FIDELITY OF RNA-POLYMERASE-II TRANSCRIPTION CONTROLLED BY ELONGATION-FACTOR TFIIS

Fidelity of DNA and protein synthesis is regulated by a proofreading m echanism but function of a similar mechanism during RNA synthesis has not been demonstrated. Analysis of transcriptional fidelity and its co ntrol has been hampered by the necessity to employ complex DNA templat es requiring either a promoter and initiation factors or 3'-extended t emplates. To circumvent this difficulty, we have created an RNA-DNA du mbbell template that can be recognized as a template-primer and extend ed by RNA polymerase II, By employing this system, we demonstrate that RNA polymerase II can misincorporate a nucleotide and carry out templ ate-dependent elongation at the mispaired end, The transcripts contain ing misincorporated residues can be cleaved by the very slow 3' --> 5' ribonuclease activity of the RNA polymerase II, but enhancement of th is activity by the elongation factor TFIIS generates RNA with a high d egree of fidelity. This enhanced preferential cleavage of misincorpora ted transcripts suggests an important role for TFIIS in maintaining tr anscriptional fidelity.