From the ¶ Department of Chemistry and the
Department of Biology, Indiana University, Bloomington,
Indiana 47405
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ABSTRACT |
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The in vitro function of the
coliphage tR1 Rho-dependent terminator is governed
primarily by a tripartite upstream sequence element designated
rut. To determine the contribution of the different components of the rut site to terminator function in the
normal context of coupled translation of the nascent cro
message, tR1 variants lacking different rut site sequences
were tested for terminator function in vivo. Intact
rutA and rutB sequences were both necessary for
efficient termination. However, deletion of the upstream
rutA was far more detrimental than deletion of
rutB. The intervening boxB, which encodes a
short RNA stem and loop, could be deleted without reducing termination
or detectably altering Rho's interaction with the corresponding
cro transcript. The relative importance of these sequence
elements was also the same in a minimal in vitro
termination assay system. Rut sequences are therefore essential for terminator function in vivo and
rutA contributes substantially more to tR1 function than
does rutB. The relative contribution of these elements can
be ascribed to differences in Rho's binding affinity for the encoded
transcripts. If other cellular factors also bind the rut
element RNA, they do not alter the relative contribution of its two
regions to Rho-dependent transcription termination in
vivo.
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INTRODUCTION |
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The bacteriophage relies on interactions with the
Escherichia coli host cell transcription apparatus and
regulatory systems for the orderly expression of its genes. The
temporal regulation of
gene expression is achieved in part by
control of transcript elongation, termination, and anti-termination
(1-3). This type of transcriptional regulation in
and other
viruses (4, 5) occurs both through the interaction of accessory factors
with the elongating RNA polymerase complex and by the alteration of interactions within the transcription complex that occur in response to
sequences in the template DNA and newly transcribed RNA (6).
An accessory factor with a central role in the life cycle is the
host Rho protein (7). Rho causes transcription termination in a
multistep interaction with the elongating RNA polymerase complex that
involves an initial high affinity binding to the nascent transcript and
subsequent secondary RNA interactions, which lead to the release of the
RNA and disassociation of the transcription complex (8). These two RNA
interactions are thought to involve two distinct types of RNA binding
sites on the Rho hexamer (9). Little is known about the
corresponding regions in the RNA, beyond a requirement for low
secondary structure (10) and at least a minimum cytosine content (11,
12).
The Lambda tR1 Rho-dependent terminator limits transcription initiated at the PR promoter to the proximal cro gene relative to cII and other downstream genes of the major rightward operon. Like other Rho-dependent terminators, tR1 is composed of two parts, an upstream rut sequence that encodes regions of the nascent transcript to which Rho binds tightly, and a downstream tsp sequence containing points at which paused polymerase complexes are disassociated by Rho (13). The tR1 rut site is interrupted by the N-utilization element, boxB, with the upstream 17 nucleotides designated rutA, and the only partially defined downstream element designated rutB (14). Transcription terminated by Rho at tR1 produces RNAs with heterogeneous 3' ends corresponding to the pauses in transcription elongation at transcription stop point subsites I, II, and III (15). Recently, Richardson and Richardson (13) showed that the upstream rut sequences are the major determinants of termination at tR1, as substitution of the transcription stop point region with foreign sequences did not preclude efficient termination at pause sites in the substituted sequence.
The tR1 rut sequences were identified by studies of transcription termination in vitro using highly purified RNA polymerase and Rho without other accessory protein factors (14, 16). To characterize the role of rut site sequences in transcription termination in vivo, where identified and as yet unidentified (3, 17) factors potentially influence Rho's interaction with the transcription complex, we developed a termination assay system with specific features that address several problems with previous assay methods (18-20). Here we report the effects of deletions of rut site sequences on the function of tR1 in vivo and in vitro and on the abilities of modified cro gene transcripts to bind to Rho and activate Rho ATPase activity. We show that tR1 terminators lacking rutA are more defective than those lacking rutB or boxB, and that the relative importance of these parts for in vivo function is the same as in a purified system with no factors other than Rho.
