SHINE-DALGARNO-LIKE SEQUENCES ARE NOT REQUIRED FOR TRANSLATION OF CHLOROPLAST MESSENGER-RNAS IN CHLAMYDOMONAS-REINHARDTII CHLOROPLASTS OR IN ESCHERICHIA-COLI

Initiation of translation in Escherichia coli and related eubacteria i nvolves well-defined interactions between a conserved Shine-Dalgarno ( SD) sequence immediately upstream of the initiation codon in the mRNA leader and an equally conserved anti-SD sequence at the 3' end of the 16S rRNA. SD-like sequences found in the leaders of many, but not all, mRNAs from cyanobacteria and chloroplasts are hypervariable in locati on, size, and base composition compared to those in E. coli, while ant i-SD sequences in the respective 16S rRNAs remain highly conserved. We have examined the function of the SD-like sequences found in the lead ers of four chloroplast genes of the green alga Chlamydomonas reinhard tii using replacement mutagenesis to eliminate complementarity with th e anti-SD sequences and insertion of canonical SD sequences (CGAGG) at positions -9 to -5 relative to the initiation codon. Promoter-leader regions of the atpB, atpE, rps4, and rps7 genes representing the diver sity of chloroplast SD-like sequences were fused to aadA and uidA repo rter genes encoding spectinomycin resistance and GUS activity respecti vely. Analysis of chloroplast transformants of C. reinhardtii and tran sformants of E. coli carrying the wild-type and mutant reporter constr ucts revealed that mutagenic replacement of the putative SD sequences had no effect on the expression of either the aadA or uidA reporter ge nes. Chloroplast transformants with the canonical SD sequence also sho wed no differences in reporter gene expression, whereas expression of the reporter genes was increased by 10 to 30% in the E. coli transform ants. Collectively our results suggest that even though SD-dependent i nitiation predominates in E. coli, this bacterium also has the capacit y to initiate translation by an SD-independent mechanism. In contrast, plant chloroplasts, and very probably their cyanobacterial ancestors, appear to have adopted the SD-independent mechanism for translational initiation of most mRNAs.