ALANINE-SCANNING MUTAGENESIS OF HUMAN TRANSCRIPT ELONGATION-FACTOR TFIIS

TFIIS is a transcription elongation factor that binds to RNA polymeras e II and allows it to transcribe through a variety of transcriptional blockages by inducing cleavage near the 3' end of the nascent transcri pt. Although this cleavage reaction plays a key role in the process of reactivation of transcription by TFIIS, the exact mechanism by which TFIIS promotes readthrough by RNA polymerase IT is not completely unde rstood. We therefore undertook a systematic mutagenesis of the C-termi nal half of TFIIS (Delta TFIIS) to evaluate the contribution of charge d residues in this region to induce transcript cleavage and promote re adthrough in vitro. Twenty-two Delta TFIIS alanine-scanning mutants we re constructed by substitution of alanine for each amino acid in clust ers of charged residues in the C-terminal half of HeLa TFIIS. The abil ity to induce transcript cleavage and readthrough of these mutants was tested in vitro using RNA polymerase II ternary elongation complexes arrested at a block to elongation. This alanine-scanning mutagenesis a nalysis allowed the identification of regions or residues important fo r the activity of TFIIS. Many of the mutants were reduced alike in bot h cleavage and readthrough activities. However, in several cases there was no simple correlation between these activity reductions.