title: Translation initiation of the replication initiator repB gene of promiscuous plasmid pMV158 is led by an extended non-SD sequence.
abstract: RepB is the pMV158-encoded protein that initiates rolling-circle replication of this promiscuous plasmid. Availability of RepB is rate-limiting for the plasmid replication process, and therefore the repB gene encoding the protein is subjected to strict control. Two trans-acting plasmid elements, CopG and the antisense RNAII, are involved in controlling the synthesis of the initiator at the transcriptional and translational level, respectively. In addition to this dual control of repB expression that senses and corrects fluctuations in plasmid copy number, proper availability of RepB also relies on the adequate functionality of the transcription and translation initiation regulatory signals. Translation of repB has been postulated to depend on an atypical ribosome binding site that precedes its start codon, although such a hypothesis has never been proved. To define sequences involved in translation of repB, several mutations in the translation initiation region of the repB mRNA have been characterized by using an Escherichia coli in vitro expression system wherein the synthesis of RepB was detected and quantified. We showed that translation of repB is not coupled to that of copG and depends only on its own initiation signals. The atypical ribosome binding site, as it was defined, is not involved in translation initiation. However, the sequence just upstream of the repB start codon, encompassing the proximal box of the atypical ribosome binding site and the four bases immediately downstream of it, is indeed important for efficient translation of repB. The high degree of conservation of this sequence among the rep genes of plasmids of the same pMV158 family supports its relevancy as a translation initiation signal in mRNAs without a recognizable Shine-Dalgarno sequence.
authors: Molecular Microbiology and Infection Biology Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E28040 Madrid, Spain.
authors: Celeste
authors: C
authors: López-Aguilar
authors: José A
authors: JA
authors: Ruiz-Masó
authors: Tania Samir
authors: TS
authors: Rubio-Lepe
authors: Marta
authors: M
authors: Sanz
authors: Gloria
authors: G
authors: del Solar
mesh_terms: D016375:Antisense Elements (Genetics) / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D054680:Ribosome Subunits, Small, Bacterial / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014158:Transcription, Genetic
chemical_list: D016375:Antisense Elements (Genetics); D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; C074125:RepB' protein, E coli; D012319:RNA Polymerase II