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EXPERIMENTAL PROCEDURES |
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Materials--
Enzymes used for DNA manipulations were obtained
from New England Biolabs and RNAsin from Promega. Exonuclease III was
from Bethesda Research Labs, T7 DNA polymerase from U. S. Biochemical and proteinase K from Beckman. E. coli RNA polymerase was
from Epicentre Technologies or purified according to Andrews and
Richardson (21). E. coli Rho was purified from
BL21(DE3)[pCB111, pLys] by Lislott Richardson as described by
Nowatzke et al. (22). T7 RNA polymerase was also prepared by
Lislott Richardson as described by Tabor and Richardson (23).
Nitrocellulose Bio-Trace NT filters for Rho RNA binding studies
were purchased from Gellman Scientific and polyethylenimine thin layer
chromatography plates from Brinkman. Ribonucleotides and
deoxyribonucleotides were from Amersham Pharmacia Biotech and
[-32P]ATP and [
-32P]UTP were obtained
from ICN Radiochemicals.
Bacterial Strains--
Hosts for plasmids containing the
PR promoter were lysogenized by
+ to
repress transcription and increase plasmid stability. Strain JG20
(F'{
(lacZ)H220
lacY+ proAB+}
lacpro supE thi
recA938(cmR)::Tn9-200
(ara)174), the host for transcriptional tR1 fusions, was
transduced to
(ara)174,
recA938(cmR)::Tn9-200 by two sequential transductions (24) of strain CSH2O (25) with phage P1
vir.
Plasmids--
To construct plasmid pJGLC, a
560-bp1 HinfI
fragment from pCYC2 (16) was made blunt-ended by T4 DNA polymerase and
ligated into the SmaI site of pBluescriptII KS+ (Stratagene)
such that cro and lacZ
shared the same
orientation. Plasmid pJGBTZ was constructed from pJGLC in a somewhat
indirect series of ligations involving an initial placement of the
pTac promoter from pKK233-3 (Amersham Pharmacia Biotech)
upstream of the cro-lac fusion. A 589-bp
HindIII-BamHI fragment from pJGLC was ligated to
the HindIII-BamHI vector fragment of the
lac fusion plasmid pTL61T of Linn and St. Pierre (26). After
excising a 70-bp EcoRI fragment from the resulting
construct, an HgiAI-BamHI fragment containing the
pTac promoter from pKK233-3 was ligated to the 8856-bp
PstI-BglII vector fragment. A 7062-bp fragment
containing the cro-lac fusion region generated by
BspHI cleavage and partial BspMI digestion was
then made blunt-ended and ligated into the SmaI site of a
modified low-copy plasmid pCL1921 (27) from which the lac
promoter containing EcoRI fragment had been deleted. The
resulting cro-lac fusion was oriented in the
opposite direction from the remaining pCL1921 lacZ
fragment. Upstream cro sequence beginning with the fifth nucleotide of the cro transcript was then restored on the
plasmid by replacing the 210-bp SmaI-NsiI
cro fragment with a 280-bp RsaI-NsiI fragment from pJGLC. Construction of pJGBTZ was completed by the addition of the F1 phage origin of replication from plasmid pUCF1 (Amersham Pharmacia Biotech) as a HincII-KpnI DNA
fragment between unique BalI and KpnI sites, and
ligation of an EcoRI-NsiI fragment containing the
araBAD promoter and divergent araC coding region from plasmid pBAD182 in place
of the 1057-bp PstI-EcoRI tac promoter
fragment.
tR1 Deletions--
Plasmids deleted for rut site
sequences as shown in Fig. 2 were constructed by a variety of methods
depending on the availability of appropriate restriction endonuclease
cleavage sites. Plasmids pJGLCrutA-B and
pJGLC
boxB were created by annealing deoxyoligonucleotides to single-stranded pJGLC templates so that specific residues were "looped-out" and therefore excluded from the complementary DNA strand synthesized in vitro (28). In plasmid
pJGLC
rutA, 33 base pairs immediately downstream of the
cro stop codon were removed by the sequential action of
EcoNI, E. coli exonuclease III, mung bean
exonuclease, and T4 DNA ligase. In pJGLC
rutB, a 26-bp
deletion was created by removing an EcoNI-BstXI
restriction fragment from pJGLC and recircularizing the vector fragment
after T4 DNA polymerase treatment. Deletions were transferred from
pJGLC to reporter gene fusion plasmid pJGBTZ as
BglII-NsiI restriction fragments containing the
altered regions. The entire tR1 rut site region was removed in the derivation of pJGBZ from plasmid pJGBTZ by replacement of a
184-bp AvaI-BamHI fragment with a linker fragment
obtained from the digestion of the annealed and extended
oligodeoxynucleotides CCTTCCCGAGTAACAAAAAAACAACAGC and
GTTAGGATCCTTATGCTGTTGT.
