Regulation of protein synthesis by minigene expression

Peptidyl-tRNA hydrolase (Pth), an enzyme essential for Escherichia coli viability, scavenges peptidyl-tRNA released during abortive polypeptide chain elongation. Bacterial strains of E coli partially defective in Pth activity are unable to maintain bacteriophage lambda growth. Phage mutations that overcome the bacterial defect have been located to several regions in the lambda genome named bar. Plasmid constructs expressing just the bar region are toxic and cause a general arrest of protein synthesis in Pth-defective cells. Inspection of the nucleotide sequence from two bar regions reveals the short coding sequence AUG AUA Stop, spaced by an AT-rich segment from a Shine Dalgarno-like sequence (S-D). These sequences have been named minigenes. Base changes altering the putative S-D, the two sense codons, or the stop codon have been found to reduce Bar-toxicity. Transcripts containing bar function as mRNA. Upon expression in pth mutants, wild-type (bar+) transcripts are found associated with ribosomes. In addition, bar+ RNA forms ternary complexes with the 30S ribosomal subunit and the initiator tRNA and can be released upon run-off translation in the same way as an authentic mRNA. A cell free system for protein synthesis reproduces the in vivo effects: bar+ expression inhibits protein synthesis, bar+ RNA sequences are associated with ribosomes in the inhibited extracts, addition of purified Pth restores synthesis, and excess of tRNA(Lys), specific for the last sense codon in a mutant toxic minigene, prevents protein synthesis inhibition. Also, bar expression promotes association of methionine with ribosomes possibly in a translation complex. These results are consistent with a model proposing tRNA starvation to explain the behaviour of a pth mutant, thermosensitive for protein synthesis.