(Received for publication, August 14, 1995; and in revised form, October 2, 1995)
From the
The Escherichia coli mutant rho201 was
originally isolated in a genetic screen for defects in rho-dependent
termination. Cloning and sequencing of this gene reveals a single
phenylalanine to cysteine mutation at residue 232 in the ATP binding
domain of the protein. This mutation significantly alters its RNA
binding properties so that it binds trp t` RNA 100-fold weaker
than the wild type protein, with a K of
approximately 1.3 nM. Rho201 binds nonspecific RNA only
3-4-fold less tightly than it binds trp t`, while the
wild type differential for these same RNAs is 10-20-fold.
Curiously, rho201 displays increased secondary site RNA activation,
with a K
for ribo(C)
of 0.6
µM, compared to the wild type value of 3-4
µM. Although rho201 and the wild type protein hydrolyze
ATP similarly with poly(C), or trp t` RNA, as cofactors,
rho201 has a higher ATPase activity when activated by nonspecific RNA.
Physically, rho201 displays an abnormal conformation detectable by mild
trypsin digestion. Despite effective ATP hydrolysis, the rho201 mutant
is a poor RNA:DNA helicase and terminates inefficiently on trp
t`. The single F232C mutation thus appears to uncouple the
protein's ATPase activity from its helicase function, so rho can
no longer harness available energy for use in subsequent reactions.
Rho is an essential cellular protein required for certain transcription termination events in Escherichia coli. It is a highly complex molecule with multiple activities that are essential to its role as a transcription terminator. It has both primary and secondary nucleic acid binding sites, which differ in the specificity and the relative affinity with which they bind polynucleotides (Richardson, 1982). The RNA-dependent ATPase activity of rho (Lowery-Goldhammer and Richardson, 1976; Galluppi et al., 1976) is essential for its 5`-3` RNA:DNA helicase activity (Brennan et al., 1987). Models for transcription termination propose that rho binds target sites on a nascent RNA, translocates in a 5`-3` direction, and disrupts any RNA:DNA helix present at the transcription bubble, utilizing the energy released by ATP hydrolysis to facilitate release of the RNA (for reviews, see Platt and Richardson (1992) and Platt(1994)).
Rho's target RNA substrates, such as the
promoter proximal region in the lacZ cistron (Richardson and
Ruteshouser, 1986), trp t` at the end of the tryptophan operon
(Galloway and Platt, 1988), the tR1 terminator (Chen and
Richardson, 1987) in E. coli, and cryptic sites within the hisG and C cistrons of Salmonella typhimurium (Ciampi et al., 1989) share no obvious sequence similarity. Their only
common characteristics appear to be a relative lack of secondary
structure (Morgan et al., 1985), the contribution of cytosine
residues (Zalatan and Platt, 1992; Richardson and Richardson, 1992) and
the possibility that a high C/G ratio in the target RNA might be
important (Alifano et al., 1991). There exists no direct
correlation, however, between termination and either the K
or the ability of these substrates to
elicit ATPase activity (Zalatan and Platt, 1992).
Rho functions as a
hexamer of identical 419 amino acid subunits (Finger and Richardson,
1982), and has a distinct domain structure that has been partially
elucidated (Dombroski et al. 1988a, 1988b; Dombroski and
Platt, 1988, 1989; Dolan et al., 1990). The two functionally
distinct sites that rho uses for RNA binding have been well
characterized, but efforts to distinguish and separate them have not
been successful (Richardson, 1982; Dolan et al., 1990). The
occupation of the tight primary binding site, which can bind either
single stranded DNA or RNA, is directly measurable using cross-linking
and nitrocellulose filter binding assays (Brennan and Platt, 1991;
Zalatan and Platt, 1992; Modrak and Richardson, 1994). The
amino-terminal domain of rho was initially implicated in primary site
RNA binding by localizing uv cross-links between trp t` and
poly(C) RNA to within the first 151 amino acids (Dombroski and Platt,
1988), and more recently by uv cross-linking ribo(C) to a
smaller fragment comprising the first 116 amino acids (Modrak and
Richardson, 1994). Site-directed mutagenesis of amino acids 62 and 64,
which are part of the RNP1 consensus sequence found in a number of
RNA-binding proteins (Kenan et al., 1991), resulted in
substantially decreased RNA binding activity (Brennan and Platt, 1991).
