Plastid run-on transcription. Application to determine the transcriptional regulation of spinach plastid genes

Abstract

We have developed a spinach plastid run-on transcription system to determine the extent of transcriptional regulation of chloroplast genes during morphogenetic changes of the organelle. In contrast to transcription in a spinach chloroplast extract, which requires initiation of exogenously added genes (Gruissem, W., Greenberg, B. M., Zurawski, G., and Hallick, R. B. (1983) Cell 35, 815-828), RNA synthesis in the run-on system is not affected by heparin or different salt concentrations. Transcription is asymmetric, and the size of the run-on transcripts varies between 75 nucleotides and 8 kilobases. Quantitative filter hybridization studies included gene-specific probes for the ribosomal RNA genes and nine protein-coding genes. Based on the amounts of hybridizable run-on transcripts, these genes can be ordered according to their respective transcriptional activities. The relative transcriptional activities of psbA, rbcL, and atpB in the run-on assay correlate closely with their reported promoter strengths in vitro. The plastid run-on transcription assay has been applied to determine the transcriptional regulation of plastid genes. Hybridization of run-on transcripts to regions of the spinach chloroplast genome containing at least nine tRNA genes indicates that most or all loci are highly transcribed. No significant qualitative and quantitative differences are detected when run-on transcripts from plastids of etiolated and greening cotyledons are hybridized to total, restriction enzyme-digested chloroplast DNA, demonstrating limited transcriptional regulation during chloroplast development.