Alanine-scanning mutagenesis of human transcript elongation factor TFIIS.
TFIIS is a transcription elongation factor that binds to RNA polymerase II and allows it to transcribe through a variety of transcriptional blockages by inducing cleavage near the 3' end of the nascent transcript. 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 II is not completely understood. We therefore undertook a systematic mutagenesis of the C-terminal half of TFIIS (delta TFIIS) to evaluate the contribution of charged residues in this region to induce transcript cleavage and promote readthrough in vitro. Twenty-two delta TFIIS alanine-scanning mutants were constructed by substitution of alanine for each amino acid in clusters of charged residues in the C-terminal half of HeLa TFIIS. The ability 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 analysis allowed the identification of regions or residues important for the activity of TFIIS. Many of the mutants were reduced alike in both cleavage and readthrough activities. However, in several cases there was no simple correlation between these activity reductions.
Cipres-Palacin
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Department of Molecular and Cell Biology, University of California, Berkeley 94720-3202, USA.
Kane
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CM
D000409:Alanine; D000595:Amino Acid Sequence; D004563:Electrochemistry; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D012319:RNA Polymerase II / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors
D010446:Peptide Fragments; D012333:RNA, Messenger; D014157:Transcription Factors; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; D012319:RNA Polymerase II; D000409:Alanine