FIG. 5.

UV cross-linking and EMSA experiments to compare protein binding to the HPV-16 NRE and HPV-31 NLE. (A) UV cross-linking of ³²P-labeled NRE and NLE probes to HeLa cell nuclear extracts. Lane 1, full-length (HPV-16) NRE; lane 2, full-length (HPV-31) NLE; lane 3, 5′ NRE (49 nt); lane 4, 5′ NLE (46 nt); lane 5, 3′ NRE (30 nt); lane 6, 3′ NLE (56 nt). (B) EMSA using ³²P-labeled RNA probes and HeLa cell nuclear extracts very similar to those used for panel A run on a nondenaturing polyacrylamide gel. RNA-protein complexes and free probes are bracketed. Arrows, RNA-protein complexes. NE, HeLa cell nuclear extracts. (C) EMSA competition assay using a ³²P-labeled NLE probe (1.5 pmol) and HeLa nuclear extracts. Lane 1, no extracts; lane 2, no competitor RNA; lanes 3 to 7, 1- to 16-fold molar excess of specific competitor, i.e., 1.5 to 24 pmol of in vitro-transcribed, unlabeled NLE RNA; lane 8, no competitor RNA; lanes 9 to 13, 1- to 16-fold molar excess of nonspecific competitor, i.e., 1.5 to 24 pmol of in vitro-transcribed pBluescript KS(+) polylinker RNA; lane 14, no competitor RNA; lanes 15 to 18, nonspecific competitor, i.e., 500 ng to 4 μg of E. coli tRNA. (D) EMSA competition assay using ³²P-labeled NLE and NRE probes and HeLa nuclear extracts. Lanes 1 to 7, NLE probe (1.5 pmol); lane 1, no extracts; lane 2, no competitor; lanes 3 to 7, 1- to 16-fold molar excess, i.e., 1.5 to 24 pmol of in vitro-transcribed, unlabeled NRE RNA; lanes 8 to 14, NRE probe (1.5 pmol); lane 8, no extracts; lane 9, no competitor; lanes 10 to 14, 1- to 16-fold molar excess, i.e., 1.5 to 24 pmol of in vitro-transcribed, unlabeled NLE RNA.