Cleavage of the nascent transcript induced by TFIIS is insufficient to promote read-through of intrinsic blocks to elongation by RNA polymerase II.

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RESUMO

RNA polymerases encounter a variety of types of blocks to elongation during transcription in eukaryotic cells. At least one protein, TFIIS, can promote read-through of many types of blocks to elongation by RNA polymerase II, and this protein stimulates cleavage of the nascent transcript in stalled elongation complexes as a prelude to read-through. The C-terminal half of the TFIIS protein is sufficient for stimulating the cleavage and read-through reactions in vitro. To study how TFIIS changes the response of RNA polymerase II elongation complexes to such blocks, targeted amino acids in the C terminus of HeLa TFIIS were mutated to alanines. Two mutant TFIIS proteins as well as the unmutated C-terminal half of the TFIIS protein were purified following overexpression in Escherichia coli. Each protein was examined for read-through activity and ability to stimulate transcript cleavage in ternary elongation complexes. Mutant TFIIS5 (E174A, E175A) was reduced in read-through and cleavage activities relative to the unmutated, truncated TFIIS (delta TFIIS). Mutant TFIIS7 (K187A, K189A) was able to stimulate cleavage nearly at the rate and to the extent of the TFIIS5 mutant. In contrast to what was observed with TFIIS5, no detectable read-through was observed in the presence of the TFIIS7 mutant during the course of the reaction. Thus, there is no simple, direct correlation between the ability of TFIIS to promote cleavage and its ability to promote read-through by RNA polymerase II. These results suggest that although TFIIS is necessary to mediate the cleavage reaction that precedes the read-through event, the cleavage event itself is not sufficient to allow read-through by RNA polymerase II.

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