Rho-dependent transcription termination in the tryptophanase operon leader region of Escherichia coli K-12

Stewart, Valley; Landick, Robert; Yanofsky, Charles

doi:10.1128/jb.166.1.217-223.1986

Cited by 78 publications

(94 citation statements)

References 41 publications

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“…The theoretical structures depend on the number of nucleotides included, which are limited to the intergenic regions in our cases and are reliable in calculations with the Zuker program. Yanofsky and colleagues (8,9) have also drawn simpler structures of this region using a more limited number of downstream nucleotides than we have (Fig. 2C).…”

Section: Regions Of Interestmentioning

confidence: 99%

A specific endoribonuclease, RNase P, affects gene expression of polycistronic operon mRNAs

Altman

2003

Proc. Natl. Acad. Sci. U.S.A.

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The rnpA mutation, A49, in Escherichia coli reduces the level of RNase P at 43°C because of a temperature-sensitive mutation in C5 protein, the protein subunit of the enzyme. Microarray analysis reveals the expression of several noncoding intergenic regions that are increased at 43°C compared with 30°C. These regions are substrates for RNase P, and they are cleaved less efficiently than, for example, tRNA precursors. An analysis of the tna, secG, rbs, and his operons, all of which contain RNase P cleavage sites, indicates that RNase P affects gene expression for regions downstream of its cleavage sites.S everal years ago, it was reported that two of the enzymes in the lac operon were produced in different molar quantities, but no direct explanation was provided for this result (1). General speculative hypotheses for differing molar concentrations of proteins coded for in operons have also been suggested (2). Intergenic noncoding RNAs in operon mRNAs in Escherichia coli may provide endoribonuclease sites as one method of allowing production of operon mRNAs and proteins with differing molar concentrations. Products of endoribonuclease cleavage could be degraded rather quickly by an mRNA nuclease degradation mechanism involving RNase E. The small intergenic RNAs could be substrates for RNase P, and they would indicate that RNase P might process many more RNAs than was originally thought.RNase P itself is an essential enzyme responsible for the processing of the 5Ј ends of precursors to tRNA and other naturally occurring stable RNA molecules in E. coli (3). In Salmonella, there is one region in an operon mRNA that appears to be cleaved by RNase P (4). In this report, the noncoding intergenic regions and genes coding for proteins (ORFs) in several operons were cloned separately and examined as substrates for RNase P. Several of the intergenic transcripts were substrates for RNase P. These transcripts accumulate at the restrictive temperature in an E. coli mutant strain thermosensitive for RNase P function. ORFs in the relevant operons were not substrates. A mechanism of determining gene expression is described in which the action of a specific endoribonuclease controls regions downstream of its cleavage site in polycistronic operon mRNAs. Materials and MethodsStrains of E. coli. E. coli A49 and several derivatives of this strain are described in ref. 5 and references therein.Cloning and Nomenclature of Intergenic RNAs. Several intergenic regions, the expression of which was high at 43°C in A49 (5), were chosen for cloning. Clones were amplified by PCR starting generally with the first nucleotide after the stop codon of an upstream gene and the last nucleotide before the start codon of a downstream gene. Clones were numbered according to the length of the sequences cloned and w or c, depending on whether transcription was in the clockwise (w) or counterclockwise (c) direction on the chromosome. The start and stop sites for each fragment on the chromosome are listed below, with the numerology based on that of Blattner ...

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Section: Regions Of Interestmentioning

confidence: 99%

A specific endoribonuclease, RNase P, affects gene expression of polycistronic operon mRNAs

Altman

2003

Proc. Natl. Acad. Sci. U.S.A.

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“…An analogous situation may exist at the end of tnaC in the tna operon. Immediately after the translation termination codon of tnaC (included in a boxA-like sequence) there is a C-rich region that does not appear to be in any base-paired structure (32).…”

Section: Rolementioning

confidence: 99%

“…The precise mechanism of tryptophan induction is not known. However, the consequence of induction is antitermination at one or more Rho factor-dependent transcription termination sites located between tnaC and tnaA (32).…”

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tRNA(Trp) translation of leader peptide codon 12 and other factors that regulate expression of the tryptophanase operon

