Ribosome-binding sites on chloroplast rbcL and psbA mRNAs and light-induced initiation of D1 translation.

Abstract

Chloroplast ribosome-binding sites were identified on the plastid rbcL and psbA mRNAs using toeprint analysis. The rbcL translation initiation domain is highly conserved and contains a prokaryotic Shine-Dalgarno (SD) sequence (GGAGG) located 4 to 12 nucleotides upstream of the initiator AUG. Toeprint analysis of rbcL mRNA associated with plastid polysomes revealed strong toeprint signals 15 nucleotides downstream from the AUG indicating ribosome binding at the translation initiation site. Escherichia coli 30S ribosomes generated similar toeprint signals when mixed with rbcL mRNA in the presence of initiator tRNA. These results indicate that plastid SD sequences are functional in chloroplast translation initiation. The psbA initiator region lacks a SD sequence within 12 nucleotides of the initiator AUG. However, toeprint analysis of soluble and membrane polysome-associated psbA mRNA revealed ribosomes bound to the initiator region. E. coli 30S ribosomes did not associate with the psbA translation initiation region. E. coli and chloroplast ribosomes bind to an upstream region which contains a conserved SD-like sequence. Therefore, translation initiation on psbA mRNA may involve the transient binding of chloroplast ribosomes to this upstream SD-like sequence followed by scanning to localize the initiator AUG. Illumination 8-day-old dark-grown barley seedlings caused an increase in polysome-associated psbA mRNA and the abundance of initiation complexes bound to psbA mRNA. These results demonstrate that light modulates D1 translation initiation in plastids of older dark-grown barley seedlings.