Mutations altering the predicted secondary structure of a chloroplast 5 ' untranslated region affect its physical and biochemical properties as well as its ability to promote translation of reporter mRNAs both in the Chlamydomonas reinhardtii chloroplast and in Escherichia coli

Random mutations were generated in the sequence for the 5' untranslated reg ion (5'UTR) of the Chlamydomonas reinhardtii chloroplast rps7 mRNA by PCR, the coding sequence for the mutant leaders fused upstream of the lacZ' repo rter in pUC18, and transformed into Escherichia coli, and white colonies we re selected. Twelve single base pair changes were found at different positi ons in the rps7 5'UTR in 207 white colonies examined. Seven of the 12 mutan t leaders allowed accumulation of abundant lacZ' message. These mutant rps7 leaders were ligated into an aadA expression cassette and transformed into the chloroplast of C. reinhardtii and into E. coli, In vivo spectinomycin- resistant growth rates and in vitro aminoglycoside adenyltransferase enzyme activity varied considerably between different mutants but were remarkably similar for a given mutant expressed in the Chlamydomonas chloroplast and in E. coli. The variable effect of the mutants an aadA reporter expression and their complete abolition of lacZ' reporter expression in E. coli sugges ts differences in the interaction between the 5'UTR of rps7 and aadA or lac Z' coding regions. Several rps7 5'UTR mutations affected the predicted fold ing pattern of the 5'UTR by weakening the stability of stem structures, Sit e-directed secondary mutations generated to restore these structures in the second stem suppressed the loss of reporter activity caused by the origina l mutations. Additional site-directed mutations that were predicted to furt her strengthen (A-U-->G-C) or weaken (G-C-->A-U) the second stem of the rps 7 leader both resulted in reduced reporter expression This genetic evidence combined with differences between mutant and wild-type UV melting profiles and RNase T1 protection gel shifts further indicate that the predicted wil d-type folding pattern in the 5'UTR is likely to play an essential role in translation initiation.