Translation initiation of the replication initiator repB gene of 1 promiscuous plasmid pMV 158 is led by intrinsic non - SD 2 sequences 3

34 35 RepB is the pMV158-encoded protein that initiates rolling-circle 36 replication of this promiscuous plasmid. Availability of RepB is rate-limiting for 37 the plasmid replication process, and therefore the repB gene encoding the 38 protein is subjected to strict control. Two trans-acting plasmid elements, CopG 39 and the antisense RNAII, are involved in controlling the synthesis of the initiator 40 at the transcriptional and translational level, respectively. In addition to this dual 41 control of repB expression that senses and corrects fluctuations in plasmid copy 42 number, proper availability of RepB also relies on the adequate functionality of 43 the transcription and translation initiation regulatory signals. Translation of repB 44 has been postulated to depend on an atypical ribosome binding site that 45 precedes its start codon, although such a hypothesis has never been proved. 46 To define sequences involved in translation of repB, several mutations in the 47 translation initiation region of the repB mRNA have been characterized by using 48 an Escherichia coli in vitro expression system wherein the synthesis of RepB 49 was detected and quantified. We showed that translation of repB is not coupled 50 to that of copG and depends only on its own initiation signals. The atypical 51 ribosome binding site, as it was defined, is not involved in translation initiation. 52 However, the sequence just upstream of the repB start codon, encompassing 53 the proximal box of the atypical ribosome binding site and the four bases 54 immediately downstream of it, is indeed important for efficient translation of 55 repB. The high degree of conservation of this sequence among the rep genes of 56 plasmids of the same pMV158 family supports its relevancy as a translation 57 initiation signal in mRNAs without a recognizable Shine-Dalgarno sequence. 58