Footprinting RNA-protein complexes following gel retardation assays: application to the R-17-procoat-RNA and tat--TAR interactions.
RNA-protein complexes isolated following a gel retardation assay can be footprinted within the gel matrix using the chemical nuclease activities of 4,7-dimethyl-, 5,6-dimethyl-, and 3,4,7,8-tetramethyl-1,10-phenanthroline-copper. These complexes are more reactive than 1,10-phenanthroline-copper but share its reaction preference for bulges and loops. The interaction of the coat protein of R-17 with its viral RNA target and tat- and tat-derived peptides with HIV TAR RNA have been studied. In both cases, the RNA sequence opposite a 2-3 nucleotide bulge are protected. Tat-derived peptides inhibit cleavage at sites which intact tat does not protect. These results are consistent with transcription studies which have suggested that truncation of tat increases nonspecific binding.
Pearson
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Department of Biological Chemistry, School of Medicine, University of California, Los Angeles 90024-1570.
Chen
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Gaynor
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Sigman
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins; D002460:Cell Line; D002628:Chemoreceptor Cells; D003300:Copper / Q000378:metabolism; D004247:DNA; D029968:Escherichia coli Proteins; D015696:Gene Products, tat / Q000378:metabolism*; D005821:Genetic Techniques; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010618:Phenanthrolines; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011956:Receptors, Cell Surface; D012260:Ribonucleases / Q000378:metabolism
D001426:Bacterial Proteins; D036022:Capsid Proteins; D029968:Escherichia coli Proteins; D015696:Gene Products, tat; D008565:Membrane Proteins; D010618:Phenanthrolines; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011956:Receptors, Cell Surface; C057331:Tar protein, E coli; D003300:Copper; D004247:DNA; D012260:Ribonucleases; C025205:1,10-phenanthroline