Nucleotide sequence of ilvGEDA operon attenuator region of Escherichia coli.

Nargang, Frank E.; Subrahmanyam, C S; Umbarger, H. E.

doi:10.1073/pnas.77.4.1823

Cited by 95 publications

(72 citation statements)

References 19 publications

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“…No regulatory protein has been identified that modulates expression of the operon in response to changes in the availability of the three amino acids. A leader-attenuator precedes the structural genes, and the proposed leader peptide includes residues of all three amino acids, consistent with the multivalent regulation of the operon (2,25,26,35). The data accumulated for the response of this operon to amino acid limitation are consistent with its being mediated via the leader-attenuator (26).…”

supporting

confidence: 64%

Analysis of regulation of the ilvGMEDA operon by using leader-attenuator-galK gene fusions

et al. 1990

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Five of the genes for the biosynthesis of isoleucine and valine form the ilvGMEDA operon of Escherichia coli K-12. Expression of the operon responds to changes in the availability of isoleucine, leucine, and valine (ILV). Addition of an excess of all three amino acids results in reduced expression of the operon, whereas limitation for one of the three amino acids causes an increase in expression. The operon is preceded by a leader-attenuator which clearly regulates the increased expression that occurs due to reduced aminoacylation of tRNA. To assess the factors that result in the reduced expression of this operon upon the addition of ILV, a series of plasmids were constructed in which the ilv regulatory region was fused to galK. In response to addition of the amino acids, expression of the galK gene fused to the leader-attenuator decreased five-to sevenfold, instead of the twofold observed for the chromosomal operon. A deletion analysis with these plasmids indicated that the ILV-specific decrease in expression required an intact leader-attenuator but not ilvGp2 or the DNA that precedes this promoter. This conclusion was supported by both Sl nuclease analysis of transcription initiation and determination of galK mRNA levels by RNA-RNA hybridization.Wild-type Escherichia coli K-12 can synthesize all its molecular components when grown on a minimal salts medium containing suitable carbon and nitrogen sources. During steady-state or exponential growth, the cells presumably synthesize the appropriate quantity of each metabolite so as to achieve optimal growth. This is facilitated by the ability of the cell to increase or decrease the synthesis of enzymes or alter the catalytic activity of specific enzymes in response to changes in the environment in which the cell is growing. For several amino acid-biosynthetic systems, the cell responds to an exogenous source of an amino acid (i.e., an excess) by decreasing (i.e., repressing) the expression of the relevant genes. Alternatively, if the amount of an amino acid becomes growth limiting (i.e., starvation), the bacteria will increase expression of the genes required for the biosynthesis of that amino acid.The trp operon is probably the most thoroughly studied set of genes for the biosynthesis of an amino acid. For the trp operon, the molecular mechanisms (34, 50) that result in the change in expression of this operon in response to either an excess or limitation for tryptophan are distinct. The trp repressor protein regulates the operon by mediating the initiation of transcription by RNA polymerase (RNAP) in response to an excess of free tryptophan within the cell.When growth is limited by tryptophan, increased expression of the operon occurs as a result of reduced aminoacylation of trp tRNA. This change in trp operon expression is mediated by the leader-attenuator. As described by Yanofsky et al. (50), the combination of the repressor-operator and the leader-attenuator results in a 500-to 600-fold range in expression of the trp operon.The ilvGMEDA operon encodes four of...

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supporting

confidence: 64%

Analysis of regulation of the ilvGMEDA operon by using leader-attenuator-galK gene fusions

et al. 1990

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“…This ilv-specific regulation appears to be exclusively a consequence of an attenuation mechanism ( Fig. 1) (4,32,38). In principle, the attenuation mechanism controlling the ilv operon appears to be quite similar to that found to control operons involved in tryptophan (35), leucine (19), phenylalanine (61), histidine (3), and threonine (17) biosynthesis: the leader region specifies a short peptide containing a disproportionate frequency of the amino acids that are found to regulate the operon.…”

mentioning

confidence: 99%

Specificity of attenuation control in the ilvGMEDA operon of Escherichia coli K-12

