Multisite Ribosomal Stalling: A Unique Mode of Regulatory Nascent Chain Action Revealed for MifM

Bacillus subtilis MifM uses polypeptide-instructed ribosomal stalling to control translation of YidC2, a membrane protein biogenesis factor. In contrast to other stalling systems involving a single arrest point, our in vitro translation/toeprint experiments show that the B. subtilis ribosome stalls consecutively at multiple codons of MifM. This mode of elongation arrest depends on nascent chain residues at the middle of the ribosomal exit tunnel and a few (four for the maximum functionality) negative charges residing proximally to the arrest points. The latter element does not require exact amino acid sequence, and this feature may underlie the multisite stalling. The arrested nascent chains were not efficiently transferred to puromycin, suggesting that growing MifM nascent chains inhibit peptidyl transferase center after acquiring an acidic residue(s). Multisite stalling seems to provide a unique means for MifM to achieve a sufficient duration of ribosomal stalling required for the regulatory function.