Different Processing of an mRNA Species in Bacillus subtilis and Escherichia coli

Abstract

Expression of the Bacillus subtilis glpD gene, which encodes glycerol-3-phosphate (G3P) dehydrogenase, is controlled by termination or antitermination of transcription. The untranslated leader sequence of glpD contains an inverted repeat that gives rise to a transcription terminator. In the presence of G3P, the antiterminator protein GlpP binds to glpD leader mRNA and promotes readthrough of the terminator. Certain mutations in the inverted repeat of the glpD leader result in GlpP-independent, temperature-sensitive (TS) expression of glpD. The TS phenotype is due to temperature-dependent degradation of the glpD mRNA. In the presence of GlpP, the glpD mRNA is stabilized. glpD leader-lacZ fusions were integrated into the chromosomes of B. subtilis and Escherichia coli. Determination of steady-state levels of fusion mRNA in B. subtilis showed that the stability of the fusion mRNA is determined by the glpD leader part. Comparison of steady-state levels and half-lives of glpD leader-lacZ fusion mRNA in B. subtilis and E. coli revealed significant differences. A glpD leader-lacZ fusion transcript that was unstable in B. subtilis was considerably more stable in E. coli. GlpP, which stabilizes the transcript in B. subtilis, did not affect its stability in E. coli. Primer extension analysis showed that the glpD leader-lacZ fusion transcript is processed differently in B. subtilis and in E. coli. The dominating cleavage site in E. coli was barely detectable in B. subtilis. This site was shown to be a target of E. coli RNase III.The steady-state level of mRNA in a cell is a function of the rate of mRNA synthesis and the rate of its decay. For bacteria, there is a wealth of information on the regulation of mRNA synthesis (see, e.g., reference 27), while much less is known about mechanisms of mRNA decay (5,6,35).Most of our knowledge about bacterial mRNA decay is based on studies of Escherichia coli (26,34). In a simple model, an initial endoribonucleolytic attack at the 5Ј end of an mRNA opens up the molecule for internal downstream cleavages and the fragments generated are subsequently degraded by exoribonucleases (12). The initial cleavage is performed by one of two endoribonucleases, RNase E, encoded by the rne gene (7), or RNase III, encoded by the rnc gene (3,8). The endonucleolytic activity of RNase E is localized to the N-terminal half of the protein, which, unlike the C-terminal half, is essential for E. coli viability (29, 31). RNase III is primarily involved in maturation of stable RNA but also in degradation of some mRNA species. The hydrolytic exoribonuclease RNase II and the phosphorolytic exoribonuclease polynucleotide phosphorylase (PNPase) are important for the final (3Ј-to-5Ј) degradation of an mRNA to mono-and oligonucleotides. For a few E. coli mRNA species, binding of specific proteins has been found to have a decisive influence on mRNA half-life (28, 43).Much less is known about mRNA degradation in Bacillus subtilis. In several bacterial species, but not B. subtilis, sequence homologues to the N-termina...

The aprE leader is a determinant of extreme mRNA stability in Bacillus subtilis

“…3b, c). This represents the 5h part of the full-length transcript and has been found also in other types of transcriptionaltranslational fusions with lacZ in B. subtilis (Persson et al, 2000). The truncated transcript decayed at the same rate as the full-length transcript, which further supports the notion that the aprE leader is the major stability determinant also in the fusion transcript.…”

“…Twenty micrograms of RNA was added to each well unless otherwise indicated. Single-stranded (ss) DNA probes were generated as described before (Persson et al, 2000) with the following primers : lacZ60 for lacZ constructs, aprEBam2 for aprE, and amyESeq for amyE. To generate templates for the ssPCR, specific fragments were amplified with PCR using the following primers and templates : glpDBam1 and lacZ60 with pLUM1041, aprESubTT and aprEBam2 with pLUS1, and amyE1 and amyESeq with chromosomal B. subtilis DNA.…”

“…The general model for mRNA degradation probably applies to other bacteria but with important modifications in details. For instance, no RNase E homologue has been found in Bacillus subtilis (Kunst et al, 1997) and different patterns of degradation have been found for the same mRNA species in B. subtilis and E. coli (Persson et al, 2000).…”

RNA Processing and Degradation inBacillus subtilis

“…Wild-type and mutant (temperature sensitive) translational fusions of the glpD leader to lacZ were integrated into the chromosomes of both organisms. In B. subtilis, the mutant fusion transcript showed the same temperature dependency as the native glpD mRNA in the absence of GlpP, showing that the glpD leader is the major stability determinant for both transcripts (187). In E. coli, however, both the wildtype and mutant glpD-lacZ mRNAs were relatively stable (half-lives, 3 to 4 min) at 42°C.…”

A Mutation in the 5′ Untranslated Region Increases Stability of norA mRNA, Encoding a Multidrug Resistance Transporter of Staphylococcus aureus

“…4). Although cleavage sites for RNase III in S. aureus have not been defined, in Bacillus subtilis this sequence has been shown to be a target for RNase III (13,20).…”