Saturation mutagenesis of residues 49-67 reveals that specific
mutations in residues 61-66 lead to significantly decreased
cross-linking of the altered rho protein to ribo(C)
. (
)Mutations within rho that affect primary site RNA
interactions have all been localized to amino acid changes within the
NH
-terminal 116 residues of the protein (Mori et
al., 1989; Tsurushita et al., 1989; Brennan and Platt,
1991), consistent with this region forming the ``core'' RNA
binding domain of the protein.
The secondary site interactions
require that the primary binding site be occupied with single stranded
nucleic acid and are presumably transient, since it has not been
possible to measure them by conventional means (Dolan et al.,
1990). The affinity of the secondary site for RNA is estimated through
an indirect functional assay, by saturating the primary binding site
with poly(dC) (which is inactive as a cofactor for ATPase activity),
and measuring the ribo(C) concentration at which
half-maximal ATPase activity can be elicited (Richardson, 1982). The
location of the secondary site within the protein has not been
determined. Steinmetz et al.(1990) proposed the existence of a
single RNA binding site on each subunit, which could alternate between
primary and secondary site character dependent on conformational state.
Such a ``two-state'' model for a single site would account
for the inability to separate chemically the primary and secondary RNA
binding sites in wild type rho (Dolan et al., 1990).
A
second domain of rho extends from amino acids 160 to 340 and shows
extensive sequence similarity with the E. coli F ATPase
and
subunits as well as adenylate kinase,
proteins known to be involved in nucleotide binding and hydrolysis. The
similarity strongly suggested that these elements within rho were
involved in NTP binding (Dombroski and Platt, 1988), a prediction that
was supported by affinity labeling studies with the ATP analog PLP-AMP, (
)which demonstrated Lys
to be the site of
modification (Dombroski et al., 1988b). Furthermore, it was
observed that rho proteins carrying mutations in this domain, at amino
acids 181, 184, and 265 were affected in reactions pertaining to and
dependent on ATP hydrolysis, without altering the ability of the
protein to bind RNA at its primary binding site (Dombroski et
al., 1988a).
The carboxyl-terminal remainder of the protein from amino acids 340 to 419 has no definite function ascribed to it. The rho mutant ts15, which harbors an IS1 insertion causing the COOH-terminal nine amino acids to be changed, has been shown to form hexamers less readily than the wild type, suggesting that these residues might be involved in the assembly of rho subunits (Opperman et al., 1995). Furthermore, studies with the known secondary site mutant, rho suA1, have identified its single mutation as a lysine to glutamic acid change at amino acid 352 (K352E), raising the possibility that this region may also play a role in rho's secondary site interactions (Zheng and Friedman, 1994; Pereira and Platt, 1995).
Finally, rho itself appears to assemble as a trimer of asymmetric dimers, with two types of both ATP and RNA binding sites within the active hexamer (Stitt, 1988; Geiselmann and von Hippel, 1992; Geiselmann et al., 1992; Wang and von Hippel, 1993). Current models for rho action thus require: 1) binding of RNA to both primary and secondary sites in order to elicit ATP hydrolysis by the ATP-binding domain (Richardson, 1982; Seifried et al., 1992); and 2) ``coupling'' of this hydrolysis, in turn, back to an RNA bind-and-release cycle to obtain directed helicase/termination function (Geiselmann et al., 1993; Platt, 1994). Communication between the monomers of the rho hexamer is thus an essential component of the coupling reactions between ATP hydrolysis and the subsequent translocation, helicase, and termination activities (Seifried et al., 1992; Geiselmann et al., 1993). Although rho factor has been extensively characterized, the elements within the protein that are involved in this ``coupling reaction'' remain unknown.
Here we report the sequencing of the gene and the characterization of the mutant rho201 protein, which displays significantly altered primary site RNA binding although its single F232C mutation lies outside the core RNA binding amino-terminal third of the protein. Despite a much weaker affinity for RNA than wild type protein, this mutant has an efficient RNA-dependent ATPase activity. Nevertheless its helicase activity is significantly lower than that of the wild type, suggesting that the mutation has uncoupled these two activities.
Figure 1:
Estimation of the
binding affinity of the wild type and rho201 proteins for trp t` RNA. Uniformly labeled trp t` RNA at 0.005 nM concentration was titrated against increasing concentrations of
wild type rho and rho201. The amount of RNA bound at each rho
concentration was determined by a nitrocellulose filter binding assay
as described (``Materials and Methods'') and the K values estimated from Scatchard plots
of the data. A, Scatchard plot of the wild type protein
binding to trp t`. B, Scatchard plot of rho201
binding to trp t`. Note that the scale of the y axis
differs in the two graphs.