1991

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Three different approaches were used to examine the regulatory effects of the amino acids specified by the peptide-coding region of the leader transcript of the ilvGMEDA operon of Escherichia coli K-12. Gene expression was examined in strains carrying an ilvGMED'-lac operon fusion. In one approach, auxotrophic derivatives were starved of single amino acids for brief periods, and the burst of j-galactosidase synthesis upon adding the missing amino acid was determined. Auxotrophic derivatives were also grown for brief periods with a limited supply of one amino acid (derepression experiments). Finally, prototrophic strains were grown in minimal medium supplemented with single and multiple supplements of the chosen amino acids. Although codons for arginine, serine, and proline are interspersed among the codons for the three branched-chain (regulatory) amino acids, they appeared to have no effect when added in excess to prototrophs or when supplied in restricted amounts to auxotrophs. Deletions removing the terminator stem from the leader removed all ilvspecific control, indicating that the attenuation mechanism is the sole mechanism for ilv-specific control.The specific control of expression of the ilvGMEDA operon in the enteric bacteria is characterized by repression of the operon when the three branched-chain amino acids are in ample supply but derepression when the supply of any one of the three is limited (14,30). This ilv-specific regulation appears to be exclusively a consequence of an attenuation mechanism ( Fig. 1) (4,32,38). In principle, the attenuation mechanism controlling the ilv operon appears to be quite similar to that found to control operons involved in tryptophan (35), leucine (19), phenylalanine (61), histidine (3), and threonine (17) biosynthesis: the leader region specifies a short peptide containing a disproportionate frequency of the amino acids that are found to regulate the operon. Thus, the rate at which the leader can be translated serves to sense whether the regulatory amino acid is in short or ample supply.As emphasized by Landick and Yanofsky (31), an important feature of the attenuation mechanism is the pausing of RNA polymerase at a certain site in the leader region. The pause allows time for the ribosome to initiate translation, and as translation proceeds, the base pairing in the protector (1:2 stem, Fig. 2) is progressively disrupted. Should ribosome movement be retarded, presumably by stalling at a regulatory codon, long enough for the bases in the upstream arm of the terminator (bases 157 to 164) to emerge on the nascent transcript, these bases can pair with those in the downstream arm of the protector (bases 98 to 105) in the antiterminator (2:3 stem, Fig. 3). It is assumed that, in the absence of ribosome stalling (repressing conditions), translation proceeds rapidly enough that the bases in the downstream arm of the protector will also be covered by the ribosome or otherwise be sterically prevented from antiterminator formation and the terminator will be formed instead. Altho...

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“…Many of the amino acid biosynthetic operons of bacteria are regulated by attenuation. Each of these operons has a transcribed leader region containing a short peptide-encoding segment rich in codons for the regulatory amino acid(s) (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). The presumptive leader peptides specified by these coding regions have been sought but have eluded detection.…”

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confidence: 99%

In vitro synthesis of the tryptophan operon leader peptides of Escherichia coli, Serratia marcescens, and Salmonella typhimurium.

Das¹,

Urbanowski²,

Weissbach³

et al. 1983

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

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We used an in vitro DNA-dependent proteinsynthesizing system to demonstrate de novo synthesis of the leader peptide specified by the tryptophan (trp) operons of several bacterial species. Peptide synthesis was directed by self-ligated short restriction fragments containing the trp promoter and leader regions. Synthesis of leader peptides was established by demonstrating that they were labeled in vitro only by those amino acids predicted to be present in the peptides. Leader peptide synthesis was abolished by the addition of the Escherichia coli trp repressor. The E. coli trp leader peptide was found to be extremely labile in vitro; it had a half-life of 3-4 min. In a highly purified DNAdependent peptide-synthesizing system, synthesis of the di-and tripeptides predicted from the Salmonella typhimurium trp operon leader sequence, fMet-Ala and fMet-Ala-Ala, also was observed. Using this dipeptide synthesis system, we demonstrated that translation initiation at the ribosome binding site used for trp leader peptide synthesis was reduced 10-fold when the transcript contained a segment complementary to the ribosome binding site. Many of the amino acid biosynthetic operons of bacteria are regulated by attenuation. Each of these operons has a transcribed leader region containing a short peptide-encoding segment rich in codons for the regulatory amino acid(s) (1-10). The presumptive leader peptides specified by these coding regions have been sought but have eluded detection. Nevertheless, gene fusion studies have demonstrated that the ribosome binding site of the trp operon leader region is an efficient site for translation initiation (11,12). We considered two explanations for our inability to detect the trp leader peptide. First, because synthesis of the peptide but not its survival was presumably of regulatory significance, we thought that the peptide might be rapidly degraded both in vivo and in vitro (11, 13). Second, scrutiny of the sequences of the trp leader transcripts of seven enteric bacterial species revealed that the distal segment of each leader transcript was complementary to the leader peptide ribosome binding site and conceivably could pair with that site and prevent multiple rounds of synthesis of the trp leader peptide (refs. 14-16; unpublished observations). The approach that was successful in the initial detection of the trp leader peptide, described herein, was suggested by Roberto Kolter of our laboratory.We performed in vitro coupled transcription/translation analyses, using as template a short restriction fragment containing the trp promoter-operator and leader peptide-encoding region and lacking the distal portion of the trp leader region that is complementary to the leader peptide ribosome binding marscens, and S. typhimurium. Nucleotide numbering is from the site oftranscription initiation (residue + 1). Negative numbers indicate the number ofbase pairs preceding the transcription start point. Numbers withinparentheses indicate that the exact site is notknown. TheE. coli template has a...

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Nucleotide sequence of ilvGEDA operon attenuator region of Escherichia coli.

Cited by 95 publications

References 19 publications

Analysis of regulation of the ilvGMEDA operon by using leader-attenuator-galK gene fusions

Analysis of regulation of the ilvGMEDA operon by using leader-attenuator-galK gene fusions

Specificity of attenuation control in the ilvGMEDA operon of Escherichia coli K-12

In vitro synthesis of the tryptophan operon leader peptides of Escherichia coli, Serratia marcescens, and Salmonella typhimurium.

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