Function and Assembly of a Chromatin-Associated RNase P that Is Required for Efficient Transcription by RNA Polymerase I
Figure 5
Pol I components copurify and associate with human RNase P.
A. A DEAE-purified RNase P was loaded (L, load) into a Sephacryl S-100 gel filtration column and eluted fractions were assayed for RNase P activity in processing of 32P-labeled precursor tRNATyr (S) to mature tRNA (3′) and 5′ leader sequence (5′). Substrate cleavage was analyzed as in Figure 4C with control HeLa RNase P (Ctrl). B. Western blot analysis of Sephacryl S-100 eluted fractions using antibodies against RPA194, RPA43, TAFI110, UBF, Rpp29 and Rpp25. Preparations of DEAE-purified RNase P (first lane) was used as control for input. Proteins were separated in 12% SDS/PAGE and the positions of the corresponding proteins and the protein size markers are shown. C. Fraction F28 described in A was subjected to immunoprecipitation analysis using antibodies against Rpp29 (lane 3), RPA43 (lane 4), UBF (lane 5), TAFI110 (lane 6), RPB8 (lane 7) and IgG (lane 8) in the presence of 150 mM KCl (IP150). For Rpp29 and RPA43 immunoprecipitation was also performed at 300 mM KCl (lanes 9 and 10). Immunoprecipitates obtained were tested for RNase P activity in processing of a 32P-labeled precursor tRNATyr (S). RNase P in fraction F28 was tested as control for enzyme activity (lane 2) and cleavage products were analyzed as in A.