A single alteration 20 nt 5 ' to an editing target inhibits chloroplast RNA editing in vivo

Transcripts of typical dicot plant plastid genes undergo C-->U RNA editing at approximately 30 locations, but there is no consensus sequence surroundi ng the C targets of editing. The cis-acting elements required for editing o f the C located at tobacco rpoB editing site II were investigated by introd ucing translatable chimeric minigenes containing sequence -20 to +6 surroun ding the C target of editing. When the -20 to +6 sequence specified by the homologous region present in the black pine chloroplast genome was incorpor ated, virtually no editing of the transcripts occurred in transgenic tobacc o plastids, Nucleotides that differ between the black pine and tobacco sequ ence were tested for their role in C-->U editing by designing chimeric gene s containing one or more of these divergent nucleotides, Surprisingly, the divergent nucleotide that had the strongest negative effect on editing of t he minigene transcript was located -20 nt 5' to the C target of editing. Ex pression of transgene transcripts carrying the 27 nt sequence did not affec t the editing extent of the endogenous rpoB transcripts, even though the ch imeric transcripts were much more abundant than those of the endogenous gen e. In plants carrying a 93 nt rpoB editing site sequence, transgene transcr ipts accumulated to a level three times greater than transgene transcripts in the plants carrying the 27 nt rpoB editing sites and resulted in editing of the endogenous transcripts from 100 to 50%. Both a lower affinity of th e 27 nt site for a trans-acting factor and lower abundance of the transcrip t could explain why expression of minigene transcripts containing the 27 nt sequence did not affect endogenous editing.