The deleterious effect of an Insertion Sequence removing the last 20% of the essential E. coli rpsA gene, is due to mRNA destabilization, not protein truncation

École normale supérieure, Laboratoire de Génétique Moléculaire-CNRS UMR8541, Paris, F-75230

^* To whom correspondence should be addressed. Email: sylvie.hermann-ledenmat{at}igmors.u-psud.fr.

	Abstract

Ribosomal protein S1, the product of the essential rpsA gene, consists of six imperfect repeats of the same motif. Besides playing a critical role in translation initiation on most mRNAs, S1 also specifically autoregulates the translation of its own messenger. SsyF29 is a viable rpsA allele that carries an IS10R insertion within the coding sequence, resulting in a protein lacking the last motif (S1^C). Growth of ssyF29 cells is slower than wild type. Moreover, translation of a reporter rpsA-lacZ fusion is specifically stimulated, suggesting that the last motif is necessary for autoregulation. However, in ssyF29 cells, the rpsA mRNA is also strongly destabilized; this destabilization, by causing S1^C shortage, might also explain the observed slow growth and autoregulation defect. To fix this ambiguity, we have introduced an early stop codon in the rpsA chromosomal gene, resulting in the synthesis of the S1^C protein without IS10R insertion (rpsA^C allele). RpsA^C cells grow much faster than their ssyF29 counterparts; moreover, in these cells, S1 autoregulation and mRNA stability are normal. In vitro, the S1^C protein binds mRNAs (including its own) almost as avidly as wild-type S1. These results demonstrate that the last S1 motif is dispensable for translation and autoregulation: the defects seen with ssyF29 cells reflect an IS10R-mediated destabilization of the rpsA mRNA, probably due to facilitated exonucleolytic degradation.