Ribosomes inhibit an RNase E cleavage which induces the decay of the rpsO mRNA of Escherichia coli.

The hypothesis generally proposed to explain the stabilizing effect of translation on many bacterial mRNAs is that ribosomes mask endoribonuclease sites which control the mRNA decay rate. We present the first demonstration that ribosomes interfere with a particular RNase E processing event responsible for mRNA decay. These experiments used an rpsO mRNA deleted of the translational operator where ribosomal protein S15 autoregulates its synthesis. We demonstrate that ribosomes inhibit the RNase E cleavage, 10 nucleotides downstream of the rpsO coding sequence, responsible for triggering the exonucleolytic decay of the message mediated by polynucleotide phosphorylase. Early termination codons and insertions which increase the length of ribosome-free mRNA between the UAA termination codon and this RNase E site destabilize the translated mRNA and facilitate RNase E cleavage, suggesting that ribosomes sterically inhibit RNase E access to the processing site. Accordingly, a mutation which reduces the distance between these two sites stabilizes the mRNA. Moreover, an experiment showing that a 10 nucleotide insertion which destabilizes the untranslated mRNA does not affect mRNA stability when it is inserted in the coding sequence of a translated mRNA demonstrates that ribosomes can mask an RNA feature, 10-20 nucleotides upstream of the processing site, which contributes to the RNase E cleavage efficiency.     

Both temperature and medium composition regulate RNase E processing efficiency of the rpsO mRNA coding for ribosomal protein S15 of Escherichia coli

Cleavage by RNase E is believed to be the rate-limiting step in the degradation of many RNAs. These cleavages are modulated by 5' end-phosphorylation, folding and translation of the mRNA in question. Here, we present data suggesting that these cleavages are also regulated by environmental conditions. We report that rpsO mRNA, 15 minutes after a shift to 44 degrees C, is stabilized in cells grown in minimal medium. This stabilization is correlated with a reduction in the efficiency of the RNase E cleavage which initiates its decay. We also observe the appearance of RNA fragments previously detected following RNase E inactivation and a defect in the adaptation of RNase E concentration. These observations, coupled to the fact that RNase E overproduction slightly reduces the accumulation of the rpsO mRNA, suggest that this stabilization is caused in part by a limitation in RNase E concentration. An increase in the steady-state level of rpsT mRNA is also observed following a shift to 44 degrees C in minimal medium; however, processing of the 9 S rRNA precursor is not affected under these conditions. We thus propose that RNase E concentration changes in the cell in response to environmental conditions and that these changes can selectively affect the processing and the stability of individual mRNAs. Our data also indicate that the efficiency of cleavage of the rpsO mRNA by RNase E is modified by other factor(s) which remain to be identified.     

Expression of the rpsO and pnp genes: structural analysis of a DNA fragment carrying their control regions.

Precise physical mapping of the genes rpsO and pnp coding respectively for ribosomal protein S15 and polynucleotide phosphorylase together with regions involved in the regulation of their expression has been obtained by the analysis of in vitro deletion mutants. The results suggest that each gene has its own promotor, but that there is coexpression of rpsO and pnp. The nucleotide sequence of rpsO and of the beginning of pnp is presented and includes the presumed regulatory regions of these genes. Several features of the sequence support the mapping experiments and are discussed in relation to the expression of the ribosomal and pnp genes.     

Physical localisation and direction of transcription of the structural gene for Escherichia coli ribosomal protein S15

The coding sequence for the Escherichia coli ribosomal protein S15 (rpsO) has been shown to lie immediately adjacent to the structural gene for polynucleotide phosphorylase (pnp). Based on DNA sequencing data, it is deduced that rpsO is transcribed counterclockwise with respect to the standard E. coli genetic map.