From the Department of Biological Sciences, Stanford University, Stanford, California 94305
Received for publication, September 28, 2000, and in revised form, October 19, 2000
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ABSTRACT |
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Expression of the tryptophanase (tna)
operon of Escherichia coli is regulated by catabolite
repression and tryptophan-induced transcription antitermination.
Catabolite repression regulates transcription initiation, whereas
excess tryptophan induces antitermination at Rho
factor-dependent termination sites in the leader region of
the operon. Synthesis of the leader peptide, TnaC, is essential for
antitermination. BoxA and rut sites in the
immediate vicinity of the tnaC stop codon are required for
termination. In this paper we use an in vitro S-30
cell-free system to analyze the features of tna operon
regulation. We show that transcription initiation is cyclic AMP
(cAMP)-dependent and is not influenced by tryptophan. Continuation of transcription beyond the leader region requires the
presence of inducing levels of tryptophan and synthesis of the TnaC
leader peptide. Using a tnaA'-'trpE fusion, we demonstrate that induction results in a 15-20-fold increase in synthesis of the
tryptophan-free TnaA-TrpE fusion protein. Replacing Trp codon 12 of
tnaC by an Arg codon, or changing the tnaC
start codon to a stop codon, eliminates induction. Addition of
bicyclomycin, a specific inhibitor of Rho factor action, substantially
increases basal level expression. Analyses of tna mRNA
synthesis in vitro demonstrate that, in the absence of
inducer transcription is terminated and the terminated transcripts are
degraded. In the presence of inducer, antitermination increases the
synthesis of the read-through transcript. TnaC synthesis is observed in
the cell-free system. However, in the presence of tryptophan, a
peptidyl-tRNA also appears, TnaC-tRNAPro. Our findings
suggest that inducer acts by preventing cleavage of TnaC peptidyl-tRNA.
The ribosome associated with this newly synthesized peptidyl-tRNA
presumably stalls at the tnaC stop codon, blocking Rho's
access to the BoxA and rut sites, thereby
preventing termination. 1-Methyltryptophan also is an effective inducer
in vitro. This tryptophan analog is not incorporated into TnaC.
The enzyme tryptophanase catalyzes the degradation of
L-tryptophan to indole, pyruvate, and ammonia. Degradation
allows organisms to utilize tryptophan as a source of carbon, nitrogen,
and energy (1, 2). The tryptophanase reaction is reversible; thus, L-tryptophan can be formed from indole and
L-serine, L-cysteine, or pyruvate and ammonia
(3, 4). The tna operon of Escherichia coli
contains two major structural genes, tnaA, encoding
tryptophanase, and tnaB, specifying a low affinity
tryptophan permease (5, 6). The tna operon promoter is
separated from the tnaA structural gene by a 319-nucleotide
leader region. This region encodes a 24-residue peptide, TnaC, that is
essential for induction. Transcription of the tna operon is
regulated by the combined action of catabolite repression and
tryptophan-induced transcription antitermination. Regulation by
catabolite repression requires the catabolite gene activator protein
plus cyclic AMP, and is tryptophan-independent (5, 7, 8). Transcription
termination/antitermination in the leader region of the operon is
regulated in response to high levels of tryptophan. Studies in
vivo and in vitro have shown that in the absence of
tryptophan, transcription is subject to Rho-dependent
termination at several transcription pause sites located between
tnaC and tnaA (9, 10). In the presence of inducing levels of tryptophan, termination at these sites is prevented (10). Translation of tnaC is essential for tryptophan
induction (11). Specifically, inactivating the tnaC start
codon, replacing the single Trp residue of TnaC with a different amino
acid, or introducing several other amino acid changes in TnaC,
prevents induction (13).1
BoxA and rut sites located immediately adjacent
to the tnaC stop codon are essential for
Rho-dependent termination; alteration of either of these
sites reduces termination (14). On the basis of these and other
findings, it has been proposed that, in the presence of inducer, TnaC
acts in cis on its associated translating ribosome to
inhibit its release at the tnaC stop codon. Inhibition of
ribosome release could block Rho's access to the BoxA or
rut sites of the transcript and thereby prevent
transcription termination (15-17).
To define the roles of TnaC, tryptophan, and Rho factor in regulation
of tna operon expression, we developed a cell-free S-30 system in which regulation of tna operon could be studied.
In this paper we show that the major regulatory features of
tna operon observed in vivo can be duplicated
in vitro. We examined transcription under inducing and
noninducing conditions and demonstrate that in the absence of inducer
transcription terminates at several sites in the leader region, and the
terminated transcripts are then degraded. In the presence of tryptophan
transcription pausing is observed, but the paused transcripts
subsequently are elongated. We also detected and analyzed the synthesis
and fate of the TnaC peptide. We show that tryptophan induction leads
to the accumulation of TnaC-tRNAPro. This finding
implicates tryptophan-induced inhibition of cleavage of
TnaC-peptidyl-tRNAPro as the event crucial to induction.
Bacterial Strains and Plasmids--
A derivative of the E. coli A19 RNaseI
Plasmid pGF4 was constructed by inserting a
HindIII-BamHI fragment containing a
tnaA'-'trpE translational fusion from pKG3 (constructed by
Kirt Gish) into pBR322. pGF14 and pGF71 were constructed by replacing
the HindIII-NsiI fragment of pGF4 containing the tna promoter through the ATG start codon of tnaA
by an homologous fragment from pPDG14 and pPDG71 (constructed by P. Gollnick), respectively. In pPDG14, tnaC Trp codon 12 is
replaced by an Arg codon; in pPDG71 the tnaC ATG start codon
is replaced by TAG. Plasmid pPDG52 (constructed by P. Gollnick)
contains a tnaC'-'lacZ translational fusion.
