Multivalent translational control of transcription termination at attenuator of ilvGEDA operon of Escherichia coli K-12.

Abstract

The regulatory region for the ivGEDA operon of Escherichia coli K-12 has been located and characterized. ilv leader RNA transcribed from this region is described, and the DNA sequence of the region is presented. This DNA sequence contains a transcription promoter, a region coding for a 32-amino-acid polypeptide containing multiple isoleucine, valine, and leucine codons, and a transcription termination site preceding the first structural gene. The mutually exclusive secondary structures of the leader RNA have been analyzed. On the basis of these data, a model for the multivalent attenuation of the ilvGEDA operon is proposed.The ilvGEDA genes of Escherichia col' K-12 are multivalently regulated by the percent aminoacylation of tRNAVal, tRNAIle, and tRNALeu (1, 2), and strains containing altered Rho factor exhibit increased expression of this operon (3,4). It has, therefore, been suggested that this operon might be regulated by an attenuator mechanism (3-5) similar to that proposed for several other amino acid biosynthetic operons (6-11). We have now defined the location of the regulatory region for the ilvGEDA operon as being promoter proximal to the livG gene. We show that a previously described short RNA transcript is an ilv leader RNA which is transcribed from the regulatory region. We designate the probable sites of transcriptional initiation and termination of this leader RNA by in vitro transcription. We also report here the DNA sequence of the IlvGEDA promoter-attenuator region and show that it contains multiple codons for isoleucine, valine, and leucine within a potential coding region for a short leader polypeptide. On the basis of these results and by analysis of the possible secondary structures of the ilv leader RNA, we conclude that this operon is regulated by translational control of transcription termination at an attenuator site (12, 13). MATERIALS AND METHODSRestriction enzymes were obtained from New England Biolabs, were prepared by standard methods, or were a gift of Charles Yanofsky. E. coli DNA-dependent RNA polymerase was obtained from New England Biolabs. Plasmid pVH153, containing the regulatory-region of the trp operonwith a G-to-A transition at base pair 130 of the attenuator (14), was a generous gift of G. Zurawski and C. Yanofsky. The plasmid, pRL5, was constructed and DNA was prepared as described (15).In vitro RNA transcription was performed as described (6,15) with the addition of heparin as described by Majors (16). The DNA sequence analysis was performed as described by Maxam and Gilbert (17). RESULTSThe in vitro transcription of two plasmids that contain the regulatory region for the ilvGEDA operon. yields two short leaderlike RNAs 180 and 250 nucleotides long (see Fig. 4, lane 1; ref. 15). This in vitro labeled RNA was hybridized to a Southern blot (18) of a restriction endonuclease digest of the regulatory region. Both transcripts hybridized to the 300-base-pair Hae III/HinfI fragment (see Fig. 2) located 1.8 kilobase pairs in front of the DNA sequence encoding the ilv...

Branched-chain amino acid biosynthesis genes in Lactococcus lactis subsp. lactis

“…4). This peptide is very similar to the leader peptides of the E. coli and S. typhimurium leu operons (18,66) but differs from those of the E. coli ilvBN (16) and ilvGMEDA (35) operons.…”

Metabolic engineering of Escherichia coli for the production of l -valine based on transcriptome analysis and in silico gene knockout simulation

“…First, feedback inhibition and transcriptional attenuation were removed by site-directed mutagenesis. The sequences involved in these regulatory mechanisms were identified from previously published data or patents (13,14,(21)(22)(23). Feedback inhibition in the small subunit of AHAS III encoded by the ilvH gene was removed by displacing the 41st base G with A and the 50th base C with T. Transcriptional attenuation sites were removed by displacing the attenuator leader region with the tac promoter by homologous recombination.…”

Transcriptional regulation of the ilv-leu operon of Bacillus subtilis

“…In E. coli and Salmonella typhimunium, operons involved in branched-chain amino acid biosynthesis, including ilv GMEDA, ilvBN, and leuABCD, are controlled by transcription attenuation mechanisms (5,8,10). In each of these cases, most transcription initiated in vitro at the promoter is terminated at an attenuator site located upstream of the structural genes of the operon.…”