Nucleic Acids Research, 1991, Vol. 19, No. 18 4849-4855
© 1991
MOLECULAR BIOLOGY |
Photoaffinity labelling of the pea chloroplast transcriptional complex by nascent RNA in vitro
International Centre for Genetic Engineering and Biotechnology PO NII Campus, Shaheedjit Singh Marg, New Delhi 110 067, India 1Department of Molecular Biology and Biochemistry, University of California Irvine, CA 92717, USA
Received July 12, 1991. Accepted August 27, 1991.
We have used photoaffinity labelling to examine the chloroplast RNA polymerase components which come into contact with nascent transcripts during the in vitro transcription of plastld DNA. The transcripts were synthesized in the presence of a photoactive analogue (4-thio UTP) and 
-32P-ATP, using enriched pea chloroplast RNA polymerase preparation and a recombinant plasmid containing the plastid 16S rRNA promoter. Brief Irradiation of the transcriptional complex crosslinked the photoactive nascent RNA to proximal proteins. Labelling of the transcriptional complex was dependent on 4-thlo UTP and template DNA. Two polypeptides of 51 and 54 kDa were consistently crosslinked to the nascent transcripts; about 60% of the total radioactivity of the crosslinked RNA was associated with these polypeptides. In some experiments, two additional polypeptides of 38 and 75 kDa were also found to be associated with about 13% and 17% of the total crosslinked RNA radioactivity, respectively. The UV-crosslinked transcriptional complexes were stable to either DNase or S1 nuclease hydrolysis but partially sensitive to RNase T1. Insensltivity of the complex to hydrolysis with RNase H suggested that the nascent transcripts were not crosslinked to the template. The complexes could also be hydrolysed by proteinase K and thermolysin. No crosslinkage was observed when labelled RNA molecules containing 4-thio UMP residues were added after synthesis to the polymerase preparation. This suggested that the method Identified only those polypeptides which came into close contact with the transcript during its synthesis. Antibodies raised against the RNA-proteln complex confirmed the presence of the polypeptides in the chloroplast RNA polymerase preparation on Western blots. Preincubatlon of these antibodies with the chloroplast RNA polymerase inhibited plastid DNA transcription. These data showed that the transcript-binding polypeptides were functional components of the chloroplast transcriptional complex.