Use of circular permutation and end modification to position photoaffinity probes for analysis of RNA structure.
Photocrosslinking allows first-order structural analysis with relatively small amounts of biological material and can be applied in complex in vitro systems. In this article we describe methods for positioning both arylazide and thionucleotide photoagents within an RNA of interest by end modification of circularly permuted RNAs. Application of this technique provided a library of constraints that, together with biochemical and phylogenetic comparative data, were used to develop a structure model of the bacterial ribonuclease P ribozyme-substrate complex. Circularly permuted genes for in vitro transcription are generated by PCR from tandem genes. Circularly permuted RNA transcripts can be modified with high efficiency at both the 5' and 3' termini with arylazide crosslinking reagents, or transcription can be primed with photoactive nucleotide analog monophosphates such as 6-thioguanosine. These crosslinking agents can be used over a wide range of experimental conditions but remain inert until they are activated by UV light. Crosslinked sites are subsequently mapped by reverse transcriptase primer extension of gel-purified crosslinked species. In addition to providing basic protocols for these methods, we discuss approaches for establishing the relevance of crosslinking data to native RNA structure.
Harris
M E
ME
Center for RNA Molecular Biology, Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA. meh2@pop.cwru.edu
Christian
E L
EL
D000628:Aminophylline / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D004155:Diphenhydramine / Q000378:metabolism; D004338:Drug Combinations; D004722:Endoribonucleases / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000378:metabolism; D008694:Methamphetamine / Q000031:analogs & derivatives / Q000378:metabolism; D008956:Models, Chemical; D008957:Models, Genetic; D008958:Models, Molecular; D008967:Molecular Biology / Q000379:methods*; D009690:Nucleic Acid Conformation; D019920:Photoaffinity Labels / Q000032:analysis*; D012313:RNA / Q000032:analysis* / Q000737:chemistry; D016337:RNA, Catalytic / Q000378:metabolism; D015203:Reproducibility of Results; D043262:Ribonuclease P; D013873:Thionucleotides / Q000378:metabolism
D003432:Cross-Linking Reagents; D004338:Drug Combinations; D019920:Photoaffinity Labels; D016337:RNA, Catalytic; D013873:Thionucleotides; D000628:Aminophylline; D008694:Methamphetamine; D012313:RNA; C066524:guanosine 5'-monophosphorothioate; D006157:Guanosine Monophosphate; D004155:Diphenhydramine; D004722:Endoribonucleases; D043262:Ribonuclease P