Identification of cis-acting RNA leader elements required for chloroplast psbD gene expression in chlamydomonas

The psbD mRNA of Chlamydomonas reinhardtii is one of the most abundant chlo roplast transcripts and encodes the photosystem II reaction center polypept ide D2. This RNA exists in two forms with 5' untranslated regions of 74 and 47 nucleotides. The shorter form, which is associated with polysomes, is l ikely to result from processing of the larger RNA. Using site-directed muta genesis and biolistic transformation, we have identified two major RNA stab ility determinants within the first 12 nucleotides at the 5' end and near p osition -30 relative to the AUG initiation codon of psbD. Insertion of a po lyguanosine tract at position -60 did not appreciably interfere with transl ation of psbD mRNA. The same poly(G) insertion in the nac2-26 mutant, which is known to be deficient in psbD mRNA accumulation, stabilized the psbD RN A, However, the shorter psbD RNA did not accumulate, and the other psbD RNA s were not translated. Two other elements were found to affect translation but not RNA stability. The first comprises a highly U-rich sequence (positi ons -20 to -15), and the second, called PRB1 (positions -14 to -11), is com plementary to the 3' end of the 16S rRNA, Changing the PRB1 sequence from G GAG to AAAG had no detectable effect on psbD mRNA translation. However, cha nging this sequence to CCUC led to a fourfold diminished rate of D2 synthes is and accumulation, When the psbD initiation codon was changed to AUA or A UU, D2 synthesis was no longer detected, and psbD RNA accumulated to wild-t ype levels, The singular organization of the psbD 5' untranslated region co uld play an important role in the control of initiation of psbD mRNA transl ation.