Translational coupling in the pyrF operon of Salmonella typhimurium

Abstract

The pyrF gene, encoding the sixth enzyme of pyrimidine biosynthesis in Salmonella typhirmurium, appears to be the first gene of an operon. The second gene, orfF, encodes a 11.5 kDa polypeptide of unknown function. To study the regulation of orfF expression directly, transcriptional and translational fusions of orfF to galK and lacZ, respectively, were constructed and the level of expression of the reporter genes was determined under different growth conditions. The results obtained show that the synthesis of OrfF and orotidine 5'-phosphate decarboxylase is coordinately controlled by pyrimidines, and that this control occurs at the level of transcription. The orfF translational start codon overlaps the pyrF translational stop codon, suggesting that the two genes are translationally coupled. This was investigated by studying how frameshift mutations, which cause premature termination of pyrF translation at different points, affect orfF expression. All mutations reduced orfF expression markedly without interfering with transcription of the gene. Thus, expression of pyrF and orfF are translationally coupled. Inspection of the nucleotide sequence of the pyrF/orfF junction region suggests that formation of secondary structures on the naked mRNA may explain the low level of orfF expression in the absence of translation of the pyrF terminal region.