Bacillus subtilis RNase III cleaves both 5'- and 3'-sites of the small cytoplasmic RNA precursor

Abstract

Bacillus subtilis small cytoplasmic RNA (scRNA) is a member of the signal recognition particle RNA family. It is transcribed as a 354-nucleotide primary transcript and processed to a 271-nucleotide mature scRNA. In the precursor, the 5'- and 3'-flanking regions form a stable double-stranded structure based on their complementary sequence. This structure is similar to those of substrates for the double-stranded RNA processing enzyme, RNase III. The B. subtilis enzyme that has similar activity to Escherichia coli RNase III has been purified and is designated Bs-RNase III. Recently, B. subtilis rncS has been shown to encode Bs-RNase III (Wang, W., and Bechhofer, D. H. (1997) J. Bacteriol. 179, 7379-7385). We show here that Bs-RNase III and the purified His-tagged product of rncS cleave pre-scRNA at both 5'- and 3'-sites to produce an intermediate scRNA (scRNA-275), although processing at the 3'-site is less efficient. The 5'-end of scRNA-275 was identical to that of the mature scRNA, whereas it contains four excess nucleotides at the 3'-end. Bs-RNase III cleavage yields a two-base 3'-overhang, which is consistent with the manner in which E. coli RNase III cleaves. We also show that truncation of the rncS gene affected processing, and significant amounts of an intermediate scRNA (scRNA-275) were found to accumulate in the rncS-truncated mutant. It is concluded that Bs-RNase III is an enzyme that processes pre-scRNA.