pGF24 was constructed by cloning the PstI-BamHI
fragment containing E. coli rpoBC"t" from plasmid pSVS24
(constructed by V. Stewart) into pUC18. pGF1 was constructed by
ligating a PCR2 fragment
(with an engineered HindIII site at its 5' end and a PstI site at its 3' end) containing the region from
To facilitate clean transcription and translation, we prepared circular
DNA templates. We performed PCR on plasmid pGF25-00 and amplified the
region from In Vitro Protein Synthesis--
S-30 extracts were prepared
according to Zubay (18). Unless otherwise specified, the standard
coupled transcription/translation reaction mixture (with a total volume
of 50 µl) contains 35 mM Tris acetate, pH 8.0, 10 mM magnesium acetate, 200 mM potassium glutamate, 30 mM ammonium acetate, 2 mM
dithiothreitol, 2 mM ATP, 0.5 mM each of CTP,
UTP, and GTP, 20 mM phosphoenol pyruvate (trisodium salt),
0.25 mg/ml E. coli tRNA (Sigma), 0.3 unit/ml pyruvate
kinase, 35 mg/ml polyethylene glycol (8000), 20 µg/ml folinic acid, 1 mM cyclic AMP, 200 µM each of 19 amino acids,
with varying amounts of added L-tryptophan, and plasmid DNA
or a circularized DNA fragment. All reactions were performed at
37 °C. To measure plasmid-directed
To measure RNA synthesis, unlabeled UTP was omitted from the incubation
mixture. After 10 min of preincubation at 37 °C, 20 µCi of
[ Identification of
TnaC-peptidyl-tRNAPro--
After CF-tna+306rpoBC"t"
directed cell-free transcription and translation (20 µCi of
[35S]methionine, labeling for 20 min), reactions were
stopped by acetone precipitation, centrifuged, and the pellets boiled
in SDS gel loading buffer for 3 min. Samples were separated
electrophoretically on a 10% Tricine-SDS-PAGE. The 25-kDa band was
localized by autoradiography, excised, and recovered by immersion in a
low pH buffer (20 mM Tris-HCl, pH 6.8, 50 mM
NaCl) overnight at 4 °C. The recovered 25-kDa molecules were used as
RT-PCR template with an Access RT-PCR kit (Promega, Madison, WI). Two
oligonucleotides specific for tRNAPro (tRNApro-Plus,
5'-CGGCACGTAGCGCAGCCTGGTAGC-3'; tRNApro-Minus, 5'-TGGTCGGCACGAGAGGATTTGAAC-3') were used. A S-30 reaction without the
CF-tna+306rpoBC"t" template was also loaded on the same gel, and
the corresponding band was recovered as a negative control for
RT-PCR.
For RNase digestion and proteinase K treatment, the recovered 25-kDa
molecules were incubated for 10 min at 37 °C with 10 µg of RNase
A/ml or 50 µg of proteinase K/ml, mixed with an equal volume of 2×
SDS gel loading buffer, and separated on a 10% Tricine-SDS protein gel.
Enzyme Assays--
Anthranilate synthase activity was determined fluorometrically by
measuring the conversion of chorismate plus glutamine to anthranilate
(21). Specific activity was calculated as fluorometer units produced
per 50 µl of cell-free reaction mixture following a 20-min incubation
at 37 °C. Fluorometer units were based on the following standard; 5 µl of a 1 mM solution of anthranilic acid were added to a
tube containing the standard 0.5-ml reaction mixture, and the tube was
incubated and processed with the assay tubes. Following acidification
and extraction of anthranilate with ethyl acetate, the ethyl acetate
extract from the standard tube was set at 100 fluorometer units. A
blank reaction tube was treated similarly, and the ethyl acetate
extract was set at zero. Fluorometer units were then determined for
each assayed sample.
Cyclic AMP-dependent Expression of a tnaC'-'lacZ Fusion
in an S-30 System--
In previous studies it was shown that
initiation of transcription of the tna operon is regulated
by catabolite repression (8). A presumed cAMP-CAP complex binding site
is located just upstream of the Basal and Induced Expression with the tnaA'-'trpE
Construct--
To study tryptophan induction of tna operon
expression in vitro, we prepared a special construct, pGF4,
containing the intact tna promoter-leader region followed by
a translational fusion of the initial segment of tnaA fused
to trpE of E. coli (Fig. 1B). The
resulting fusion protein TnaA-TrpE would be tryptophan-free; thus,
in vitro synthesis could be examined in the absence as well as the presence of tryptophan. The strain used to prepare the S-30
extract carried a trpR mutation and a trpE
deletion; this extract has high levels of TrpG-D but no TrpE enzyme
activity. As expected, expression of the tnaA'-'trpE fusion
was cAMP-dependent; without cAMP, very low levels of
TnaA-TrpE enzyme activity were detected either in the absence or
presence of tryptophan (Table I). In the presence of cAMP, addition of
300 µM tryptophan led to a 17-fold increase in synthesis
of the TnaA-TrpE protein. Addition of a tryptophan analog, 1MT, also
led to a 20-fold increase (Table I). We believe that the increase in
tryptophan-free TnaA-TrpE protein synthesis in the presence of
tryptophan, or 1MT, is due to their action as inducers of
antitermination in the leader region.
We also examined the effects of tryptophan addition on TnaA-TrpE
synthesis by measuring [35S]methionine incorporation into
the TnaA-TrpE protein, following SDS-polyacrylamide gel electrophoresis
(Fig. 2). Template plasmid pGF4 contains
a Changing the tnaC Start Codon to TAG, or Replacing the tnaC Trp
Codon 12 by an Arg Codon, Prevents Tryptophan Induction--
It was
shown previously, in vivo, that synthesis of the intact TnaC
peptide containing its crucial Trp residue is essential for tryptophan
induction of tna operon expression (13).1 To
verify the role of tnaC translation in induction of the
tna operon in the S-30 system, Trp codon 12 (TGG) and
tnaC start codon (ATG) in the tnaA'-'trpE
construct were replaced by an Arg codon (CGG) and a stop codon (TAG),
respectively. Replacing the tnaC start codon by a stop codon
(pGF71, Fig. 1B) should eliminate leader peptide synthesis.
Basal level expression by this plasmid was decreased 5-fold relative to
that of the wild type plasmid pGF4, and induction by tryptophan was
abolished (Table II). Replacing Trp codon
12 by an Arg codon (pGF14, Fig. 1B) should allow peptide synthesis to proceed to the normal TGA stop codon of tnaC,
generating an altered peptide. TnaA-TrpE production directed by pGF14
was decreased 3-4-fold compared with that of the wild type
tnaA'-'trpE fusion plasmid pGF4, and was not inducible by
tryptophan (Table II). Addition of arginine had no effect on expression
of pGF14 (TnaC Trp12 Bicyclomycin Increases Basal Level Expression of All tnaA'-'trpE
Constructs--
Bicyclomycin is a specific inhibitor of E. coli Rho factor. This antibiotic inhibits the poly(C)-stimulated
ATPase activity of E. coli Rho factor (22, 23). Consistent
with this finding, it has been shown that bicyclomycin increases basal
level expression of the tna operon in vivo (24).
Addition of an appropriate concentration of this antibiotic directly to
the S-30 system should therefore inhibit Rho factor activity and
increase basal level expression of the tna operon. To
examine this possibility, 50 µg/ml bicyclomycin was added in
vitro with tnaA'-'trpE plasmids that did or did not have tnaC mutations. Bicyclomycin relieved
Rho-dependent termination in the tna operon,
irrespective of whether an intact TnaC peptide could be synthesized
(Table II).
