A cold-sensitive mutation in 16S rRNA provides evidence for helical switching in ribosome assembly.

Abstract

A C-->U substitution at position 23 of 16S rRNA confers a dominant, cold-sensitive phenotype. The mutation changes the G11-C23 base pair of the 5' terminal pseudoknot helix to a G-U pair, which is predicted to cause significant weakening of the helix. Ribosomes containing mutant RNA are impaired in assembly and function at low temperature. Cells expressing the C23U mutation have decreased polysome levels and accumulate free 30S and 50S subunits and particles that resemble those previously observed in cold-sensitive alleles of ribosomal protein S5 and in in vitro reconstitution of 30S subunits carried out at low temperature. Three second-site suppressor mutations suggest that cold sensitivity is caused by competition between the 5' helix and an alternative helix formed by base-pairing of the upstream precursor sequence with one strand of the mature helix. Cold sensitivity appears to be relieved by destabilization of the competing precursor helix relative to the mature helix.