Determination of tR1 Termination Efficiency in Vivo--
JG20
strains containing transcriptional fusions were grown in a defined
medium (M9 salts, 0.2% glucose, 0.4% casamino acids, 40 µg/ml
L-leucine, 10 µg/ml thiamine, 0.2% arabinose, and 70 µg/ml spectinomycin), and -galactosidase activity representing transcriptional readthrough of tR1 was determined. For
-galactosidase assays, frozen stocks were inoculated into defined
medium lacking arabinose and grown 4 h before the addition of
arabinose. Cultures were then grown overnight before inoculation at
0.15% (v/v) into 2 ml of complete defined media. Experimental cultures
were grown for 4 h (approximately 5 doublings) on a tube roller at
37 °C to an A600 of 0.3-0.4 and placed on
ice for exactly 20 min before the determination of
o-nitrophenyl-
-D-galactopyranoside hydrolysis rates in cells permeabilized by SDS and chloroform (24).
In Vitro Transcription--
DNA fragments equivalent to
the 560-bp HinfI fragment containing PR and
tR1 (15) were synthesized by 17 cycles of PCR (29). Transcription
templates were prepared by addition of proteinase K (50 µg/ml) to PCR
reactions and incubation for 20 min at 37 °C followed by a single
chloroform-isoamyl alcohol (24:1) extraction. Aqueous phases were then
loaded directly onto 0.8% low-melting agarose gels before recovery of
the desired DNAs by a modified freeze-thaw extraction (30). Templates
were then desalted on Sephadex G50 spin columns (31) and equilibrated
with 10 mM Tris acetate (pH 7.8).
Rho Binding and ATPase Cofactor Activity Assays--
Templates
for synthesis of microgram quantities of tR1 containing RNAs were
prepared by incorporating a T7 promoter upstream of the cro
coding sequence by PCR. Primers T7croG
(GCAAGAATTTAATACGACTCACTATAGGGATCTACTAAGGAGGAGGTTGTATGG) and L
(CGATTCGTAGAGCCTCGTTG) were extended on pJGLC and deletion plasmid templates for the synthesis of wild-type and mutant
cro RNAs. These RNAs are identical to those synthesized by
E. coli RNA polymerase on the 560-bp HinfI
fragment except for replacement of "AUG" by "GGAUC" at the 5'
termini. Such 5'-modified full-length cro RNAs showed very
similar Rho binding and maximum ATPase cofactor activities to
unmodified cro RNAs and RNAs with different 5'-terminal base
modifications (33). Templates encoding truncated cro RNAs used in some experiments were synthesized with alternate downstream primers rather than L. Rho binding and ATPase assays were performed as
described previously (13, 33). The binding data were fitted to
theoretical binding curves using the curve-fitting program GraFit
(Erithacus Software, Ltd. Staines, United Kingdom).
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RESULTS |
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Rut Sites Mediate Termination in Vivo--
To test the role of the
rut site and its sequence elements in termination at tR1
in vivo, we constructed a transcriptional fusion plasmid
that is well suited for quantitative analysis of transcription
termination. Plasmid pJGBTZ (Fig. 1)
contains a complete translated cro coding sequence and tR1
terminator positioned between the tightly regulated araBAD
promoter and the lacZ reporter gene on a low-copy
pSC101-derived replicon. The fusion junction contains an RNase III
processing site allowing a uniform -galactosidase message to be
generated in all tR1 constructs, and the entire region is insulated
from transcription initiated outside the region by flanking
rrnB terminators, features developed by Linn and St. Pierre
(26). The araBAD promoter allows adjustment of
-galactosidase expression levels to those readily tolerated by the
host cell.