Figure 2:
Characterization of the affinity of the
secondary site of the wild type and rho201 proteins for RNA. The
efficiency of secondary site binding of the two rho proteins was
determined by estimating the amount of ATP hydrolyzed by each protein
(5.0 nM) when its primary binding site was saturated with 0.6
µM poly(dC) and its secondary site activated
with varying concentrations of oligo rC
, as described
under ``Materials and Methods.'' The K
values for the wild type (
) and
rho201 (
) proteins were calculated from Lineweaver-Burk plots of
the resultant data. Note that the V
values
(obtained from the y intercept) are similar for the two
proteins.
Figure 4: Coomassie Blue staining of the trypsin digestion products of wild type and rho201 proteins. Rho concentrations were at 0.225 mg/ml. Freshly made trypsin was added to 0.003 mg/ml (see ``Materials and Methods'' for details). Lanes 1 and 2 consist of the two proteins without trypsin; lanes 3-6 are the proteins incubated with trypsin for the indicated lengths of time; lanes 7-10 represent digestions in the presence of 1 mM ATP and 0.1 mg/ml poly(C). The positions of full-length rho and the F1 and F2 fragments are indicated. Both proteins show larger amounts of undigested protein at the end of 60 min (lanes 5, 9, and 10) as compared to 30 min (lanes 3, 7, and 8), this was because the aliquot removed from the reaction at the 30-min time point consisted of slightly less than half the reaction mix (see ``Materials and Methods'').
The results of these experiments are shown in Fig. 3. With trp t`, the wild type protein (lane
2) produces terminated products 130-155 nucleotides in
length. Rho201, in keeping with its characteristics as a defective
termination factor in vivo, does not terminate trp t` as efficiently. It is defective at causing termination at the
earlier end points; allowing RNA polymerase to transcribe further along
the DNA before termination occurs (lane 3). Neither the wild
type nor the 201 protein terminates very well on templates carrying
either 2T/H or 11a (lanes 5, 6, 8 and 9). In 2T/H the
wild type protein is found to enhance termination at an unmapped site ()within the second copy of T/H in the tandem construct (Fig. 3, lane 5). Rho201 seems defective at this site
as well. Therefore, despite an increased ability to use substrate or
nonspecific RNA as cofactors in the ATPase reaction, rho201 cannot
terminate efficiently on wild type or defective templates in
vitro, as occurs in vivo with trp t`. This
suggests that the rho201 defect in primary site binding cannot be
compensated for by either efficient ATPase activity or enhanced
secondary site binding in order to bring about transcription
termination.
Figure 3: Characterization of the in vitro transcription termination activities of the wild type and rho201 proteins with wild type and defective substrates. Wild type rho and rho201 were tested for in vitro transcription termination activities with trp t` (wild type RNA substrate), 2T/H which contains random E. coli vector sequences, and 11a which consists of a mutated defective substrate carrying the first 104 nucleotides of trp t` with 11 C to U changes. Transcription was initiated from the strong T7A1 promoter. RT indicates readthrough transcription by RNA polymerase. The boxed in area represents rho-dependent termination giving rise to truncated transcripts. The first 104 nucleotides of trp t` are terminated by rho at the same end points as the entire 220 nucleotides of trp t` so that truncated transcripts of the same length are produced in both cases (data not shown).
In order to test if the change in rho201's conformation would affect its capacity to assemble normally, the wild type and the rho201 proteins were subjected to sedimentation on a 5-20% sucrose gradient (Dombroski et al., 1988a) alongside molecular weight markers. Both proteins sedimented in the fractions corresponding to the hexameric form of the protein (data not shown), indicating that the conformational change does not interfere with rho201's ability to form active hexamers. From the tryptic digestion pattern of the two proteins, we conclude that the F232C mutation in rho201 causes a conformational change in the protein significant enough to be detected by mild trypsin degradation.