Characterization of tna Operon Regulation in the S-30
System--
To optimize reaction conditions, we examined three
variables: reaction time, plasmid pGF4 concentration, and the
concentration of inducer. Fig.
3A presents the results of a
time-course experiment performed in the presence of inducer. TnaA-TrpE
enzyme activity was observed to increase linearly for 40 min, whereupon
it began to level off. In the absence of inducer, there was no
significant increase in TnaA-TrpE activity. Plasmid concentration was
varied, as shown in Fig. 3B. When 5 nM pGF4 was
present, there was 18-fold induction by 300 µM
tryptophan. Increasing the pGF4 plasmid concentration to 10 nM elevated basal level expression appreciably, and there was only 1.2-fold induction by added tryptophan. When the pGF4 plasmid
concentration was increased to 15 nM or greater, basal level expression increased further and there was no significant effect
of added tryptophan. These results are consistent with the intensities
of the TnaA-TrpE bands observed on a SDS-PAGE gel (Fig. 2, compare
lanes 7 and 8); when 15 nM
pGF4 was used, the [35S]methionine-labeled TnaA-TrpE
protein bands in lane 7 (without added
tryptophan) and lane 8 (with 300 µM
tryptophan) were indistinguishable. These data suggest that the S-30
system employed is limiting for some component required for efficient
transcription termination under noninducing condition.
1MT, an analog of tryptophan, effectively induces tna operon
expression in vivo (25). It is thought that 1MT induces
tna operon without being charged onto tRNATrp or
being incorporated into protein (see below) (13). The effect of varying
1MT concentration on TnaA-TrpE production in our S-30 system is shown
in Fig. 3C. A concentration of 0.5-1 mM was
sufficient to fully induce tna operon expression; maximum
induction provided a 23-fold increase in ASase-specific activity over
that observed in the absence of added 1MT. Increasing the tryptophan
concentration from 50 µM to 300 µM (Fig. 2,
lane 4 versus lane 5) also
led to a significant increase in TnaA-TrpE production. Since
tryptophanyl-tRNA synthase has a relatively high affinity for
tryptophan (Km of 5 × 10 Rho Is Limiting in Our Cell-free System--
One unexpected
finding was that, when the concentration of plasmid pGF4 was increased
from 5 to 15 nM, basal level expression of
tnaA'-'trpE increased dramatically (Fig. 3B).
This observation suggests that a factor(s) responsible for (or
participating in) basal level expression in the S-30 system might be
limiting, or titrated out. To determine whether increased translation
of tnaC is responsible for this presumed titration effect,
constructs with mutations in tnaC that prevent induction
were tested. It can be seen in Table III
that, with 15 nM pGF71 (or pGF14) (Fig. 1B) as
template, the results obtained are similar to those observed with pGF4
(Fig. 3B). These findings indicate that translation of
tnaC is not responsible for the apparent titration
effect.
300 µM Trp was added, as indicated.
ND, not determined.
A second reasonable explanation is that Rho may become limiting because
the additional tna transcripts produced contribute BoxA and rut binding sites that sequester much of
the available Rho. If this is the case, addition of purified Rho
protein should reverse the titration effect. As expected, when 6 µM Rho was added to the reaction mixture (Table III),
both the basal and induced levels of tna operon expression
were lowered, and now 6-fold tryptophan induction was observed with
wild type tnaC plasmid pGF4. No induction was observed with
either pGF14 or pGF71, as expected. These findings support the
interpretation that Rho is responsible for setting the basal level of
expression of the tna operon.
Measurement of tna mRNA Synthesis--
To demonstrate directly
that tryptophan has no effect on cAMP-dependent
transcription initiation at the tna promoter, a circularized DNA template was constructed that contains the intact tna
promoter but lacks the leader region beyond bp +6,
CF-tna+6rpoBC"t". This circular template was used to direct the
S-30 system (Fig. 4A). The
predicted ~130-nucleotide transcript would contain only the first 6 nucleotides of the tna leader region, immediately followed by the E. coli rpoBC terminator (rpoBC"t").
In Fig. 4B it can be seen that with this template
transcription initiation from the tna promoter was highly
efficient and was dependent on the presence of cyclic AMP in the
reaction mixture. No effect of added L-tryptophan was
evident.
To confirm that the tryptophan induction observed with the
tnaA'-'trpE translational fusion construct was due to relief
from Rho-dependent termination, another circularized
construct, CF-tna+306rpoBC"t", was examined (Fig.
5A). This construct contains
the intact tna promoter, tna leader region to
+306, followed by the rpoBC transcription terminator.
Transcripts produced from this template that escape Rho-dependent termination should terminate at the
rpoBC terminator, yielding a ~430-nucleotide read-through
transcript (Fig. 5A). No transcripts were observed when cAMP
or the template was omitted from the S-30 system (Fig. 5B,
Single-round Transcription Analyses in the S-30 System--
In the
experiment described in Fig. 5B (
In other experiments, we tested templates analogous to
CF-tna+306rpoBC"t" but bearing various tnaC mutations
(ATG to TAG, or Trp12 Synthesis of the TnaC Peptide in Vitro--
Previous attempts to
synthesize TnaC of E. coli in an S-30 system were
unsuccessful (28). When we used the tnaA'-'trpE construct pGF4 to program protein synthesis, we also did not observe TnaC peptide
production. Synthesis was examined using a Tricine-SDS protein gel (see
Fig. 2). To improve our ability to detect TnaC, we decided to eliminate
competition for transcription or translation. Therefore, we changed our
strategy and used a circularized small PCR fragment,
CF-tna+306rpoBC"t", as template. This circularized template
contains only the tna promoter, the tna leader
region to bp +306, followed by the rpoBC terminator. The
only transcripts expected would be those initiated at the
tna promoter, and the resulting peptide product would be
TnaC. In Fig. 7A it can be seen that, with [35S]methionine as label, two new bands
(a ~3-kDa band and a ~25-kDa band) appear in the presence of added
tryptophan. The ~3-kDa band (but not the ~25-kDa band) is present
in the absence of added tryptophan. Neither of these bands is present
when the CF-tna+306rpoBC"t" template is omitted from the S-30
reaction (Fig. 7A, lane 1). The ~3-kDa band
could be TnaC, and the ~25-kDa band could be TnaC-peptidyl-tRNA. Some
nonspecific products were also detected in all the reactions.
