Mutations that alter the higher-order structure of its 5 ' untranslated region affect the stability of chloroplast rps7 mRNA

In this paper, we examine the effects of mutations in the 5'UTR of the chlo roplast rps7 transcript of Chlamydomonas reinhardtii that reduce the stabil ity of the mRNA. Five point mutants in the rps7 5'UTR were selected on the basis of their failure to accumulate reporter mRNA in Escherichia coli. Eac h of these mutations produces alterations in the predicted higher-order str uctures of the rps7 5'UTR that destabilize the mRNA. Cis-acting suppressors of these mutations have been selected in E. coli and in the C. reinhardtii chloroplast that restore message stability and function. No differences in RNA melting and reannealing profiles have been observed between wild type, original mutant, and suppressor 5'UTRs transcribed in vitro. Proteins of 3 2 kDa and 47 kDa that bind to the wild-type rps7 5'UTR are not detected by UV cross-linking assays performed with any of the mutant rps7 5'UTRs. Howev er, binding of the 32-kDa protein is restored in the six suppressor mutants examined. This suggests that the 32-kDa protein may be involved in protect ing the rps7 5'UTR and the attached coding region from digestion by ribonuc leases. Alternatively, the binding site for the 32-kDa protein may be indep endently lost in the rearranged tertiary structure of the mutant 5'UTR that exposes the RNA to degradation and is restored in the suppressor mutants.