Genome-wide analysis of TFIIB’s role in termination of transcription

Abstract

AbstractApart from its well-established role in initiation of transcription, the general transcription factor TFIIB has been implicated in the termination step as well. The ubiquity of TFIIB involvement in termination as well as mechanistic details of its termination function, however, remains largely unexplored. To determine the prevalence of TFIIB’s role in termination, we performed GRO-seq analyses insua7-1mutant (TFIIBsua7-1) and the isogenic wild type (TFIIBWT) strains of yeast. Almost a three-fold increase in readthrough of the poly(A)-termination signal was observed in TFIIBsua7-1mutant compared to the TFIIBWTcells. Of all genes analyzed in this study, nearly 74% genes exhibited a statistically significant increase in terminator readthrough in the mutant. To gain an understanding of the mechanistic basis of TFIIB involvement in termination, we performed mass spectrometry of TFIIB, affinity purified from chromatin and soluble cellular fractions, from TFIIBsua7-1and TFIIBWTcells. TFIIB purified from the chromatin fraction of TFIIBWTcells exhibited significant enrichment of CF1A and Rat1 termination complexes. There was, however, a drastic decrease in TFIIB interaction with both CF1A and Rat1 termination complexes in TFIIBsua7-1mutant. ChIP assay revealed that the recruitment of Pta1 subunit of CPF complex, Rna15 subunit of CF1 complex and Rat1 subunit of Rat1 complex registered nearly 90% decline in the mutant over wild type cells. The overall conclusion of these results is that TFIIB affects termination of transcription on a genome-wide scale, and TFIIB-termination factor interaction may play a crucial role in the process.