The 2.9-kDa 24-residue TnaC peptide predicted from the tnaC
nucleotide sequence should contain 1 Trp, 4 Ile, 1 Pro, and no Tyr
residues (5). Accordingly, [3H]Trp,
[3H]Ile, and [3H]Pro should label TnaC
while [3H]Tyr should not. In vitro synthesis
of the leader peptide was performed in the presence of
[3H]Trp, [3H]Ile, [3H]Pro, or
[3H]Tyr (Fig. 7B). As expected, when
[3H]Tyr was used, no protein band was labeled, either in
the absence or presence of tryptophan (lanes 5 and 6). The ~3-kDa peptide band was detected when
[3H]Ile or [3H]Pro was used
(lanes 1-4). [3H]Trp can label the
~3-kDa peptide in the absence of added unlabeled tryptophan
(lane 7), and in the presence of 1MT as inducer
(lane 8). No [3H]Trp-labeled band
was visible when unlabeled tryptophan was added (lane
9). We also observed that, when a near-identical construct was used in which the tnaC start codon was replaced by TAG,
no ~3-kDa or ~25-kDa product was detected (data not shown). Taken together, these data suggest that the ~3-kDa peptide band is TnaC. These findings also indicate that 1MT, an effective inducer, does not
compete significantly with [3H]Trp incorporation into the
TnaC peptide.
Identification of TnaC-peptidyl-tRNAPro--
The
~25-kDa band, observed only in the presence of added inducer (Fig. 7,
A and B), has the same labeling characteristics as TnaC. Thus, [3H]Tyr did not label the ~25-kDa band
(Fig. 7B, lane 6), whereas [3H]Pro (lane 2),
[3H]Ile (lane 4), and
[3H]Trp (lane 8) did. Since the
read-through transcript produced from our template should be about 430 nucleotides in length (Fig. 5, A and B), this
~25-kDa molecule cannot simply be a protein product resulting from
translation of this read-through transcript. Thus, we considered it
likely that the ~25-kDa band was a peptidyl-tRNA. To prove that this
~25-kDa molecule is indeed TnaC linked to another molecule, and to
further characterize this molecule, the 25-kDa band was excised from
the gel and treated with RNase A, proteinase K (Fig.
8A), and DNase. After RNase A
treatment, the ~25-kDa band disappeared and the labeled product
shifted to the TnaC position. The ~25-kDa molecule disappeared after
proteinase K digestion (Fig. 8A). DNase treatment had no
effect (data not shown). These data suggest that this ~25-kDa
molecule contains TnaC linked to a RNA.
The most likely identity of the ~25-kDa band is
TnaC-peptidyl-tRNAPro (the C-terminal residue of TnaC is
Pro). To examine this possibility, the ~25-kDa molecule was separated
from E. coli total tRNA by long distance Tricine-SDS-PAGE,
located by autoradiography, excised, and used as template for RT-PCR
using primers based on the sequence of tRNAPro. A S-30
sample that had not been incubated with the circularized DNA template
was also loaded on the same gel, and the corresponding band was
recovered as a control to test for tRNA contamination. The S-30 control
did not yield a product (Fig. 8B, lane 2), and the lane in which read-through was omitted also lacked a product (lane 3), while a 70-80 bp PCR product was
observed using the ~25-kDa molecule as template (lane
4). As a positive control, total tRNA from E. coli (Sigma) was used as template. We observed the same 70-80-bp
product plus a shorter product (lane 1). These findings indicate that the ~25-kDa molecule observed in the presence of tryptophan is TnaC linked to tRNAPro.
One question raised by these results is whether the peptidyl-tRNA
accumulated in the presence of tryptophan is only full-length TnaC-tRNAPro or whether other peptidyl-tRNAs are present in
the peptidyl-tRNA band? Clearly TnaC-tRNAPro is present
because the band is labeled by [3H]Pro and there is only
a single Pro in TnaC (Fig. 7B). If the ~25-kDa band is a
mixture of peptidyl-tRNAs, we might expect to see a ladder or smeared
band on SDS gels. To the contrary, we ran long 10% and 15% SDS gels
and always observed only a sharp ~25-kDa band (data not shown). Taken
together, our results indicate that the ~25-kDa species observed when
tryptophan is present is TnaC-peptidyl-tRNAPro. In contrast
to our findings with the template containing the wild type
tnaC coding region, when we used template
CF-tna+306rpoBC"t"bearing a tnaC mutation
(Trp codon 12 changed to an Arg codon), no ~25-kDa TnaC(W12R)-tRNA
accumulation was observed (data not shown). This finding suggests that
Trp at TnaC position 12 is required for the inhibition of
TnaC-peptidyl-tRNAPro cleavage under inducing condition.
The Effect of Tryptophan Concentration on the Accumulation of
TnaC, TnaC-tRNAPro, and the Read-through
Transcript--
As shown above, tryptophan, the inducer of the
tna operon, plays a crucial role in the accumulation of
peptidyl-tRNA and the production of the read-through transcript. To
determine the tryptophan concentration dependence of the events leading
to peptidyl-tRNA and read-through transcript production, we performed
experiments with our circularized CF-tna+306ropBC"t" DNA template,
and varied the tryptophan concentration (Fig.
9). As the tryptophan concentration in
the S-30 reaction was increased, stronger TnaC peptide and TnaC-tRNAPro signals were detected, with an apparent
maximum at 1 mM L-tryptophan (Fig.
9A). When the read-through transcript level was determined, using [ Tryptophan-mediated induction of tna operon expression
proceeds by preventing transcription termination at Rho
factor-dependent termination sites in the leader region of
the operon (9-11). The simplest model consistent with all of our
previous experimental findings is that, in the presence of inducing
levels of tryptophan, the nascent TnaC peptide acts in cis
on its translating ribosome to inhibit its release at the
tnaC stop codon. The stalled ribosome would presumably block
Rho's access to the BoxA and rut site adjacent to the tnaC stop codon, and thereby prevent Rho-mediated
transcription termination (29).
In this study we successfully reproduced all the in vivo
features of tna operon regulation by tryptophan-induced
transcription antitermination using a coupled transcription/translation
S-30 system from E. coli. In addition, we provide the first
demonstration of E. coli TnaC peptide synthesis in the S-30
system (in vitro synthesis of Proteus vulgaris
TnaC has been shown (Ref. 28)). Most importantly, we show that the
presence of inducing levels of tryptophan leads to the accumulation of
TnaC-tRNAPro. We also show that moderately high tryptophan
concentrations are required for induction and for peptidyl-tRNA
accumulation. Our results provide direct support for the hypothesis
that, in the presence of inducing levels of tryptophan,
TnaC-tRNAPro is not cleaved. This peptidyl-tRNA presumably
blocks release of the translating ribosome at the tnaC stop
codon, and prevents Rho action. Our preliminary findings (data not
shown) demonstrate that the peptidyl-tRNAPro is associated
with the translating ribosome.
