Cloning and analysis of the Bacillus subtilis rpsD gene, encoding ribosomal protein S4

Abstract

The rpsD gene, encoding ribosomal protein S4, was isolated from Bacillus subtilis by hybridization with oligonucleotide probes derived from the S4 amino-terminal protein sequence. Sequence analysis of the cloned DNA indicated that rpsD is likely to be monocistronic, in contrast to Escherichia coli rpsD, which is located in the alpha operon and is the translational regulator for alpha operon ribosomal protein gene expression in E. coli. The cloned gene was shown to map at position 263 degrees on the B. subtilis chromosome, at the position to which mutations conferring alterations in the electrophoretic mobility of protein S4 were localized. A promoter was identified upstream of the rpsD coding sequence; initiation of transcription at this promoter would result in a transcript containing a leader region 180 bases in length. Immediately downstream of the rpsD coding region were two sequences resembling transcriptional terminators. An open reading frame homologous to tyrosyl-tRNA synthetase (tyrS) genes was identified downstream of rpsD but in the opposite orientation. The leader region of rpsD mRNA is predicted to have extensive secondary structure, resembling a region of B. subtilis 16S rRNA where S4 is likely to bind; similar mRNA features have been found to be important in ribosomal gene regulation in E. coli. These results provide the first steps toward analysis of the regulation of rpsD gene expression in B. subtilis.