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In Vitro Termination--
To determine whether intracellular
factors other than Rho contributed to the different relative importance
of rutA and rutB in termination, the efficiency
of Rho-dependent transcription termination in
vitro with purified RNA polymerase was measured for these
terminator deletions. Fig. 4 shows the
products of a single round of transcription initiated at PR
where transcripts were elongated in the presence of relatively high
levels of all four nucleoside triphosphates. Under conditions in which
a nearly saturating level of Rho caused termination at a level of
73%,3 the efficiencies of
termination with the rutA,
rutB, and
boxB templates were 27, 48, and 70%, respectively,
indicating that rutA, rutB, and boxB
contribute 63, 34, and 4%, respectively, to the level of termination
of the complete sequence. Within experimental error, these values are
the same as those found with the in vivo measurement. Thus,
the relative contributions appear to be intrinsic to the basic
interaction between Rho and the transcription complex and are not
dependent on other cellular factors, including those known to interact
with the overlapping nutR region (3).
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Rut Site Deletions and Transcriptional Pausing-- Because the efficiency of transcription termination is dependent on Rho's ability to interact with a paused transcription complex, we analyzed the kinetic progress of transcript elongation on the tR1 deletion templates described. Fig. 5 shows the products of an in vitro transcription reaction in which the progress of the transcription complex can be followed in terms of the discrete RNA products synthesized at particular intervals. Although analysis of transcriptional pausing is approximate at best on such templates of unequal length in which different multiple early pause sites lead to asynchronous later pausing at relevant transcription stop points, it can be seen that polymerases continued to make substantial pauses at all three tR1 subsites on each deletion template.
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RNA Binding--
A critical aspect of Rho's termination activity
is the ability to make stable ATP-independent interactions with nascent
transcripts. A filter binding assay developed by Ceruzzi and Richardson
(36) was used to determine the nature of the defects in termination associated with the tR1 deletions described. Rho bound wild-type cro messages extended to the third tR1 subsite with the
previously established high affinity (Table
I) (33). The rutA deletion reduced Rho's apparent Ka for the RNA approximately
9-fold, whereas
rutB resulted in a more modest 3-fold
decrease (Table I). Deletion of boxB had little effect on
Rho's ability to bind the corresponding transcript, whereas the
rutA-B deletion reduced Rho binding approximately
13-fold, similar to a more extensive rut deletion previously
characterized (
AR70; Ref. 33). As well as being required for a high
affinity Rho-cro interaction, addition of the 23 rutA nucleotides in their native context immediately downstream of the cro coding sequence was sufficient to
allow high affinity Rho binding of transcripts truncated at the
cro stop codon (data not shown).
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Rho-ATPase Activation--
In addition to binding to the nascent
transcript, interactions between Rho and RNA that are coupled to ATP
hydrolysis are necessary for Rho-dependent transcription
termination (37, 38). The ability of a transcript to elicit this type
of productive interaction can be assessed by the degree to which it can
activate ATP hydrolysis by Rho when the RNA is present at a
concentration well above the Kd for dissociation of
the Rho-RNA complex. As shown by the data in Table I, Rho had virtually
no ATPase activity with the rutA-B RNA as a cofactor and
only partial activity with other rut site deletion RNAs as
cofactors; again the reduction in ATPase activity was well correlated
with the reduced termination activity of the corresponding mutant tR1
terminators.
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DISCUSSION |
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We have shown that the rut sequences of tR1 are
necessary for efficient terminator function in vivo. As
these sequence elements were identified from studies of transcription
termination in vitro, our results establish their central
role in transcription termination in E. coli in the presence
of all accessory transcription factors and in the normal context of
coupled translation of the nascent cro mRNA.
With our assay system, we found that the in vivo termination efficiency at tR1 was 70-80%, which is at the high end of what has been previously reported when various other methods were used to measure in vivo tR1 function (18-20, 39). Although the relatively greater efficiency of termination at tR1 as we determined it may represent a more accurate assessment with our assay system, the possibility remains that differences at the 5'-end of the cro mRNA related to the substitution of araPBAD for PR influenced terminator function. Although all fusions were made such that transcripts contained the entire cro open reading frame and native Shine-Dalgarno sequence, possible upstream structural differences would still have the potential to affect the efficiency of translation initiation and therefore interactions between Rho and the nascent cro transcript. However, whereas these differences, as well as the specific choice of host genetic background and culture conditions, have the potential to alter the absolute level of termination at wild-type tR1, such effects would not be expected to prevent the determination of the relative termination defects in the deletion mutants studied.
