![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
and Charles Yanofsky*
Department of Biological Sciences, Stanford University, Stanford, California 94305-5020
Received 16 June 2003/ Accepted 12 August 2003
 |
ABSTRACT |
|---|
 Top Abstract TextReferences |
|---|
|
TEXT |
|---|
|
TopAbstractTextReferences |
|---|
The tryptophanase (tna) operon of Escherichia coli contains two major structural genes, a promoter-proximal gene, tnaA, encoding tryptophanase, and a distal gene, tnaB, encoding a low-affinity tryptophan permease (1, 17). Preceding tnaA in the tna operon is a 319-nucleotide (nt) transcribed regulatory region that contains the coding region for a 24-residue leader peptide, TnaC. The 220-nt spacer sequence that separates the tnaC stop codon from the start codon of tnaA contains several transcription pause sites that can serve as regulated sites of Rho-dependent transcription termination (16, 17). The RNA sequence in the vicinity of the tnaC stop codon is rich in cytidylate residues (Fig. 1A). Such a C-rich sequence, termed a Rho utilization site (rut), is required for efficient Rho-dependent termination in the leader region of the tna operon (3, 5, 9, 12, 13).
|
For tryptophan induction of the tna operon to occur, transcription and translation of the operon must be coordinated so that a translating ribosome may stall at the tnaC stop codon before RNA polymerase completes transcription of the leader region. This stalling would prevent Rho factor's access to the rut site in the leader transcript, thereby preventing transcription termination. The findings in previous studies (6) mentioned above do not explain how transcription and translation are coupled during transcription of the tna leader region. Of potential relevance, it was established in studies with the biosynthetic trp operon of E. coli that transcription pausing and ribosome release of the paused polymerase complex are the crucial events that establish that ribosome movement on the trp operon transcript and polymerase movement in the leader region of the operon proceed in unison (10). We reasoned that a similar mechanism might be operating in the tna operon; this would ensure that a ribosome would be engaged in translating tnaC before the transcribing RNA polymerase resumed transcription and synthesized the transcript segment that serves as the Rho factor binding site. Early studies using a purified transcription system did demonstrate the existence of several transcription pause sites in the tna leader region (16). One of these pause sites generated a major short pause RNA species, 103 nt in length (16). Nucleotides at positions 52 through 99 of this tna leader transcript were predicted to form a relatively stable hairpin structure (
G = -9.6 kcal/mol; Fig. 1B); this hairpin structure was proposed to be responsible for the transcription pause producing the 103-nt RNA species (16). Since the nucleotides forming this hairpin structure are located at the distal end of the tnaC coding region (Fig. 1A), if the paused RNA polymerase remained at this site until released by the ribosome translating tnaC, synchronization of transcription and translation would be achieved. The present study was performed to examine this possibility.
An S-30 system permitting transcription and translation of the tna operon and its regulation was employed in these analyses; the S-30 reaction conditions used have been described elsewhere (7). An appropriate tna operon promoter-leader region template was constructed. This DNA template was a self-ligated PCR fragment containing the region from positions -195 to +115 of the tna operon (relative to the tna transcription initiation site), followed by the attached rpoBC transcription terminator sequence (Fig. 1A) (7). Thus, in this template, the intact tna promoter was present, but the multiple transcription pause sites (7, 16) located between tnaC and tnaA coding sequences were removed and replaced by the rpoBC terminator and adjacent sequences. This template allowed us to focus on the role of the transcription pause that occurs at the end of tnaC; this presumed RNA hairpin structure is shown in Fig. 1B.
To monitor RNA synthesis in the S-30 extract, UTP was replaced by [
-33P]UTP. To enhance transcription pausing, neither CTP nor GTP was added to the S-30 system; low levels of CTP and GTP are present in the S-30 extract. We compared the rate of transcription under these conditions with the rate under the conditions we used before in reconstituting regulation of the tna operon expression in vitro (7). Previously 200 µM (each) CTP and GTP was present in the S-30 reaction mixture, and only a low concentration of labeled UTP was added. We observed no significant difference in the rate of transcription when we performed reactions with or without added CTP and GTP, suggesting that the low CTP and GTP concentrations in our S-30 extract are sufficient to allow transcription to proceed. Presumably, this is so because the labeled UTP is limiting in both reactions. Bicyclomycin, at 50 µg/ml, was generally added to each reaction mixture, as a specific inhibitor of transcription termination factor Rho (11). Since the rpoBC terminator is an intrinsic terminator, neither Rho factor nor bicyclomycin should have an effect on termination at the rpoBC site. Reaction mixtures were preincubated with bicyclomycin at 37°C for 10 min, and then 100 µg of rifampin (an inhibitor of transcription initiation) per ml and 2 µl of [-33P]UTP (10 µCi/µl; Perkin-Elmer) were added. Time course samples were then taken to monitor transcription and transcription pausing. When used, the translation inhibitor chloramphenicol or puromycin was added together with the [-33P]UTP.
