Summary: Most prokaryotic mRNAs contain within the 5′ untranslated region (UTR), a Shine‐Dalgarno (SD) sequence, which is complementary to the 3′ end of 16S rRNA and serves as a major determinant for correct translational initiation. The tobacco chloroplast rps2 mRNA possesses an SD‐like sequence (GGAG) at a proper position (positions −8 to −5 from the start codon). Using an in vitro translation system from isolated tobacco chloroplasts, the role of this sequence in translation was examined. Unexpectedly, the mutation of the SD‐like element resulted in a large increase in translation. Internal and external deletions within the 5′ UTR revealed that the region from −20 to −5 was involved in the negative regulation of translation. Scanning mutagenesis assays confirmed the above result. Competition assays suggested the existence of a trans‐acting factor(s) involved in translational regulation. In this study, we discuss a possible mechanism for the negative regulation of rps2 mRNA translation.

Summary: Genes encoding chlorophyll a/b‐binding proteins of photosystem II (Lhcb) constitute a multigene family. Nine Lhcb1 genes have previously been isolated from the tobacco species, Nicotiana sylvestris, and the transcription initiation sites in vivo have been mapped. Reaction conditions from a previously developed in vitro transcription system from tobacco cultured cells were optimized for the Lhcb1 genes. Transcription initiation sites in vitro predominantly coincided with those found in vivo and were typically cytidines, a system unique to N. sylvestris Lhcb1 genes. CTC^*A (C^* for initiation site) was a consensus motif for the initiation region in vitro, as reported in vivo. Mutation analysis defined functionally that the TATA box is essential for transcription initiation and that the CTCA motif is a determinant of transcription initiation sites but not transcript levels. Polyadenylation sites were determined from in vivo transcripts, located 12–21 nt downstream from likely poly(A) signals. Four major 3′‐ends of in vitro transcripts from Lhcb1^*6 were detected, 40–300 nt downstream of the poly(A) site, suggestive of multiple, discrete transcription termination sites. These 3′‐ends are mapped in or nearby T‐rich sequences.