The S-30 Cell-free System--
E. coli strain
RNaseI The Level of Tryptophanyl-tRNATrp--
How added
tryptophan serves as the signal that leads to inhibition of
Rho-dependent termination is a basic unanswered question. Translation of Trp codon 12 of tnaC by tRNATrp
is believed to be essential for tryptophan-induced antitermination with
the wild type tna operon (13). It has been suggested that the TnaC peptide might be modified in some manner when excess tryptophan is present (15). Since full induction of the tna operon in the S-30 system is also observed with 1 mM 1MT as
inducer, and since unlabeled 1MT does not appear to reduce
incorporation of labeled tryptophan into TnaC or
TnaC-tRNAPro (Fig. 7B), it now seems unlikely
that the Trp residue at position 12 is modified. Similarly, induction
appears to be independent of the level of
tryptophanyl-tRNATrp, since we find, in agreement with
in vivo data, that the tryptophan concentration required for
appreciable tna operon induction is higher than for general
protein synthesis. This is logical for an amino acid catabolic enzyme,
since the degradative operon should only be expressed when its
substrate is well in excess of the level required for tRNA charging and
protein synthesis. It seems likely, therefore, that there is a specific
binding site for tryptophan in the translating ribosome, and that
tryptophan must be at this site for induction to occur. Whether
TnaC-tRNAPro contributes to this binding site is a question
that requires experimental attention. In any event TnaC peptide
synthesized in the presence and absence of inducer should be sequenced
to be certain that induction does not involve a modification of one of
the residues in the TnaC peptide. This is now feasible because we can
produce the TnaC peptide in vitro.
Rho-dependent Termination--
Rho action is largely
responsible for the low level of expression observed in vivo
in the absence of inducer. Rho-dependent transcription
termination at sites located in the tna leader region has
been characterized previously, both in vivo and in an
in vitro purified transcription system (9). Bicyclomycin
inhibition of termination (Table II and Fig. 6B) and Rho
titration (Table III) are consistent with this conclusion. The
antibiotic bicyclomycin is known to interact with Rho and inhibit its
action (22-24). Addition of bicyclomycin to our in vitro
system led to a 15-fold increase in basal level expression (Table II).
In a recent study, it has been shown that a BoxA site and a
rut site (both are adjacent to the tnaC stop
codon) are crucial for Rho action in vivo (14).
TnaC-tRNAPro Synthesis, Ribosome Stalling at the tnaC
Stop Codon, and the RNA Doublet--
The observation that
TnaC-tRNAPro accumulates in the presence of inducing levels
of tryptophan suggests that the translating ribosome may be stalled at
the tnaC stop codon. If this is correct, the associated
transcript should not be available for subsequent rounds of
translation. In the absence of added tryptophan, TnaC would presumably
be synthesized and released. Thus, one might expect to detect a higher
level of TnaC peptide in the absence versus the presence of
added tryptophan. However, in the presence of tryptophan, TnaC would
exist as two species, free and as peptidyl-tRNA. Unfortunately, the
TnaC peptide is labile in the S-30 system (data not shown); therefore,
we could not reliably measure the level of TnaC that is synthesized.
Appearance of the RNA doublet only in the presence of tryptophan
provides additional support for the interpretation that induction results in ribosome stalling at the tnaC stop codon. It is
very likely that the RNA doublet arises from RNA processing. When we employ a template with a 5-bp deletion at the 5'-end of the
tna leader region instead of the wild type template, a
corresponding decrease is observed in the length of RNA doublet (data
not shown). This establishes that the 3' end of the doublet is
identical using either template. The normal length RNA doublet was also
observed when a template was used that has the rut region
deleted (from bp +101 to +123, just beyond the stop codon of
tnaC), suggesting that the RNA secondary structure that
forms just beyond the tnaC stop codon (9) is not responsible
for formation of the doublet (data not shown). When streptolydigin was
added to an incubation mixture to inhibit all ongoing transcription,
RNA doublet accumulation was still observed, consistent with doublet
formation resulting from RNA processing.
Our combined results show that tna operon regulation can be
reconstituted in an S-30-coupled transcription/translation system, with
expression dependent on catabolite repression,
Rho-dependent transcription termination, and
tryptophan-induced antitermination. We also conclude that, in the
presence of inducer, the newly synthesized TnaC-peptidyl-tRNAPro is resistant to cleavage. We assume
that TnaC-peptidyl-tRNAPro is in the P site of the
translating ribosome, and that the tnaC UGA stop codon is in
the ribosomal A site. We previously reported that inactivation or
overproduction of release factor 3 affects both basal level expression
and induction of the tna operon (31). These findings imply
that the release factor 3-mediated event in normal ribosome release can
influence the unusual events associated with induction. A schematic
representation of the hypothetical stalled translation termination
complex that forms in the presence of tryptophan is shown in Fig.
10. In this representation the peptidyl portion of TnaC-peptidyl-tRNAPro is placed in the
polypeptide exit tunnel of the 50 S ribosomal subunit; however, the
presumed active segment of this TnaC-peptidyl-tRNAPro could
interact with a surface of the peptide tunnel or with a region of the
ribosomal A site (32, 33). Wherever TnaC-peptidyl-tRNAPro
is located, its presence, plus inducing levels of tryptophan, appear to
prevent release factor 2 from mediating cleavage of TnaC-peptidyl-tRNAPro. Similar findings have been described
by Cao and Geballe (34-36) in studies on translational regulation of
gene expression in the cytomegalovirus. They have shown that
translation of an upstream open reading frame, uORF2, regulates
translation of a downstream coding region, corresponding to gene
UL4. The uORF2 polypeptide, 22 residues in length, like
TnaC, accumulates as a peptidyl-tRNAPro, at the stop codon
of the uORF coding region. This peptidyl-tRNA also is resistant to
cleavage, resulting in ribosome stalling and blockage of translation of
the downstream coding region for UL4 (35). The features of
this example also suggest that certain amino acid sequences in a
nascent peptide can interfere with peptidyl-tRNA cleavage when the
translating ribosome is reading a stop codon. Addition of puromycin
does not result in cleavage of this peptidyl-tRNA, and its release from
the ribosome (37). By contrast, TnaC-peptidyl-tRNAPro can
be released and cleaved in response to
puromycin.3 Somewhat related
examples in bacteria concern antibiotic inhibition of peptide chain
elongation, leading to ribosome stalling. This influences the
availability of a downstream nucleotide sequence needed for translation
initiation. Chloramphenicol action during translational attenuation in
the CAT operon is perhaps the best understood example of this type
(12).