Using a modified single-round transcription approach (19) with our S-30 system, a short RNA species (Fig. 2A, pause I) was evident, particularly in the presence of a translation inhibitor (Fig. 2). This pause RNA species comigrated with a 100-nt RNA marker (Ambion, Austin, Tex.) run on the same urea-polyacrylamide gel (marker not shown). Also apparent was the 
120-nt doublet RNA species produced only when inducing levels of tryptophan were present (7) (Fig. 2A, doublet). The RNA doublet is thought to be produced by nuclease attack on the mRNA, 3' of the stalled ribosome, and partial degradation of the ribosome-bearing terminated transcripts (7). Consistent with this view, the intensity of the RNA doublet on the gel increased with time in this single-round transcription assay. In contrast, the intensity of the pause I RNA species decreased with time, as the RNA species terminating at the rpoBC terminator (Fig. 2A, T band) became more prominent. The half-life of pause I RNA detected in the reaction performed without added chloramphenicol was estimated as 1.1 min (Fig. 2A). Due to the presence of low levels of nucleoside triphosphates in our S-30 extract, we were unable to synchronize transcription initiation in our cell-free system. Thus, the modified single-round transcription approach used in our experiments differs from the single-round transcription procedure that allows synchronized transcription initiation (19). Thus, the apparent RNA pause half-life that can be estimated from our data is likely to be an overestimate of the true pause half-life.
|
Similar results were obtained with another translation inhibitor, puromycin, in comparable single-round transcription analyses performed in the absence of added tryptophan. Figure 2B shows the effect of puromycin addition on the stability of the pause I complex; its half-life increased from 0.9 min without puromycin to 7 min with puromycin. The RNA doublet band was not evident (Fig. 2B versus Fig. 2A), because tryptophan was not added to these reaction mixtures. We noticed a prominent 150-nt pause RNA species, pause II, migrating between pause I and the terminated transcripts, with either chloramphenicol or puromycin as an inhibitor (Fig. 2). This species exhibited a pattern of formation similar to that of pause I RNA in that its presence depended on the addition of a translation inhibitor. However, the intensity of pause I RNA was reduced with time in this single-round transcription assay, while by contrast, the intensity of this pause II RNA species initially increased with time before decaying. Apparently, RNA polymerase paused at site II ultimately elongates to terminate at the rpoBC terminator. We could not calculate the stability of the pause complex producing this RNA species, because RNA polymerases producing the pause I RNA species continued elongating to this pause site. Examination of the sequence of the rpoBC terminator fragment incorporated into our template revealed that two stem-loop hairpin structures could theoretically form after the pause I RNA structure (Fig. 1A). We believe these hairpin structures are responsible for the two major bands we see on the gel in addition to pause I RNA and the terminated RNA species (Fig. 2). It is possible that the pause at site I is responsible for the pause at site II. Perhaps the formation of pause I RNA prevents the formation of a third hairpin structure that would prevent the formation of pause II RNA. This third structure might contain a segment of the 3' strand of pause I and a segment of the 5' strand of pause II. Such a theoretical hairpin structure does in fact exist (not shown). Pause II RNA (150 nt) corresponds to one of the hairpin structures predicted for the rpoBC-associated sequences (Fig. 1A).
In other experiments, we tested an analogous DNA template in which the tnaC start codon, ATG, was replaced by a stop codon, TAG. This template gave a pause I RNA complex with a half-life of 6.5 min in the single-round transcription assay. Moreover, addition of chloramphenicol or puromycin to the reaction mixture did not further increase the stability of the pause I complex (data not shown). Thus, eliminating translation of tnaC by start codon replacement also increased pause I RNA half-life, but here addition of chloramphenicol or puromycin had no effect.
The 5' end of the 100-nt RNA species of the pause I complex was determined by primer extension (14). Pause I RNA and the doublet RNA species from the gels shown in Fig. 2A were recovered, and their 5' ends were determined. The oligonucleotide used in the primer extension reaction mixtures was complementary to nts +84 to +99 of the tna transcript. The 5'-end nucleotide of the pause 1 RNA species was the same as that of the doublet. The 5' end corresponds to nucleotides at the tna transcription initiation site (Fig. 3). Therefore, we believe that the 3' end of the pause I RNA is approximately nt 103. The 3' end of what we believe is the same pause RNA species was determined previously using a purified transcription system (16).
|
In this report, we demonstrate that RNA polymerase does pause at the end of the tnaC coding region, allowing time for a ribosome to bind to the transcript and begin translation. We also show that the translating ribosome decreases the lifetime of this transcription pause complex. Pause release thus allows the paused RNA polymerase to resume transcription. This coupling of translation with transcription would therefore permit the translating ribosome to reach the tnaC stop codon and determine whether the Rho factor could gain access to the transcript. Preventing completion of translation of tnaC significantly prolonged the half-life of the critical transcription pause that occurs at the end of the tnaC coding region. We recognize that the artificial conditions we used for transcription and translation in the in vitro S-30 system are substantially different from the conditions that exist in vivo. However, the finding that the ribosome synthesizing TnaC can release the critical transcription pause complex in vitro strongly supports a role for the translating ribosome in achieving coupling of transcription and translation in the tna leader region. Thus, transcription pausing, ribosome-mediated pause release, and tryptophan-induced ribosome stalling are all essential events in the mechanism of regulation of tna operon expression. This coupling resembles the coupling of transcription with translation found to be essential in studies on attenuation regulation of the trp biosynthetic operon of E. coli (10). Transcription pausing in a transcribed leader region has also been proposed to be involved in attenuation regulation of the trp operon of Bacillus subtilis, although in this example transcription is coupled with the binding of a regulatory protein (20).
|
ACKNOWLEDGMENTS |
|---|
This work was supported in part by a grant from the National Science Foundation (MCB-0093023) (to C.Y.).
|
FOOTNOTES |
|---|
Present address: Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164-4660.
|
REFERENCES |
|---|
|
TopAbstractTextReferences |
|---|
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Appl. Environ. Microbiol. | Infect. Immun. | Eukaryot. Cell |
|---|---|---|
| Mol. Cell. Biol. | J. Virol. | Microbiol. Mol. Biol. Rev. |
| ALL ASM JOURNALS |
