Identification of a sequence motif critical for editing of a tobacco chloroplast transcript

Nucleotides are specifically and efficiently targeted for modification from C to U within transcripts of chloroplasts in higher plants. Although the enzymatic apparatus responsible for altering C to U has not been identified, the sequences surrounding editing sites are known to contain information essential for efficient editing. We set out to determine the nucleotides that are critical for editing of a particular C, NTpsbE C214, in chloroplast transcripts in tobacco. Assay of editing of substrates with different lengths of 5′ and 3′ sequence around the target C was carried out to delimit the region of sequence critical for editing in vitro. Mutated substrates were then constructed with an altered nucleotide at each position within the previously defined region around NTpsbE C214. In individual nucleotides, both 5′ and 3′ of the edited nucleotide were found to be important for editing. The sequence GCCGUU, which occurs 5′ of the editing site, was discovered to be critical for editing. Editing substrates mutated to alter the distance between the GCCGUU sequence and NTpsbE C214 resulted in the generation of a new editing target, the 3′ adjacent nucleotide. These data are consistent with a model in which the selection of the C target for editing is determined by its distance from a crucial 5′ sequence.