Four important questions remain unanswered regarding tna
operon regulation: how is tryptophan recognized, what is the role of
TnaC, what is the role of Trp at position 12, and, under inducing conditions, how is cleavage of TnaC-tRNAPro prevented?
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain containing trpR
lacZ
trpEA2 tnaAbgl::Tn10, was
constructed and used as the source of cell-free S-30 extracts. E. coli DH5
was occasionally employed for amplification of
plasmids. All plasmid DNAs were purified by banding twice in cesium
chloride-ethidium bromide gradients.
195 to
+6 (relative to tna transcription initiation site) into
pGF24 (HindIII and PstI sites). pGF25-00 was
constructed by inserting a PCR fragment (with an engineered
HindIII site at the 5' end and a PstI site at the
3' end) containing the region from
190 to +306 into pGF24 (HindIII and PstI sites).
160 upstream of tna promoter to a site just
beyond the rpoBC terminator. We used two oligonucleotides, ECOR-160 (5'-ACGGAATTCCTGTTATTCCTCAACCC-3') and RPOC-ECOR
(5'-ACGGAATTCCTTGCCGAGTTTGACTC-3'). EcoRI sites were
introduced at both the 5' and 3' ends of this fragment. The fragment
was digested with EcoRI and then circularized with T4 DNA
ligase, resulting in CF-tna+306rpoBC"t". Using plasmid pGF1 as
template and the same procedure, CF-tna+6rpoBC"t" was constructed.
2-3 µg of CF-tna+306rpoBC"t" or CF-tna+6rpoBC"t" were used
in each 50-µl S-30 reaction.
-galactosidase and TnaA-TrpE
synthesis, reaction mixtures were incubated for 40 min (except in
time-course experiment). In protein labeling experiments, the unlabeled
amino acid was replaced by 20 µCi of [35S]methionine,
[3H]isoleucine, [3H]proline,
[3H]tyrosine, or [3H]tryptophan
(PerkinElmer Life Sciences, 1000 Ci/mmol). Details are presented in the
legends. At the end of the incubation, a 5-µl aliquot was removed,
precipitated with five volumes of cold acetone, and pellets were
analyzed by Tricine-sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (Tricine-SDS-PAGE) (19).
-33P]UTP (PerkinElmer Life Sciences, 1000-3000
Ci/mmol) was added to the reaction mixture, and samples were taken
after another 10 min, and extracted with phenol. A 5-µl aliquot was
analyzed on a 6% polyacrylamide, 7 M urea gel. For
single-round transcription (coupled with translation) assays, after 10 min of incubation at 37 °C in the absence of added CTP and UTP, 50 µCi of [
-33P]UTP, 200 µM CTP, and 200 µg/ml rifampicin were added at the same time. Samples were taken at
different intervals.
-Galactosidase activity was determined as
described (20).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
35 region of the tna
promoter (5, 10). Plasmid pPDG52 contains a tnaC'-'lacZ
translational fusion (Fig.
1A). This plasmid DNA was used
as a reporter to examine catabolite repression-dependent
expression of the tna operon in vitro. In this
construct most of the tna leader region had been removed; thus, expression from the promoter could be tested independently of the
effects of the tna operon downstream region and attenuation. The data in Table I show that, in the
absence of cAMP and presence of added tryptophan,
-galactosidase
activity was almost undetectable. By contrast, in the presence of cAMP
and absence of added tryptophan, high levels of
-galactosidase were
produced. Addition of tryptophan increased
-galactosidase production
less than 2-fold. These results suggest that expression of
tnaC'-'lacZ construct in this cell-free system is
cAMP-dependent, and that tryptophan produced by protein turnover provides most of the tryptophan required for
-galactosidase synthesis. The less than 2-fold increase observed upon addition of
tryptophan is probably not regulatory; it presumably is due to
increased completion of synthesis of the 1024-residue
-galactosidase monomer, which contains 39 Trp residues.
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Fig. 1.
Translational fusion constructs used in this
study. A, schematic diagram of the
tnaC'-'lacZ gene fusion fragment in plasmid pPDG52. In this
construct, tnaC codon 20 is fused in frame to
lacZ codon 8. B, schematic diagram of the
tnaA'-'trpE gene fusion fragment in plasmids pGF4, pGF14 and
pGF71. This fragment contains the intact tna promoter,
tna leader region, and the initiation codon ATG of
tnaA in frame with the second codon of trpE. This
cloning procedure substitutes histidine for glutamine at residue 2, yet
functional TrpE is produced. In pGF14, the tnaC Trp12 codon,
TGG, was replaced by an Arg codon, CGG. In pGF71, the tnaC
start codon, ATG, was replaced by the TAG stop codon.
Expression of tnaC'-'lacZ and tnaA'-'trpE gene fusions in vitro
-lactamase gene, which also is expressed in vitro;
-lactamase has four tryptophan residues, and therefore serves as an
excellent internal reference to monitor the availability of charged
tRNATrp for protein synthesis. In Fig. 2 it can be seen
that there was no significant effect of tryptophan addition on
-lactamase production. This suggests that there are appropriate
levels of charged tRNATrp (tryptophan presumably generated
by protein turnover) for synthesis of proteins like
-lactamase, even
in the absence of added tryptophan. A 25-fold increase in TnaA-TrpE
synthesis was observed in the presence of 300 µM
tryptophan; synthesis was assessed by counting the Cerenkov radiation
of 35S-labeled TnaA-TrpE bands (Fig. 2, lane
5 versus lane 2) on the SDS-PAGE gels (data not shown). This increase agrees with the values in
Table I, and suggests that the ASase assay results accurately reflect
the levels of TnaA-TrpE protein produced in this S-30 system.
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Fig. 2.
A 10% Tricine-SDS-PAGE analysis of
pGF4-directed in vitro translation products.
In vitro reactions and Tricine-SDS-PAGE conditions are
described under "Experimental Procedures." Polypeptide bands were
labeled with [35S]methionine. The templates used were: no
plasmid (lane 1), 5 nM pGF4
(lanes 2-5), 5 nM pBR322
(lane 6), and 15 nM pGF4
(lanes 7 and 8). Protein molecular
weight standards (Low Range; Life Technologies, Inc.) were used as
markers; their molecular weights are given. The positions of TnaA-TrpE
and -lactamase are marked by arrows. *, 50 µM Trp was present in this reaction.
