Regulation of ilvEDA expression occurs upstream of ilvG in Escherichia coli: additional evidence for an ilvGEDA operon

We previously characterized a set of X dilv phages by genetic, restriction enzyme, and heteroduplex analyses and tentatively correlated isoleucine-valine gene products with specific ilv DNA segments by using cloned ilv segments in maxicells and A dilv phage infection of UV-irradiated cells. In this work, the identity of the ilvC gene product, a-acetohydroxy acid isomeroreductase, was confirmed by demonstrating its induction by the physiological inducers aacetolactate and a-acetohydroxybutyrate. The identity of the ilvE gene product, transaminase B, was confirmed by antibody precipitation of the purified enzyme. Phage derivatives with ilv regulatory mutations were found to have the predicted effect upon the ilvGEDA and ilvC protein products. The distribution of the ilvGEDA and ilvC gene products in the soluble, periplasmic, inner membrane, and outer membrane fractions was examined, and no significant membrane association was observed. The expression of the ilv genes in the X dilv phage from ilv and phage X promoters was compared in order to determine the fractional contribution of each to ilv gene expression. An additional protein of 54,000 daltons that was not detected in the previous analysis was observed to be coded by a bacterial gene but was produced only by readthrough from phage promoters.

Section: Control Of Expression Of the Uv Genes In A Dilvmentioning

confidence: 99%

“…Several hypotheses have been proposed to account for the control of expression of the ilv genes that code for the isoleucine-valine biosynthetic enzymes of Escherichia coli K-12 (6,9,14,18,20,28,29). We previously examined a related set of independently isolated A dilv phages that are useful for studies of this type (2, lOa, 12,21).…”

mentioning

confidence: 99%

Absence of significant membrane localization of the proteins coded by the ilvGEDAC genes of Escherichia coli K-12

Gray¹,

Calhoun²

1982

“…Both A dilv58 and pPU34 have been described previously (6,31,32). The fusion fragment had previously been inserted into the KpnI sites of a plasmid-borne ilv cluster by John Noti (31). This fragment was removed by digestion of pPU34 by KpnI.…”

Section: Dilv-1ac13 Inserted Intomentioning

confidence: 99%

“…Previously, a fusion of the lac operon to ilvD was constructed and placed in a multicopy plasmid (31) to allow for the convenient study of the control of expression of il/v operon. We placed this fusion in a lysogenizing lambda phage and isolated two mutant derivatives of this ilv-lac phage with increased expression of the fusion operon.…”

mentioning

confidence: 99%

Isolation and analysis of two Escherichia coli K-12 ilv attenuator deletion mutants with high-level constitutive expression of an ilv-lac fusion operon

Bennett¹,

Umbarger²

1984

Self Cite

A lysogenizing lambda phage, X dilv-lacll, was constructed to carry an ilvD-lac operon fusion. Expression from the phage of the ilvE and lacZ genes is controlled by an intact ilv control region also carried by this phage. Two spontaneous mutants of A dilv-lacll that have high-level constitutive expression of the ilv-lac fusion operon were isolated by growth on a P-chloroalanine selective medium. The mutants were shown by nucleotide sequence determination to contain large deletions (A2216, -1.6 kilobases; A2219, -1.9 kilobases), which in both cases remove the proposed ilv attenuator terminator. The rest of the ilv leader and promoter region DNA remains intact in these mutants. Deletion 2216 also removed part of the downstream ilvG gene, whereas A2219 extended through the entire ilvG gene into the ilvGE intercistronic region. A possible mechanism of deletion formation is discussed.

“…The nucleotide sequences of the fragments cloned into the M13 derivatives were obtained by the dideoxynucleotide chain termination method of Sanger et al (26). RNA polymerase binding studies were done by the method of Subrahmanyam et al (33).…”

mentioning

confidence: 99%

Comparison of the regulatory regions of ilvGEDA operons from several enteric organisms

Harms¹,

Hsu²,

Subrahmanyam³

et al. 1985

Self Cite

The nucleotide sequence preceding the ilvGEDA operon has been examined and compared in five enteric organisms. The sequence in Escherichia coli B was identical to the earlier-described strain K-12 sequence. The sequences of SalmoneUa typhimurium and Klebsiella aerogenes were remarkably similar to that of E. coli and identical in that part of the leader region that specified the putative 32-amino-acid peptide. Thus, identical secondary structures could be postulated for the leaders of all three organismst and regulation of operon expression could be like that postulated earlier for E. coli. Different secondary structures had to be postulated for the leader transcripts of Edwardsie11a tarda and Serratia marcescens. Control of attenuation of the operon in these organisms by the level of leucyl tRNA could be explained only if ribosome stalling occurred at a single leucine codon. In both organisms, that single leucine codon is the rarely used CUA rather than the CUG that is in E. coli, S. typhimurium, and K. aerogenes.The expression of the ilvGEDA operon of Escherichia coli K-12 is multivalently controlled by the three branched-chain amiino acids (for a review, see reference 36). Regulation is thought to occur by attenuation of transcription in response to the charging levels of the cognate tRNAs (15,21). According to the attenuation model, transcription of the ilvGEDA operon is usually terminated at the end of the leader (regulatory) region. The leader transcript contains 15 isoleucine, leucine, and valine codons in an open reading frame specifying 32 amino acids. The site of termination (the terminator) is typical of a p-independent termination site, a sequence of uracil residues preceded by bases capable of forming a stem-and-loop structure. If any branched-chain amino acid is limiting, the first ribosome translating the leader transcript is thought to stall at a codon for that amino acid. This condition leaves bases complementary to the upstream arm of the terminator uncovered and allows the formation of a different stem-and-loop structure, the preemptor. The formation of the preemptor precludes formation of the terminator and results in transcription into the ilvGEDA operon.As an approach to better understanding of the mechanism of attenuation and determination of the minimal features required to execute this type of control, we have compared and analyzed the nucleotide sequences of the ilvGEDA regulatory regions of five enteric organisms, i.e., E. coli B, Klebsiella aerogenes, Edwardsiella tarda, Serratia marcescens, and Salmonella typhimurium, and compared them with the sequence previously determined for the K-12 strain of E. coli. The leader region of S. typhimurium (previously published by Taillon et al. [34]) was independently examined in our laboratory and is also considered here in the compari-sQn.Because the nucleotide sequences of the ilv leader region and part of the gene for acetohydroxy acid synthase II were nearly identical in E. coli K-12 and B, we were led to examine in strain B the region homologo...