Identification of the template binding polypeptide in the pea chloroplast transcriptional complex.
We have identified the template-binding polypeptide in the pea chloroplast transcriptional complex by photoaffinity labelling. This polypeptide has an apparent molecular weight of about 150 kDa and binds to both, chloroplast ribosomal (16S rRNA) and messenger (psbA) promoters. The 16S rRNA and psbA promoters were amplified from chloroplast DNA by the polymerase chain reaction and labelled with a photoactive analogue of TTP, 5-bromodeoxy UTP, as well as with alpha-32P-dCTP. Using the filter-binding assay, the conditions for binding of the RNA polymerase complex to chloroplast promoters were optimized. The polypeptide directly interacting with the template was photo-crosslinked to it and resolved by denaturing gel electrophoresis. The photoaffinity labelling of the 150 kDa polypeptide was dependent on photoactivation by UV irradiation, and the presence of chloroplast promoters. Competition experiments showed that the protein formed a strong interaction with the plastid promoters which could not be displaced by lambda-phage DNA or synthetic polynucleotides. The photo-crosslinked and nuclease-treated promoter-polypeptide complex was resistant to further digestion with DNase and RNase, but could be hydrolyzed by Proteinase K. Binding of the promoters by the 150 kDa polypeptide could not be surpressed by transcription inhibitors like rifampicin and alpha-amanitin. However, heparin (0.001%) inhibited the formation of the enzyme-promoter complex, and interfered with the photoaffinity labelling of the 150 kDa polypeptide. The extent of photoaffinity labelling of 150 kDa polypeptide exhibits some degree of correlation to total transcriptional activity under various salt concentrations. The results demonstrate that the 150 kDa polypeptide is a functional template binding polypeptide of the pea chloroplast transcription complex.
Khanna
N C
NC
International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Lakhani
S
S
Tewari
K K
KK
D001483:Base Sequence; D002736:Chloroplasts / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004586:Electrophoresis; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism; D010944:Plants / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D004268:DNA-Binding Proteins; D010940:Plant Proteins; D012336:RNA, Ribosomal, 16S; D004247:DNA; D012321:DNA-Directed RNA Polymerases