The helix was formed by annealing the last 28 nucleotides of labeled trp t` RNA to a complementary polylinker region on single stranded M13 mp11. Both the wild type and the rho201 proteins, at a concentration of 10 nM, were incubated in separate reactions with the helicase substrate at 1 nM and the amount of RNA released over time was monitored. As shown in Fig. 5, the wild type protein shows a very fast initial rate of helix disruption, with most of the RNA being released within the first 2 min. Rho201, however, shows reduced helicase activity and incubation with the helix for increased lengths of time does not increase the activity significantly, nor does it exhibit catalytic behavior (data not shown). Thus the rho201 protein does not behave like the F62A/F64A mutant in the helicase assay, nor does it mimic it in the transcription termination assay in vitro. There is, however, a good correlation between rho201's inability to disrupt an RNA:DNA helix and its defects in transcription termination in vivo as well as in vitro.
Figure 5:
Characterization of the RNA:DNA helicase
activity of wild type rho and rho201. An RNA:DNA helix was formed by
annealing 1 nMtrp t` RNA via complementary sequence
at its 3` end to single stranded M13 mp11. Helicase activity releases
RNA from the annealed duplex, and these two species are separated and
analyzed on an agarose gel. The femtomoles of RNA released by 10 nM wild type rho (&cjs1359;) or rho201 () at 37 °C was
plotted as a function of time. Note: we commonly see only 60-70%
of the RNA in helix form at time 0 (see Pereira and Platt,
1995).
The mutant rho201 allele was isolated using a
genetic screen, which detected transcriptional readthrough into a
reporter gene due to defective termination (Guarente et al.,
1977). Our sequencing of the mutant rho gene revealed a single amino
acid change within the predicted 3 strand of the ATP binding
domain of rho (Dombroski and Platt, 1989). Although this change (F232C)
lies in the ATP domain, it weakens primary site RNA binding by
100-fold, which is accompanied by decreased discrimination between RNA
substrates. Surprisingly, the secondary RNA site activation
characteristics have become more sensitive, in responding to oligo
r(C)
stimulation at 5-fold lower concentrations than wild
type protein. The mutant protein is a highly efficient ATPase,
equalling or surpassing wild type levels, particularly with nonspecific
RNA molecules. The rho201 protein is nevertheless very defective in
RNA:DNA helicase activity and transcription termination, in agreement
with in vivo observations. This protein therefore harbors an
unusual mutation in which the energy generated by ATP hydrolysis has
become uncoupled from helicase activity, and hence termination.
In contrast to rho201, other rho proteins with amino acid changes in the ATP binding domain are relatively unaffected in their RNA binding function. For example, the K181Q, K181Q/K184Q, and D265N mutants have near normal RNA binding ability, although altered in their ATPase activities and subsequent reactions that are dependent on ATP hydrolysis (Dombroski et al., 1988a). In another case, the termination defective mutant rho ts702 has the mutation A304T within the ATP binding domain (see Platt and Richardson(1992)), and shows wild type ATPase activity with poly(C), although it is severely defective with poly(U) and poly(A) RNA (Shigesada and Imai, 1978). The mutant is postulated to be defective in its secondary site interactions with RNA. A direct measurement of primary site RNA binding has not been reported and we think it is likely that rho ts702, despite defective ATPase activity with non(C) homo-ribopolymers, binds RNA normally, similar to rho suA1 and rho G99V, which are unaffected for binding RNA at the primary binding site and have wild type ATPase activities with poly(C), but are defective with T7 and trp t` RNA (Richardson and Carey, 1982; Pereira and Platt, 1995).
Whether the alteration in the
RNA binding properties or the uncoupling between the ATPase and
helicase activities, or both, are a direct result of the conformational
change in the rho201 protein remains to be determined. Brennan et
al.(1990) observed that the trypsin pattern with wild type rho
bound to poly(C) differed depending on which NTP was present, and that
the energy coupling to RNA release in the helicase assay was two to
four times more efficient for ATP than for GTP, UTP, and CTP,
suggesting some correlation between coupling and conformational
changes. However, studies by Dombroski et al. (1988a) with the
K181Q mutant protein, which displays an altered conformation on probing
with trypsin but does not uncouple ATPase from helicase action, show
that not all conformational changes have such effects. Perhaps amino
acids in the 3 strand are part of the region involved in the
coupling reaction, and if this were true the conformational change
might be incidental to, rather than the cause of the uncoupling effect.
