Translational coupling between the ilvD and ilvA genes of Escherichia coli

Abstract

The hypothesis that translation Qf the ilvD and ilvA genes of Escherichia coli may be linked has been examined ip strains in which lacZ-ilvD protein fusions are translated in all three reading frames with respect to ilvD. In these strains, the nucleotide sequence was altered to obtain premature termination of ilvD translation, and in one strain translation termination of ilvO DNA occurred two bases downstream of the ilvA initiation codon. In the wild-type strain, the ilvD translation termination site was located two bases upstream of the ilvA start codon. In each of the mutant strains, expression of ilvA, as determined by the level of threonine deaminase activity, was strikingly lower than in the wild-type strain. The data suggest that expression of ilvD and ilvA is translationally coupled. By inserting a promoterless cat gene downstream of ilvA, it was shown that the differences in enzyme activity were not the result of differences in the amount of ilvA mRNA produced.In members of the family Enterobacteriaceae, the biosynthesis of isoleucine and valine occurs by parallel pathways with enzymes specified by a cluster of genes, ilvGMEDA YC (39). The first five genes in the cluster form the ilvGMEDA operon; ilvY and ilvC are separate transcriptional units. Blazey and Burns (4) first implicated regulation at the translational level in the ilv operon and raised the possibility of translational coupling. Their experiments showed that TnJO insertions in both orientations in the ilvD gene of Salmonella typhimurium exerted an absolute polarity on ilvA expression. In Escherichia coli, a similar strong polar effect has been demonstrated in strains containing bacteriophage Mu-1 insertions (32) or Tn5 insertions (3). Even earlier, it had been noted that the ilvlI188 ochre mutation resulted in a 60% reduction in threonine deaminase activity, although at the time, the effect was thought to be "antipolar" (42).Nucleotide s'equence analysis has revealed that the ilvD translation stop codon and'ilvA start codon are separated by 2 bases, TAA-TA-ATG (8, 18). (In this paper, we use the phrase "n bases upstream [or downstream]" to indicate that n bases separate the stop and start codons.) Proximity of translational termination and initiation signals, including overlapping signals, has been described as a feature in coupling translation of adjacent genes (11,24,33,35). Translational coupling has been reported for a number of gene pairs, including galK-galT (29), trpD-trpE (26), and trpB-trpA (1, 9). In this paper we present evidence that efficient translation of the downstream ilvA gene is dependent on translation of the upstream ilvD gene. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains used in this study and their sources are listed in Table 1. The preparation of some of the strains requires special mention. Strain CU1644 was prepared by replacing the wild-type ilv region of CU1318 with that in pPU316, which carries AU(i'G-Y)2243::aad'. The aad+ gene, inserted at the site of the ilv deletion, was derived f...

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