Analysis of the bldA tRNA gene of Streptomyces clavuligerus: Implications for mistranslation of TTA codons.

摘要

The bldA gene encodes the tRNALeuUAA that translates the rare UUA leucine codon in the high G +C (∼70%) Streptomyces mRNA. These rare TTA codons are confined to Streptomyces genes that function relatively late in growth, after the switch from primary metabolism and vegetative growth to secondary metabolism and differentiation. bldA mutant strains of S. coelicolor, S. lividans, and S. griseus are defective in sporulation and antibiotic production, as key genes required for these processes contain TTA codons and are not efficiently translated in the absence of the bldA tRNALeuUAA .; In an effort to gain evidence that sporulation and antibiotic production are similarly regulated by bldA in other (perhaps all) Streptomyces species, the bldA gene was cloned and sequenced in Streptomyces clavuligerus, and a bldA mutant strain was generated by gene replacement. The phenotype of the mutant strain was sporulation defective as expected, however, the S. clavuligerus bldA mutant was not defective in antibiotic production, as cephamycin C, and clavulanic acid were produced in the mutant strain. Subsequent analysis indicated that antibiotic production in the absence of the bldA tRNALeuUAA was due to the efficient mistranslation of the single TTA codon in the cephamycin C and clavulanic acid pathway-specific activator gene, ccaR, possibly by wobble base pairing of the tRNALeuCAA with the UUA codon.; Since a TTA-containing reporter gene was not expressed in the S. clavuligerus bldA mutant it was evident that not all UUA codons are mistranslated efficiently in the S. clavuligerus bldA mutant strain, and that there must be something unique about the context of the TTA codon in the ccaR gene that makes its expression essentially independent of the bldA tRNA. When the ccaR gene was compared to several TTA-containing genes that were shown to be dependent on bldA for their expression, it was found that ccaR contains a guanine nucleotide 3' of the TTA codon whereas most TTA codons, in other genes contain a cytidine in this position. This observation lead to the hypothesis that +1 frameshifts are introduced at TTA C and TTA T sequences by the tRNATyrGUA in bldA mutants while TTA G and TTA A sequences are mistranslated in-frame, as +1 shifts are prevented by the TAG or TAA stop codon in the +1 frame.