MifM-instructed translation arrest involves nascent chain interactions with the exterior as well as the interior of the ribosome

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.

Release factor-dependent ribosome rescue by BrfA in the Gram-positive bacterium Bacillus subtilis

“…We used DNA fragments encoding GFP but without an in-frame stop codon (GFP-ns) to direct in vitro transcription and translation with Bs hybrid PURE system and separated the translation products by neutral pH SDS-PAGE, which preserved the peptidyl-tRNA ester bond 31 . The major product, migrating between the 42 and the 55 kDa markers, represents the peptidyl-tRNA (GFP-tRNA; Fig.…”

“…One hundred microliters portions of the bacterial cultures were used for assay of β-galactosidase activity, presented in Fig. 2d as follows and also as described previously 31 . The cultures were transferred to individual wells of another 96-well plate for a Thermo Scientific Multiskan Go microplate spectrophotometer and OD 600 was recorded.…”

“…The E. coli -based coupled transcription–translation system with purified components (PUREfrex 1.0; GeneFrontier) was used for in vitro translation as described previously 29–31 with some modifications. To maximize transcription, we added 2.5 U/μL of T7 RNA polymerase (Takara) further to the reaction mixture.…”

ResQ, a release factor-dependent ribosome rescue factor in the Gram-positive bacterium Bacillus subtilis

“…The E. coli -based coupled transcription-translation system with purified components (PUREfrex 1.0; GeneFrontier) was used for in vitro translation as described previously 29,30,58 with some modifications. To maximize transcription, we added 2.5 U/μL of T7 RNA polymerase (Takara) further to the reaction mixture.…”

“…Samples for SDS-PAGE were heated at 65°C for 5 min and separated by 10% wide range gel (Nacalai Tesque). Translation products were detected by Western blotting using anti-GFP (A-6455; Thermo) or anti-DYKDDDDK (anti-FLAG tag; Wako) as described previously 58 .…”