The phenylalanine residue at position 232, while absolutely
conserved in rho homologs from evolutionarily divergent bacteria
(Opperman and Richardson, 1994), is at one of the non-conserved
positions at the end of the 3 strand when compared with other ATP
binding proteins, such as adenylate kinase and the
and
subunits of F
-ATPase (Dombroski and Platt, 1989). The
conformational change in the rho201 (F232C) protein could result from
the formation of an internal disulfide bond. According to the predicted
structure for rho's ATP binding domain (Dombroski and Platt,
1989) the only other cysteine residue within rho (at position 202) lies
at the opposite end of the ATP binding pocket in the
2 strand,
making the distance between these two cysteines too great for
intramolecular bond formation. To ask whether intersubunit disulfide
bonds might be affecting the activity, we tested both the wild type
protein and rho201 in ATPase assays with poly(C) and trp t` as
well as in helicase assays, either in the absence of dithiothreitol or
in the presence of 10 or 100 mM dithiothreitol. We saw no
difference in the results obtained (data not shown). Furthermore,
although the trypsin digestion experiments were done in the presence of
dithiothreitol, rho201 still revealed a conformational change. The
simplest interpretation of these observations is that neither the
conformational change nor the altered activities of the mutant protein
are due to the formation of any disulfide bonds.
One surprising
observation that contrasts with other mutant rho proteins tested is
that rho201's termination activity is unaffected by the presence
of the accessory E. coli protein NusG in vitro (data
not shown). NusG, originally identified as a participant in the N
mediated anti-termination complex (Horwitz et al., 1987; Mason
and Greenblatt, 1991; Li et al., 1992) is also necessary for
rho-dependent termination at some sites in vivo (Sullivan and
Gottesman, 1992) and enhances rho-dependent termination in vitro with both defective RNA substrates (Nehrke et al., 1993)
and with mutant rho proteins (Pereira and Platt, 1995). NusG acts in
part by slowing the off-rate of rho from nascent RNA in stalled
elongation complexes (Nehrke and Platt, 1994), and thus would be
predicted to improve the termination efficiency of mutants with
weakened primary site binding such as rho201 and F62A/F64A, which
display K
values of 1.3 and 1.6 nM for trp t` RNA, respectively (this work and Brennan and Platt,
1991). Indeed with the F62A/F64A mutant, NusG has such an effect. (
)In the case of rho201, since the helicase activity appears
to be uncoupled from ATP hydrolysis, even if NusG did increase the
``dwell time'' of rho on the RNA, the inability to harness
the energy obtained from ATP hydrolysis to subsequent function would
explain rho201's lack of response to NusG.
Our long-term goal is a mutational and biochemical dissection of the overall process of termination, which will entail understanding each of the individual steps along the pathway. Thus far, a number of mutations have been analyzed in our laboratory and elsewhere that address the first stages involving RNA binding and ATPase activation. These include K181Q/K184Q, K181Q, and D265N, which reduce or eliminate the ATPase activity of rho without any apparent effect on the RNA binding properties (Dombroski and Platt, 1988a). Another class of mutations, represented by K352E (rhosuA1) and G99V (derived from rho115) display defects in activating the secondary RNA site of rho, with little significant effect on primary site RNA binding (Richardson and Carey, 1982; Pereira and Platt, 1995). In these cases termination defects correlate with the deficiencies in ATPase activation at the secondary site. In all mutants tested, with the exception mentioned below, the mutational effects are manifested in the levels of ATPase activation, such that poor ATP hydrolysis correlates with poor helicase and termination activities.
The first suggestion that the energy coupling step in the pathway could be affected was provided by the rho nitA18 mutant, which carries an unmapped mutation leading to significantly higher ATPase activity than the wild type protein with different RNA substrates (Shigesada and Imai, 1978). Although its helicase activity was not determined, it had moderate termination activity ranging from 35 to 90% of the wild type efficiency depending on the template used. We have now reported and characterized here a mutational change in the rho201 protein that appears to result in a severe defect in this second critical step in the pathway, the ability to harness ATP hydrolysis to helicase and termination ability. Recently, Miwa et al.(1995) have characterized 14 mutations in the COOH-terminal 100 amino acids of rho, and found that many of these also have ``coupling'' effects on the RNA binding, ATP binding and hydrolysis, and termination activities of rho factor in vitro. The characterization of these and other mutants, in concert with biochemical analysis of rho's structure-function relationships, should help to decipher the mechanism by which RNA binding, ATP hydrolysis, and helicase activity are coupled together to bring about termination of transcription by E. coli RNA polymerase.