Arg), confirming the in
vivo finding that induction is not simply dependent on high
concentrations of the amino acid encoded by codon 12 (13). These
results suggest that translation of tnaC, and incorporation
of Trp at codon position 12, are essential in establishing the basal
level of tna operon expression, and for tryptophan
induction, in vitro.
Effect of added tryptophan and bicyclomycin on expression of
tnaA'-'trpE plasmids
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Fig. 3.
Characterization of the cell-free S-30
system. In vitro protein synthesis and ASase assays
were carried out as described under "Experimental Procedures."
Induction ratios are shown in parentheses. A, a
time-course experiment with plasmid pGF4-programmed TnaA-TrpE
synthesis. B, effect of plasmid pGF4 concentration on
TnaA-TrpE production, both in the presence and absence of added
tryptophan. C, effect of DL-1-methyl-tryptophan
(DL-1MT) concentration on the
production of TnaA-TrpE protein.
5 M; Ref. 26), the
tRNATrp in our S-30 should be fully charged in the presence
of 50 µM added tryptophan. Taken together, these findings
suggest that tna induction is not based on the extent of
charging of tRNATrp.
Addition of purified Rho lowers basal level expression when high
concentrations of tnaA'-'trpE plasmids are used
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Fig. 4.
Demonstration of the requirement for cAMP for
initiation of transcription of the tna operon.
A, schematic representation of the circularized fragment
(CF-tna+6rpoBC"t") used as template to program the cell-free
reactions illustrated in A. The first 6 nucleotides of the
tna leader transcript were followed by the E. coli
rpoBC terminator (rpoBC"t"). The source of the
predicted ~130-nucleotide transcript is shown. B, analysis
of transcription in an S-30 system. A 6% polyacrylamide, 7 M urea gel was used. Transcription initiation at the
tna promoter was cAMP-dependent and
tryptophan-independent. The S-30 reactions (50 µl each) were
incubated at 37 °C for 10 min in the presence or absence of cyclic
AMP and/or L-tryptophan. 20 µCi of
[ -33P]UTP was then added, and after a 5-min
incubation, the reactions were stopped by phenol extraction and the
contents loaded onto an RNA gel.
cyclic AMP lane). In the presence of
cAMP and added tryptophan, a prominent read-through (RT)
transcript band was observed (Fig. 5B, last
lane). Only a faint read-through transcript was produced
when tryptophan was omitted (Fig. 5B,
Trp
lane). Consistent with the translational results described above, no effect of tryptophan was observed when two templates with
different tnaC mutations (ATG start codon changed to TAG, or
Trp codon 12 changed to an Arg codon, in
CF-tna+306rpoBC"t") were used (data not shown). Addition
of bicyclomycin with cAMP increased basal level expression with all
templates (data not shown).
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Fig. 5.
Requirements for in vitro
synthesis of the read-through transcript. A,
schematic representation of the circularized DNA fragment,
CF-tna+306rpoBC"t", used as template to program the cell-free
reactions in A. This fragment contains the intact tna
promoter, the tna leader region to bp +306, followed by the
rpoBC transcription terminator. The predicted tna
transcripts in the absence or presence of tryptophan are also shown.
B, analysis of the requirements for read-through
transcription. A 6% polyacrylamide, 7 M urea gel was run
with transcription/translation assay mixtures. S-30 reaction mixtures
with 20 µCi of [ -33P]UTP (50 µl/reaction mixture)
with the indicated additions were incubated at 37 °C for 20 min. The
reactions were then stopped by phenol extraction, and the RNA samples
were loaded on RNA gels. An arrow points to the position of
the read-through transcript (RT).
Trp
lane), only low levels of terminated transcripts were
observed. Why were bands of comparable intensity to those in the
+Trp lane not also seen? In an attempt to explain
this observation, we analyzed transcription more closely using the
"single-round transcription" approach (27). Using this procedure,
template is incubated in the S-30 extract plus or minus tryptophan for
10 min at 37 °C without added CTP or UTP, and then rifampicin,
[
-33P]UTP, and CTP are added. Samples are removed at
various intervals thereafter and examined by urea gel electrophoresis.
In the absence of added tryptophan (Fig.
6A), multiple paused
transcripts are visible at 1 min; these bands subsequently disappear,
presumably as a result of RNA degradation. In the presence of added
tryptophan (Fig. 6A), comparable bands are observed at 1 min, but thereafter distinct intermediate length bands appear and the
read-through band becomes more prominent. When the identical experiment
is performed in the presence of bicyclomycin (Fig. 6B), the
mRNA banding pattern in the absence of added tryptophan more
closely resembles the pattern observed in the presence of tryptophan. It would appear, therefore, that the differences observed in the absence of bicyclomycin are the result of Rho-mediated transcription termination, and degradation of these Rho-terminated transcripts. Comparison of the
Trp and +Trp lanes
(Fig. 6A) suggests that pausing may occur at most of the
same sites under both conditions. One exception to this generalization
is evident; in the presence of added tryptophan, a ~120-nucleotide
RNA doublet can be seen near the bottom of the gel. We believe that
this doublet is produced by degradation of the ribosome-bearing
read-through transcript that is produced in the presence of tryptophan
(see "Discussion").
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Fig. 6.
Single-round transcription analyses examining
the effects of added tryptophan and bicyclomycin. A,
single-round transcription analyses (coupled with translation) were
performed in an S-30 system. Circularized fragment
CF-tna+306rpoBC"t" (see Fig. 5B) was used as template.
S-30 reactions (100 µl) without CTP and UTP, in the absence or
presence of tryptophan, were incubated at 37 °C for 10 min, then 50 µCi of [33P]UTP, 200 µM CTP, and 200 µg/ml rifampicin were added together to the reaction mixture. Samples
(10 µl) were taken at indicated time points, stopped by phenol
extraction, and loaded on an RNA gel. The read-through transcript
(RT) and the RNA doublet observed only in the presence of
tryptophan are marked by arrows. B, effect of
addition of the Rho inhibitor bicyclomycin, to a reaction mixture
incubated in the absence of tryptophan. Single-round transcription
assays (coupled to translation) were performed as described in
A. CF-tna+306rpoBC"t" was used as template. Control
reactions (+Trp) are shown on the left.
Arg). Both in the presence and
absence of tryptophan, all templates gave RNA expression patterns
similar to those obtained with the wild type template in the absence of
tryptophan (Fig. 6A,
Trp lanes); thus, we do
not show these results. We also examined the effects of adding the
translation inhibitors streptomycin, kasugamycin, or chloramphenicol.
Addition of any of these drugs eliminated the tryptophan induction
effect (data not shown). These results confirm the importance of
tnaC translation in induction of tna operon;
tnaC translation is addressed in the next section.
