Abstract
Bacillus subtilis MifM is a monitoring substrate of the YidC pathways of protein integration into the membrane and controls the expression of the YidC2 (YqjG) homolog by undergoing regulated translational elongation arrest. The elongation arrest requires interactions between the MifM nascent polypeptide and the ribosomal components near the peptidyl transferase center (PTC) as well as at the constriction site of the ribosomal exit tunnel. Here, we addressed the roles played by more N-terminal regions of MifM and found that, in addition to the previously-identified arrest-provoking elements, the MifM residues 41-60 likely located at the tunnel exit and outside the ribosome contribute to the full induction of elongation arrest. Mutational effects of the cytosolically exposed part of the ribosomal protein uL23 suggested its involvement in the elongation arrest, presumably by interacting with the extra-ribosomal portion of MifM. In vitro translation with reconstituted translation components recapitulated the effects of the mutations at the 41-60 segment, reinforcing the importance of direct molecular interactions between the nascent chain and the ribosome. These results indicate that the nascent MifM polypeptide interacts extensively with the ribosome both from within and without to direct the elongation halt and consequent up-regulation of YidC2.