1990

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Tryptophanase (tna) operon expression in Escherichia coli is induced by tryptophan. This response is mediated by features of a 319-base-pair leader region preceding the major structural genes of the operon. Translation of the coding region (tnaC) for a 24-amino-acid leader peptide is essential for induction. We have used site-directed mutagenesis to Tryptophanase is a catabolic enzyme that degrades Ltryptophan to indole, pyruvate, and ammonia. Bacterial species with this activity, such as Escherichia coli, have the capacity to grow on L-tryptophan as a sole carbon or nitrogen source. The enzyme also catalyzes a variety of related reactions (19,23,24). Most importantly, tryptophanase can synthesize tryptophan from indole and serine (35); this activity permits genetic selection for tryptophanase function in mutant strains that lack the tryptophan biosynthetic enzyme tryptophan synthetase (19,20).The tryptophanase operon (tna) of E. coli contains two major structural genes, tnaA and tnaB, encoding tryptophanase and a tryptophan permease, respectively (8, 9). These genes are preceded by a 319-base-pair transcribed leader region that has a coding region, tnaC, for a 24-residue leader peptide. Transcription of the tna operon in E. coli is induced by L-tryptophan (2) and is subject to catabolite repression (4); i.e., cyclic AMP and the catabolite gene activator protein are required for expression. Catabolite repression of tna operon expression appears to involve elements both within and distinct from the tna promoter (31; Goilnick and Yanofsky, unpublished results). The precise mechanism of tryptophan induction is not known. However, the consequence of induction is antitermination at one or more Rho factor-dependent transcription termination sites located between tnaC and tnaA (32).Studies with gene fusions joining tnaC or tnaA with lacZ, the E. coli ,-galactosidase structural gene, demonstrated that the leader region contains elements responsible for tryptophan regulation of the operon (31). Thus, a gene fusion containing the tna promoter and the entire leader region, tnaA'-lacZ, was inducible by tryptophan. An analogous fusion that deleted the nontranslated portion of the leader (tnaC'-lacZ) was insensitive to tryptophan induction but responded to catabolite repression (31). Transcription initiation at the tna promoter is therefore constitutive, regulated * Corresponding author.only by catabolite repression, whereas tryptophan induction involves transcription antitermination and is mediated by features of the leader region.Further evidence for a role of tnaC in regulating the operon comes from isolation and analysis of cis-acting constitutive mutants that synthesize high levels of tryptophanase in the absence of inducer (31). All of these mutants contain single-base-pair changes in tnaC; most of these are in a nine-base sequence homologous to the boxA sequence that is involved in antitermination control of early lytic gene expression in bacteriophage lambda (11). Translation of the tnaC coding region is required fo...

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“…TnaB, a product of the tna operon, transports tryptophan when tryptophan is used as a carbon or nitrogen source. Expression of the tna operon is subject to tryptophan-induced transcription antitermination and catabolite repression (7,32).…”

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Role of regulatory features of the trp operon of Escherichia coli in mediating a response to a nutritional shift

Yanofsky

Horn

1994

J Bacteriol

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Physiological studies were performed under nutritional stress and nonstress conditions to assess the relative importance of the various regulatory mechanisms that Escherichia coli can use to alter its rate of tryptophan synthesis. Mutants were examined in which the tip repressor was inactive, transcription termination at the trp attenuator was altered, transcription initiation at the tip promoter was reduced, or feedback inhibition of anthranilate synthase was abolished. Strains were examined in media with and without tryptophan, phenylalanine and tyrosine, or acid-hydrolyzed casein and following shifts from one medium to another. Growth rates and anthranilate synthase levels were measured. In media lacking tryptophan, each of the mutants showed relief of repression and/or attenuation and maintained a near-normal growth rate. Following a shift from a medium containing tryptophan to a trptophan-free medium containing phenylalanine and tyrosine or acid-hydrolyzed casein, mutants with abnormally low trp enzyme levels exhibited an appreciable growth lag before resuming growth. The wild-type strain displayed termination relief only under one extreme shift condition, upon transfer from a minimal medium containing tryptophan to minimal medium with only phenylalanine and tyrosine. A promoter down-mutant had difficulty adjusting to a shift from high tryptophan to low tryptophan levels in a medium containing acid-hydrolyzed casein. In all media tested, anthranilate synthase levels were lower in a feedback-resistant mutant than in the wild type. These studies demonstrate the capacity of E. coli to adjust its rate of tryptophan synthesis to maintain rapid growth following a shift to stressful nutritional conditions. Escherichia coli has the ability to adjust its rate of tryptophan biosynthesis over a several-thousand-fold range. This impressive regulatory capacity emanates from the use of a relatively strong promoter to drive tip operon expression, repression and attenuation to regulate tip operon transcription, and feedback inhibition to regulate entry of the common aromatic precursor, chorismate, into the tryptophan biosynthetic pathway (36). In addition, tryptophan influences the flow of carbon into and through the common aromatic pathway that culminates in chorismate production (23 (415) 725-8221. tion at the attenuator. Attenuation regulates transcription of the structural genes of the tip operon ca. six-to eightfold (11, 13). There is one additional regulatory feature of transcriptional attenuation in the tip operon. Cells that are incapable of initiating synthesis of the tip leader peptide experience five times greater termination at the attenuator than cells that can perform this function (24,33,43). This phenomenon has been termed superattenuation (33). Events that prevent or decrease synthesis of the tip leader peptide could cause superattenuation and lead to an additional fivefold increase in termination at the attenuator. Feedback inhibition of anthranilate synthase (ASase), the enzyme catalyzing the first r...

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Rho-dependent transcription termination in the tryptophanase operon leader region of Escherichia coli K-12

Cited by 78 publications

References 41 publications

A specific endoribonuclease, RNase P, affects gene expression of polycistronic operon mRNAs

A specific endoribonuclease, RNase P, affects gene expression of polycistronic operon mRNAs

tRNA(Trp) translation of leader peptide codon 12 and other factors that regulate expression of the tryptophanase operon

Role of regulatory features of the trp operon of Escherichia coli in mediating a response to a nutritional shift

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