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Fig. 7.
Detecting TnaC peptide synthesis in
vitro. CF-tna+306rpoBC"t" was used as template.
A, [35S]methionine-labeled products produced
in the presence and absence of added tryptophan. S-30 reaction mixtures
(50 µl) were incubated at 37 °C for 20 min with 20 µCi of
[35S]methionine; the reactions were stopped by acetone
precipitation, boiled in Tricine-SDS sample buffer for 3 min, and then
loaded on a 10% Tricine-SDS protein gel. A control sample, without
template, is shown in lane 1. Arrows
mark the positions of TnaC, TnaC-tRNAPro, and the
nonspecific products. B, selective 3H labeling
in the presence or absence of added tryptophan. S-30 reactions (50 µl) were incubated at 37 °C for 10 min (to decrease background
labeling and let the transcription/translation reaction reach steady
state), then [3H]proline, [3H]isoleucine,
[3H]tyrosine, or [3H]tryptophan, was added.
After 10 min of incubation, reactions were stopped by acetone
precipitation, boiled in Tricine-SDS sample buffer for 3 min, and then
loaded onto a 10% Tricine-SDS gel.
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Fig. 8.
Identification of
TnaC-peptidyl-tRNAPro. A, a
[35S]methionine-labeled ~25-kDa band was excised from a
gel and treated with RNase A or proteinase K (for details, see
"Experimental Procedures"), boiled in 1× Tricine-SDS sample buffer
for 3 min, and then loaded onto a 10% Tricine-SDS protein gel. RNase A
digestion shifts the ~25-kDa band to the TnaC band position.
B, RT-PCR identification of tRNAPro. E. coli total tRNA (Sigma) was used as the control RT-PCR template
(positive control, lane 1). The 25-kDa TnaC-tRNA
band was excised, recovered from a long distance Tricine-SDS protein
gel, and used as a RT-PCR template (lane 4). A
S-30 reaction performed without the CF-tna+306rpoBC"t" template was
also loaded onto the same gel, and the corresponding band was recovered
as a control and used for RT-PCR (lane 2). A
reaction mixture similar to the one used for lane
4, but without RT, was loaded in lane
3. A 10-bp DNA ladder (lane M, Life
Technologies, Inc.) is shown.
-33P]UTP to label transcripts in the presence
of increasing concentrations of tryptophan, the maximum level of
read-through transcript was observed at the highest tryptophan
concentration tested, 0.5 mM (Fig. 9B). These
findings indicate that, in the S-30 system, a tryptophan concentration
in excess of 0.25 mM is probably required to obtain 50% or
greater induction of tna operon expression.
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Fig. 9.
Tryptophan dependence of TnaC,
TnaC-tRNAPro, and read-through transcript production.
A, a 10% Tricine-SDS protein gel of S-30 reactions
performed at increasing tryptophan concentrations. S-30 reactions (50 µl each) were performed with CF-tna+306rpoBC"t" in the presence
of the indicated concentrations of L-tryptophan. Incubation
was at 37 °C for 10 min, then 20 µCi of
[35S]methionine was added for 10 min, and the reactions
were stopped by acetone precipitation, boiled in 1× Tricine-SDS sample
buffer for 3 min, and then loaded onto a 10% Tricine-SDS gel. Levels
of TnaC peptide and TnaC-tRNAPro were quantified using a
PhosphorImager. The levels of TnaC and TnaC-tRNAPro in the
1 mM tryptophan lane were set at 100%, respectively.
B, a 6% polyacrylamide, 7 M urea gel of a
transcription/translation reaction examining the effect of increasing
tryptophan concentration on the production of read-through
(RT) transcript. CF-tan+306rpoBC"t" was used as
template. The S-30 reaction mixtures (50 µl each), in the presence of
the indicated concentrations of L-tryptophan, were
incubated at 37 °C for 10 min, then 20 µCi of
[33P]UTP and 200 µg/ml rifampicin were added. After 1, 5, or 15 min of incubation, samples were taken and reactions were
stopped by phenol extraction and loaded on an RNA gel.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
trpR
lacZ
trpEA2 tnaA
bgl::Tn10 was used to prepare our S-30 extracts. This
strain was derived from the classic RNase I minus A-19 strain.
trpE and lacZ deletions were introduced into this
strain; consequently, any TrpE or LacZ activity detected in the S-30
extract would result from plasmid-programmed synthesis. This strain is
trp repressor minus; thus, S-30 extracts from this strain
have elevated levels of the TrpG-D protein (30) that are sufficient to
fully activate all the TnaA-trpE protein synthesized during the course
of an experiment. This conclusion has been confirmed by measuring
TnaA-TrpE activity in the NH3-dependent anthranilate synthase reaction that does not require TrpG-D protein (data not shown).
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Fig. 10.
Model depicting the mechanism of tryptophan
induction of tna operon expression. When a
ribosome reaches the tnaC termination codon, UGA, in the
presence of tryptophan, the 24-residue TnaC peptide (chain
of circles) remains covalently joined to
tRNAPro. Consequently the ribosome associated with the
peptidyl-tRNA stalls at the tnaC stop codon. This blocks
Rho's access to the BoxA and rut sites, and
thereby prevents transcription termination. The positions at which
amino acid residue changes have been shown to largely prevent induction
are represented by black circles; positions where
changes can result in constitutive expression are white
circles; positions where changes result in mixed effects are
indicated by white circles with a
diagonal line; positions for which there are
insufficient data are indicated as gray circles
(14). The crucial Trp codon 12 is shown as an enlarged
black circle. PTC, peptidyltransferase
center.
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ACKNOWLEDGEMENTS |
---|
We are indebted to Virginia Horn for constructing the E. coli strain used for S-30 preparations. We thank Peter von Hippel for the sample of purified E. coli Rho factor. We also thank Angela Valbuzzi, Kouacou Vincent Konan, and Ajith Kamath for critical reading of the manuscript. We thank the Fujisawa Pharmaceutical Co. Ltd., Osaka, Japan, for providing the sample of bicyclomycin.
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FOOTNOTES |
---|
* This work was supported by Grant GM09738 (to C. Y.) from the United States Public Health Service.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. Tel.: 650-725-1835;
Fax: 650-725-8221; E-mail: yanofsky@cmgm.stanford.edu.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M008892200
1 M. Eshoo and C. Yanofsky, unpublished data.
3 F. Gong and C. Yanofsky, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: PCR, polymerase chain reaction; RT, reverse transcription; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis; 1MT, 1-methyltryptophan; ASase, anthranilate synthase; uORF, upstream open reading frame; bp, base pair(s).
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REFERENCES |
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