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

Abstract

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...

A Bacterial mRNA Leader that Employs Different Mechanisms to Sense Disparate Intracellular Signals

“…Although this is lower than the increase observed in the proline auxotroph (Figure 4E), it is similar to the gene expression changes promoted by branched-chain amino acids in the attenuation-regulated ilvGMEDA operon (Chen et al, 1991). Moreover, it is higher than what has been reported for the attenuation-regulated trp operon where starvation for tryptophan does not alter trp expression in a tryptophan prototroph (Yanofsky and Horn, 1994).…”

Mutations replacing the leucine codons or altering the length of the amino acid-coding portion of the ilvGMEDA leader region of Escherichia coli

“…Under limiting valine, both enzyme activities increased 3.5-fold. As was mentioned in the preceding report (4) and was observed consistently, the fold derepression of transaminase B under limiting leucine was always about twice that of P-galactosi-dase. Fourfold derepressed P-galactosidase activity and sevenfold derepressed transaminase B activity were observed when leucine was limited in the auxotrophic ilv::lac strain with the wild-type leader.…”

“…The templates were the pBR322 or the M13mpl8 and M13mpl9 derivatives carrying the various leader mutations (Tables 4 and 5). The templates were hybridized with the primers flanking the leader region described in the preceding report (4).…”

“…In Escherichia coli, the multivalent regulation of the ilvGMEDA operon by the three branched-chain amino acids appears to be achieved exclusively via an attenuation mechanism (2,21,22,26). In the preceding report (4), the specificity of the ilvGMEDA regulatory region was examined with respect to the amino acids that, upon limitation, would lead to derepression or when added singly to minimal medium-grown cells would lead to repression. Although the 32-amino-acid peptide-coding region of the ilv leader transcript contains 14 branched-chain amino acid codons between positions 4 and 20, there are interspersed among them, at position 6, a codon for arginine, and at positions 9 and 14, two codons for serine.…”

“…Growth of cells. The media employed and the methods of growing the cells under conditions of excess and limiting amino acid supplements were those described in the preceding report (4).…”