MOLECULAR ANALYSIS OF RNAI CONTROL OF REPB TRANSLATION IN INCB PLASMIDS

The translation of RepA, the replication initiation protein of the Inc B plasmid pMU720, requires that its mRNA (RNAII) folds to form a pseud oknot immediately upstream of the repA Shine-Dalgarno sequence. The fo rmation of this pseudoknot is dependent in turn on the translation and correct termination of a leader peptide, RepB. A small countertranscr ipt RNA, RNAI, controls the replication of pMU720 by interacting with RNAII to negatively regulate the expression of repA both directly, by sequestering the proximal bases required for pseudoknot formation, and indirectly, by inhibiting the translation of repB. Inhibition of the translation of repB by RNAI was found to depend on the close proximity of the RNAI-RNAII complex to the translational initiation region of r epB, indicating that the primary mechanism of RNAI control involves st eric hindrance. Disruption of;RNAI control of repB had only a small ef fect on the copy number of the IncB plasmid, indicating that inhibitio n of the expression of repA by RNAI is achieved predominantly by inhib ition of pseudoknot formation rather than by inhibition of repB transl ation.