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.
Department of Biological Chemistry, School of Medicine, University of California, Los Angeles 90024-1570.
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Pearson
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Chen
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Gaynor
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Sigman
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