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.
Highlights
Living organisms have developed a variety of mechanisms to control gene expression in response to environmental and physiological changes
Previous genetic and structural studies of MifM showed that the elongation arrest of MifM requires interactions between the MifM nascent chain and the ribosomal components at multiple sites, from the proximity of the peptidyl transferase center (PTC) to the central constriction of the polypeptide exit tunnel
The lacZ fusion genes were integrated into the chromosomal amyE locus of B. subtilis, and β-galactosidase activities were determined for the transformants
Summary
Living organisms have developed a variety of mechanisms to control gene expression in response to environmental and physiological changes. Previous genetic and structural studies of MifM showed that the elongation arrest of MifM requires interactions between the MifM nascent chain and the ribosomal components at multiple sites, from the proximity of the peptidyl transferase center (PTC) to the central constriction of the polypeptide exit tunnel. Systematic mutagenesis, such as alanine-scanning, of the C-terminal region of MifM allowed us to identify several crucial residues that reside near the PTC-proximal and mid-tunnel regions[2,21]. Glu[88] contacts the PTC-forming nucleotides of the ribosomal RNA, resulting in an interference with the conformational changes of the A-site forming ribosomal RNA residues to accommodate the incoming aminoacyl-tRNA25
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