Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598–600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C Gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along Gram-negative and Gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored.

The 5′ untranslated region (UTR) of the long-lived Escherichia coli ompA transcript functions as an mRNA stabilizer that can prolong the cytoplasmic lifetimes of a variety of messages to which it is fused. Previous studies have identified two domains of this 5′ UTR that together are responsible for its stabilizing effect. One is a 5′-terminal stem-loop. The other is a single-stranded RNA segment (ss2) that contains a ribosome binding site highly complementary to 16S ribosomal RNA. Here we report a detailed investigation of the function of these two stabilizing elements. Our data indicate that mRNA protection by a 5′ stem-loop requires no sequence features or thermodynamic stability beyond the minimum necessary for stem-loop formation. Stabilization by ss2 appears to result not from a high frequency of translation initiation, but rather from a high degree of occupancy of this 5′ UTR segment by bound ribosomes. Although close spacing of translating ribosomes is not critical for message stabilization by the ompA 5′ UTR, mRNA longevity does require the periodic passage of ribosomes through the protein-coding region. Unlike bound ribosomes, which hinder mRNA cleavage by RNase E, the 5′ stem-loop appears to impede degradation of ompA mRNA via a distinct pathway that is RNase E-independent. These findings imply that the ompA 5′ UTR prolongs mRNA longevity by impeding multiple pathways for mRNA degradation.