Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: mature 5S rRNA is dispensable for ribosome function.

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.

Function of polypeptide chain release factor RF-3 in Escherichia coli. RF-3 action in termination is predominantly at UGA-containing stop signals.

Two protein release factors (RFs) showing codon specificity, RF-1 (UAG, UAA) and RF-2 (UAA, UGA), are required for polypeptide chain termination in Escherichia coli. We recently reported the localization and characterization of the gene encoding RF-3 (prfC), a third protein component previously described as stimulating termination without codon specificity. RF-3 is a GTP-binding protein that displays much sequence similarity to elongation factor EF-G. In a termination assay in vitro, RF-3 lowers the Km for terminator trinucleotides and is thought to act in termination signal recognition. The gene prfC was identified by transposon insertion mutagenesis leading to enhanced nonsense suppression of UGA. We report here that (i) RF-3 inactivation significantly enhances the suppression of termination in vivo only at UGA-dependent stop signals; (ii) the codon-dependent contribution to the stimulation of fMet release in vitro by RF-3 is significantly greater with UGA termination triplet than UAG termination triplet; (iii) RF-3 increases dramatically the affinity of RF-2 to the UGA termination complex in vitro but not that of RF-1 to the UAG termination complex; (iv) RF-3 inactivation leads to a positive feedback on the autoregulation of RF-2 synthesis in vivo, dependent on the competition between frameshifting and termination. These findings are discussed in terms of the mechanism of involvement of RF-3 in translation termination.

Localization and characterization of the gene encoding release factor RF3 in Escherichia coli.

Two protein release factors (RFs) showing codon specificity, RF1 and RF2, are known to be required for polypeptide chain termination in Escherichia coli. A third protein component has also been described that stimulates termination in vitro, but it has remained uncertain whether this protein, RF3, participates in termination in vivo or is essential to cell growth. We report (i) the purification and N-terminal sequencing of RF3; (ii) the isolation of transposon insertion mutants similar to miaD, a suppressor of a leaky UAA mutation affecting the gene miaA, leading to enhanced nonsense suppression; (iii) the localization of the affected gene on the physical map of the chromosome; and (iv) the cloning and sequencing of the wild-type gene, providing proof that it encodes the factor RF3. We designate the gene prfC. Two transposon insertions were shown to interrupt the coding sequence of prfC, at codons 287 and 426. The enhanced nonsense suppression in the insertion mutants shows that the product participates in termination in vivo. The isolation of such mutants strongly suggests that the gene product is not essential to cell viability, though cell growth is affected. RF3 is a protein with a molecular weight of 59,460 containing 528 amino acids and displays much similarity to elongation factor EF-G, a GTP binding protein necessary for ribosomal translocation, and other GTP binding proteins known or thought to interact with the ribosome.

Third position base changes in codons 5' and 3' adjacent UGA codons affect UGA suppression in vivo.

The base sequence around nonsense codons affects the efficiency of nonsense codon suppression. Published data, comparing different nonsense sites in a mRNA, implicate the two bases downstream of the nonsense codon as major determinants of suppression efficiency. However, the results we report here indicate that the nature of the contiguous upstream codon can also affect nonsense suppression, as can the third (wobble) base of the contiguous downstream codon. These conclusions are drawn from experiments in which the two Ser codons UCU233 and UCG235 in a nonsense mutant form (UGA234) of the trpA gene in Escherichia coli have been replaced with other Ser codons by site-directed mutagenesis. Suppression of these trpA mutants has been studied in the presence of a UGA nonsense suppressor derived from glyT. We speculate that the non-site-specific effects of the two adjacent downstream bases may be largely at the level of the termination process, whereas more site-specific or codon-specific effects may operate primarily on the activity of the suppressor tRNA.