Analysis and comparison of the internal promoter, pE, of the ilvGMEDA operons fromEscherichia coliK-12 andSalmonella typhimurium

Abstract

It was previously determined that the distal portion of the ilvGMEDA operon was expressed despite the insertion of transposons into ilvG and ilvE. This observation suggested the existence of internal promoters upstream of ilvE (pE) and ilvD (pD). The internal promoter pE, responsible for part of ilvEDA expression, has been analyzed both in vivo and in vitro. Our results indicate that: pE exists in both E. coli K-12 and S. typhimurium; pE is located in the distal end of the ilvM coding sequence; the pE sequence is highly conserved in the two bacteria; the amino acid sequence of the ilvM gene product is 93% homologous between the two bacteria; transcription from pE can be demonstrated both in vivo and in vitro; the efficiency of pE is essentially equivalent in the two bacteria.

The absence of branched-chain amino acid and growth rate control at the internal ilvEp promoter of the ilvGMEDA operon

“…The cat gene from pKK232-8 had previously been placed under the ilvEp promoter and used for a different purpose (16). The strain carrying the ilvEp-cat fusion, CU1648, offered a convenient system to examine whether ilvEp is regulated specifically by the availability of valine, isoleucine, and leucine, the end products of the pathway, or is affected by the growth rate of the cells as proposed earlier (21,33). Strain CU1648 carries the ilvEp promoter on a 100-bp HaeII-AatII fragment upstream of the promoterless cat gene, with about 50 bp upstream of the -35 region.…”

“…It has been repeatedly observed that when cells are grown in rich medium (L broth), the genes of the ilv operon are more strongly repressed than they are in cells growing in the presence of all three branched-chain amino acids (9,21,33). Furthermore, this effect is independent of the operon promoter ilvGp (11,21). This repression might reflect a more general type of regulation, i.e., a growth rate-dependent control.…”

“…The question arises as to whether ilvEp itself is subject to either amino acid-mediated or any other kind of regulation and whether any such kind of regulation contributes significantly to downstream gene expression under various growth conditions. Previous studies have been contradictory, some indicating that ilvEp might be regulated (21,33) and others proposing that it is constitutively expressed (3,37). The experiments described below address this question.…”

“…The expression of the entire operon is specifically regulated by the presence of all or the absence of any one of the branched-chain amino acids via translational control of transcription termination at an attenuator site preceding ilvG, the first structural gene of the operon (10,19,26,34). Two internal promoters, potential sites for modulation of expression, have been described (3,4,21,22). Promoter ilvEp is located upstream of ilvE within the coding region for ilvM, and promoter ilvAp is located upstream of ilvA within the coding region of ilvD (22,37).…”

“…Therefore, in E. coli K-12 grown under repressing conditions, the amount of transcription into the structural genes, initiated at ilvGp but not terminated at the attenuator site, will be further reduced at these polarity sites, upstream of UivE. Indeed, transcription of the structural genes in wildtype E. coli K-12 has been attributed mainly to transcription initiation at the internal promoter ilvEp under repressing conditions (4) and in minimal medium (21). ilvEp was shown to be a weaker promoter than the major operon promoter ilvGp (4,37).…”