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

Translational regulation plays a major role in controlling the expression of chloroplast-encoded genes (7, 13, 18, 26, 29). While the translation machinery of the chloroplast shares many functional characteristics with that of its prokaryotic relatives (14, 16), several differences are beginning to emerge with respect to the well-defined Escherichia coli model (2, 11, 27, 38). Interactions between evolutionarily conserved Shine-Dalgarno (SD) sequences (GGAGG) found in the vast majority of genes in E. coli 7 ± 2 nucleotides (nt) upstream of the initiator AUG codon and anti-SD sequences near the 3′ end of the 16S rRNA (CCUCC) are known to be essential for translational initiation in this bacterium. Although the anti-SD sequences in the chloroplast 16S rRNAs are highly conserved, SD-like sequences in the leaders of chloroplast mRNAs vary significantly in location, size, and nucleotide composition or are absent altogether (3, 8, 31). Neither elimination of the variable putative SD sequences in chloroplast leaders by deletion mutagenesis (1, 19, 22, 32) or replacement mutagenesis (8, 21) nor insertion of canonical SD sequences (8) has significant effects on chloroplast gene expression, indicating that translation initiation in the chloroplast occurs largely in an SD-independent manner. Surprisingly, the same chloroplast reporter constructs lacking SD sequences are also expressed efficiently in E. coli and addition of canonical SD sequences to the leaders of these constructs only modestly enhances translation of these mRNAs by the bacterial protein synthesizing system (8). In contrast to the majority of E. coli mRNAs, chloroplast mRNAs contain fairly long, AU-rich 5′ untranslated regions (5′UTRs) with little primary sequence conservation that appear to play a major role in the regulation of translation initiation (10, 13, 18). Analyses of representative chloroplast 5′UTRs, to determine likely secondary structure based on minimum energy models (42), predict that these leaders are highly folded, with several having large stem-loop structures directly upstream of the initiation codon (10, 18). Interactions between these structures and the translational apparatus of the chloroplast presumably allow for the regulation of translation initiation. We have carried out a random PCR mutagenesis on the sequence for the 5′UTR of the chloroplast rps7 gene from Chlamydomonas reinhardtii that specifies a protein of the small subunit of the chloroplast ribosome. The population of rps7 mutant leaders was then fused to the E. coli lacZ′ coding sequence in a pUC18 plasmid, and bacterial transformants that were unable to express β-galactosidase on X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) plates were identified as white colonies. Twelve of the 207 white colony mutants were found to have alterations in the coding sequence for the first 225 nt upstream of the initiation AUG codon of the rps7 5′UTR. Seven rps7 mutants that did not alter levels of lacZ′ mRNA accumulation affected expression of the aminoglycoside adenyltransferase (AAD) reporter protein to various degrees in both the C. reinhardtii chloroplast and in E. coli. The marked difference between the effect of a given mutant rps7 leader on translation of the lacZ′ and aadA reporter mRNAs in E. coli suggests that interactions between the coding sequences and the 5′UTR sequence play a role in the regulation of translation initiation. Three of the seven mutants altered the predicted structure of the second stem-loop upstream of the AUG initiation codon. Complementary nucleotide changes made in these mutants to reconstitute this predicted wild-type stem-loop structure restored spectinomycin-resistant growth and AAD enzyme activity. Additionally, site-directed mutants with base pair changes that strengthened or weakened the second predicted stem-loop of the rps7 5′UTR decreased growth and AAD enzyme activity in the C. reinhardtii chloroplast. These data strongly suggest that the secondary structure of this rps7 leader in vivo involves these predicted stem-loop structures that are essential for normal translational regulation. Changes in the predicted secondary and tertiary structures of the mutant 5′UTRs were also supported by differences in the RNA melting-reannealing profiles and RNase T1 gel shift protection patterns from those of wild-type and suppressed mutant strains. The combined genetic and physical evidence supports the hypothesis that the wild-type rps7 leader sequence resulting in the predicted folding pattern for the second stem-loop is essential for normal function.