Figure 6.

The role of the C-terminus of LARP7 and 7SK RNA in inhibition of MEPCE. Unless indicated, 6% TBE urea gels were used. (A) Structural domains of LARP7 and mutants indicating the two RNA binding domains of LARP7 and a predicted helical C-terminal region. (B) Stoichiometric EMSA of full length and truncated LARP7 with MEPCE on 7SK folded as indicated analyzed on a 4% native gel. Amounts of proteins and RNA are in pmol. (C) Methyltransferase assay using 5 pmol MEPCE to methylate recombinant 7SK with ¹⁴C-S-adenosylmethionine and the indicated molar equivalents of LARP7 or LARP7 mutants. (D and E) Methyltransferase assays with the indicated wild type or mutant 7SK RNAs and molar equivalents of LARP7. Gel was analyzed by ethidium bromide staining (EtBr) and phosphorimaging of ¹⁴C-labeled 7SK (7SK). 7SK RNAs are represented as cartoons with the mutated regions denoted by red arrows. (F) SHAPE analysis of the indicated mutant 7SK RNAs folded under indicated Mg conditions. GAUC motifs are shown as numbered green boxes and sites of mutations as ***.