stopped at position 205 in the tR1d
DNA (pCBC2) were prepared using the EcoRI-Gln111 method.
This site was chosen because it is the first site at which Rho alone
causes termination with the high NTP level, and it was examined in the
tR1d construct because it yielded a transcript with a size similar to
that of the lac RNA in the complex stopped in tiZ1. The
results in Fig. 6C show that release was much more rapid
from the complex with tR1 DNA than with the complex with lacZ DNA. The rate constant for the reaction without NusG
from the best fit curve is 0.10 ± 0.01 s
1, or
10-fold greater than with the lacZ DNA complex. This result thus correlates with the finding that Rho is not as kinetically limited
with tR1 as with the lacZ intragenic terminators. NusG did
cause a slight increase in the rate of release to 0.11 ± 0.02 s
1, but the error for this measurement was not
sufficiently small to conclude that the difference with this template
is significant.
DISCUSSION
tR1. In fact the rate of release at tR1 in the absence of
NusG was sufficiently high to account for the ability of Rho to act
efficiently at this site in vitro without the assistance of
NusG, even with the high NTP level. Similarly, the rate of release of
RNA from the lacZ site with the variant M. luteus
Rho also appeared to be fast enough to account for its ability to function there without NusG.
tR1 at its normal genetic location in vivo is very
dependent on NusG. Thus, our finding that the function of
tR1
in vitro was not greatly affected by the presence of NusG even during transcription from a shorter than normal transcription unit
and with in vivo levels of NTPs was surprising. However, the
conditions we have imposed in vitro were probably still not as stringent as those in the cell where ribosomes would be present, translating the RNA. The distance between the translation termination codon and the first transcription stop point site of tR1 is only 70 bp,
and the presence of a ribosome blocking the RNA in the vicinity of the
termination codon would leave only a small segment (<50 nucleotides)
of cro RNA available for interaction with Rho. Hence, the
time period for Rho to bind to that segment would be very short, less
than 1 s before RNA polymerase would traverse the termination stop
point region. In this situation the ability of NusG to enhance the rate
of release would be critical. Another possible explanation for the
discrepancy between the weak effect of NusG in vitro and the
strong effect in vivo is that NusG could be acting on some
other factor of the in vivo system which influences Rho
action such as the release of the ribosome. The findings that nusG homolog genes are present in the genomes of two
organisms that lack rho homolog genes, a bacterium,
Mycoplasma genitalium (34), and an Archaeon,
Methanococcus jannaschii (35), suggest that NusG has another
physiological function besides activating Rho-dependent
termination of transcription.
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ACKNOWLEDGEMENTS |
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We thank Barbara Stitt, Richard Burgess, Lislott Richardson, Chon Martinez, Paul Modrich, and Dorothy Erie for generously providing NusG, NusA, E. coli Rho, F62S Rho, EcoRI-Gln111 and pDE13, respectively.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Training Grant GM07227 (to C. M. B.) and by National Institutes of Health Grant AI10142 (to J. P. R.).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.
§ Present address: Nuffield Department of Clinical Biochemistry, University of Oxford, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 D9S, United Kingdom.
Present address: Washington University School of Medicine,
Laboratory Medicine Box 8118, 660 S. Euclid, St. Louis, MO 63110.
** To whom correspondence should be addressed. Tel.: 812-855-1520; Fax: 812-855-8300; E-mail: jrichard{at}bio.indiana.edu.
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ABBREVIATIONS |
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The abbreviation used is: bp, base pair(s).
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REFERENCES |
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