The tR1 rut region encodes two single-stranded RNA regions, which are interrupted by the small boxB RNA hairpin (40). Similar small hairpin structures were previously shown to be a conserved component among Rho-binding RNAs affinity-selected from a large pool of random sequence RNAs (41). In contrast, we found that boxB can be deleted with essentially no effect on termination efficiency at tR1, and without reducing Rho's affinity for cro RNA. Thus, the boxB hairpin structure is not an important component of the RNA-binding region of tR1.
Of the two remaining components of the rut region, the upstream rutA was more important than rutB for both terminator function and in terms of the contribution of each to the binding affinity of Rho for the cro RNA. The RNA region encoded by rutA is much richer in cytidylate residues than that encoded by rutB (see Fig. 2), confirming the importance of this residue in both terminator function (11, 12, 42) and binding of Rho to RNA (43, 44).
RutA also contains a boxA sequence (45), which
differs by only a single residue from a consensus motif that has been
shown to bind the NusB-S10 complex (46). Deletions of rutA
and upstream sequences that had the greatest effect on termination also
eliminated boxA, and 5' nucleotides specifically proposed as
capable of binding an unidentified "inhibitory host factor" in N-mediated antitermination (3, 47). Our results suggest that Rho is
this host factor but do not rule out the possibility that Rho is also a
competitor for the same RNA site as the NusB-S10 complex and an
unidentified inhibitor of antitermination. Although such host
factor-nutR interactions have the potential to modulate Rho
interactions at tR1, the finding that the relative contributions of
rutA and rutB to the terminator function were the
same in vitro and in vivo suggests that if
NusB-S10 or other cellular factors are binding to sites in the
rut region, they are doing so in a way that does not alter
the relative recognition of those segments by Rho.
The E. coli trp t' terminator, like tR1, is capable of
remaining partially functional despite significant deletion of upstream regions (48). This bacterial terminator was, however, much more resilient to deletions of the size that our results show drastically reduce termination at tR1, and no single region of trp t'
appeared to be required for efficient termination under the conditions examined (48). trp t' therefore appears to contain a greater redundancy of sequence necessary for productive interaction with Rho
than does tR1.
Our results confirm the previous finding of Faus and Richardson (33) that the deletion of the rut region causes a surprisingly small change in the binding affinity of Rho for the isolated full-length cro transcript, yet results in a nearly complete loss of termination and ATPase cofactor activity of the encoded transcript. One explanation for the apparently small difference in binding affinities is that Rho makes a number of different kinds of nonproductive interactions with the rut deletion RNAs that are comparable with the complexes a repressor protein makes with nonoperator DNA sites. The overall binding affinity of Rho for an RNA lacking a rut site would therefore represent the sum of separate weak interactions at a large number of different nonspecific sites. In that case, the true difference in energy between Rho complexed with a single nonspecific site and Rho binding a specific rut site is probably much greater than the 2 kcal/mol that we calculated by comparison of the ability of Rho to bind full-length cro RNAs with and without the rut sites.
cro RNAs that lack rut sequences are unable to activate significant Rho ATP hydrolysis even when they are present at a concentration that is well above the Kd for the Rho-RNA binding interaction. This observation suggests that the complexes that do form with the nonspecific sites do not appear to have the RNA positioned correctly for subsequent ATP-dependent interactions. Hence, these nonspecific interactions that do form do not lead to ATP hydrolysis or the processes coupled to ATP hydrolysis that lead to the disruption of the RNA-DNA helix in the transcription complex (49, 50). The rut site thus appears to serve two roles; it provides a strong attachment site for Rho, and it positions the RNA on the protein for the ATP-dependent reactions that result in transcription termination.
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FOOTNOTES |
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* This work was supported by Grant AI10142 NIAID, Department of Health and Human Services.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: Dept. of Biology, Campus Box 1137, Washington University, One Brookings Dr., St. Louis, MO 63130-7323.
To whom correspondence and reprint requests should be
addressed. Tel.: 812-855-1520; Fax: 812-855-8300; E-mail:
jrichard{at}bio.indiana.edu.
The abbreviations used are: bp, base pair(s); PCR, polymerase chain reaction.
2 L. Guzman, unpublished material.
3 The main reason this value is significantly lower than values we have obtained previously (14, 32) is because we purposely used higher levels of NTPs.
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REFERENCES |
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