Combination of in vitro capping and ribonuclease protection improves the detection of transcription start sites in chloroplasts.

A primary transcript from the chloroplast rpl32 gene was labelled at its 5' end using a capping enzyme and [alpha-32P]GTP followed by hybridization to a cold RNA probe. A RNase protection assay gave a clear protected band and its initiation site of transcription could thus be estimated, which had not been possible by using DNA probes. The combination of in vitro capping and RNase protection is an excellent method for mapping transcription initiation sites on the chloroplast genome and shows a high improvement relative to the DNA-employing strategies.