Identification of regions of brome mosaic virus coat protein chemically cross-linked in situ to viral RNA.
RNA-protein cross-links were introduced into brome mosaic virus in situ by using the heterobifunctional agent p-azidophenylglyoxal. An improved RNA isolation method, without phenol extraction, was used to isolate RNA cross-linked with protein. RNA of the covalently linked complex was acid-digested and the oligonucleotides still attached to protein were 5'-end-labelled with 32P. The complexes were digested with trypsin and the tryptic peptides were purified by reversed-phase high-performance liquid chromatography. Amino acid analyses of cross-linked tryptic peptides revealed that out of the total 188 amino acids of brome mosaic virus coat protein only the 80 N-terminal amino acids are involved in the interaction with viral RNA. These results are discussed in connection with a predicted secondary structure of the coat protein. Both alpha helix (for amino acids 11-19) and other structures (between amino acids 20 and 80) are implicated in the coat protein-viral RNA interactions.
D000596:Amino Acids / Q000032:analysis; D001386:Azides; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D009029:Mosaic Viruses / Q000378:metabolism*; D010658:Phenylglyoxal / Q000031:analogs & derivatives; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*; D014357:Trypsin; D014759:Viral Envelope Proteins / Q000378:metabolism*
D000596:Amino Acids; D001386:Azides; D003432:Cross-Linking Reagents; D012367:RNA, Viral; D014759:Viral Envelope Proteins; C032404:4-azidophenylglyoxal; D014357:Trypsin; D010658:Phenylglyoxal