2C-07 Intiation of translation of replication initiator protein (Rep) in ColE2 plasmid

Expression of the ColE2 Rep protein, required for intiation of plasmid DNA replication, is regulated by a small antisense RNA at a post-transcriptional level. Translation intiation of the ColE2 Rep is efficient in E coli despite the absence of a canonical Shine-Dalgarno (SD) sequence. There is GA cluster sequence immediatry upstream of initiation codon, but its function as the SD sequence is very week, if any. To define the sequence and structural elements responsible for efficient translation initiation of the Rep mRNA, a series of rep-lacZ translational fusions bearing various mutations in the plasmid-derived region was constructed and tested for beta-galactosidase activity. It was suggested that in addition to the GA culaster and initiation codon, one region containing a stem-loop structure upstream of the initiation codon and the two regions inside the Rep ORF are important for the efficient Rep translation. These regions migth be involved in interaction with the ribosome andlor in possible formation of a certain secondary or teritiay structure required for efficient initiation of translation.