A 5′ stem–loop and ribosome binding but not translation are important for the stability of Bacillus subtilis aprE leader mRNA

Abstract

The Bacillus subtilis aprE leader is a determinant of extreme mRNA stability.The authors examined what properties of the aprE leader confer stability on an mRNA. The secondary structure of the aprE leader mRNA was analysed in vitro and in vivo, and mutations were introduced into different domains of an aprE leader-lacZ fusion. The half-lives of the corresponding transcripts were determined and β-galactosidase activities were measured. Removal of a stem-loop structure at the 5' end or diminishing the strength of the RBS reduced the half-lives from more than 25 min to about 5 min. Interfering with translation by abolishing the start codon or creating an early stop codon had no or little effect on mRNA stability. The authors conclude that a 5' stem-loop and binding of ribosomes are necessary for the stability of aprE leader mRNA. The present results, together with a number of other data, suggest that translation of a B. subtilis mRNA is generally not important for its stability ; the situation seems different in Escherichia coli. It is further concluded that the calculated strength of a B. subtilis RBS cannot be used to predict the stability of the corresponding transcript.

Composability of regulatory sequences controlling transcription and translation in Escherichia coli

“…For example, several previous studies in Escherichia coli and Bacillus subtilis have shown that for particular model transcripts, increased ribosome binding or occupancy may enhance mRNA stability (36)(37)(38)(39)(40)(41)(42). Such studies have been hard to interpret due to the complex interactions between the ribosome, RNA degradation machinery, and transcript.…”

Lost in translation: the influence of ribosomes on bacterial mRNA decay: Figure 1.

“…For example, experiments in both E. coli and B. subtilis have shown that a variety of mRNAs can be significantly destabilized by mutations in the Shine-Dalgarno element that interfere with ribosome binding by markedly reducing its complementarity to 16S rRNA (Wagner et al 1994;Arnold et al 1998;Jürgen et al 1998;Hambraeus et al 2002;Sharp and Bechhofer 2003). In E. coli, the accelerated decay of such translationally impaired mRNAs is generally mediated by RNase E (Jain and Kleckner 1993;Arnold et al 1998).…”

“…This finding suggests that the proximity of the PTC to the 5Ј-most initiation codon may in some cases be more important for rapid decay than the unmasking of potential cleavage sites downstream of the PTC. Furthermore, some transcripts (e.g., B. subtilis aprE, Rhodobacter capsulatus pufBA) are not destabilized by PTCs, presumably because other characteristics of these mRNAs or their host organisms make them resistant to ribonuclease attack under these circumstances (Klug and Cohen 1991;Hambraeus et al 2002;Sharp and Bechhofer 2003).…”

“…In E. coli and B. subtilis, changing an initiation codon from AUG to a weaker initiation codon (GUG, CUG, or UUG) or to a codon that cannot function in translation initiation typically results in a marked reduction in translational efficiency, yet more often than not, such changes cause only a modest decrease in mRNA half-life (Wagner et al 1994;Matsunaga et al 1997;Arnold et al 1998;Hambraeus et al 2002;Sharp and Bechhofer 2003;Komarova et al 2005). These findings suggest that a high degree of RBS occupancy by ribosomes is usually more important for mRNA longevity than is close spacing of translating ribosomes.…”

“…The key features of these 5Ј-UTRs that enable them to protect mRNA from degradation include their ribosome-binding site (RBS; see below) and the presence of a stem-loop structure at the 5Ј terminus (Emory et al 1992;Arnold et al 1998;Hambraeus et al 2002). These findings have led to the conclusion that there is an important pathway for bacterial mRNA degradation in which bound ribosomes or base-pairing near the 5Ј end can impede access to internal endonuclease cleavage sites by a mechanism that is poorly understood.…”