Abstract
TFIIH is a general transcription factor with kinase and helicase activities. The kinase activity resides in the Kin28 subunit of TFIIH. The role of Kin28 kinase in the early steps of transcription is well established. Here we report a novel role of Kin28 in the termination of transcription. We show that RNAPII reads through a termination signal upon kinase inhibition. Furthermore, the recruitment of termination factors towards the 3′ end of a gene was compromised in the kinase mutant, thus confirming the termination defect. A concomitant decrease in crosslinking of termination factors near the 5′ end of genes was also observed in the kinase-defective mutant. Simultaneous presence of termination factors towards both the ends of a gene is indicative of gene looping; while the loss of termination factor occupancy from the distal ends suggest the abolition of a looped gene conformation. Accordingly, CCC analysis revealed that the looped architecture of genes was severely compromised in the Kin28 kinase mutant. In a looping defective sua7-1 mutant, even the enzymatically active Kin28 kinase could not rescue the termination defect. These results strongly suggest a crucial role of Kin28 kinase-dependent gene looping in the termination of transcription in budding yeast.
Highlights
The kinase subunit of TFIIH, Kin[28], phosphorylates carboxy-terminal domain (CTD) of RNAPII at serine-5 and serine-7 residues[1]
CTD-kinase activity of Kin[28] is not essential either for transcription or for the survival of yeast cells, growth of cells is severely inhibited in the kinase-defective mutant[5,12,14,15]
Our results are in disagreement with another study that did not find a significant decrease in the global mRNA level upon inhibition of Kin[28] kinase using the same kin28-as mutant[14]
Summary
The kinase subunit of TFIIH, Kin[28], phosphorylates carboxy-terminal domain (CTD) of RNAPII at serine-5 and serine-7 residues[1]. The shifting of Kin[28] mutants to the elevated temperature adversely affected the recruitment of TFIIH complex at the promoter resulting in a dramatic decrease in the CTD-serine-5 phosphorylation, and a concomitant decrease in the level of steady state mRNA level[11,12]. A global decrease in the transcript level in the absence of Kin[28] kinase activity, was observed[12] This decrease in mRNA level in the Kin28-as mutant was attributed to the effect of serine-5 phosphorylation on the capping of mRNA at the 5′ end rather than a direct role of Kin[28] kinase in transcription. We propose that Kin[28] kinase-mediated gene looping facilitates termination of transcription in at least a subset of genes in budding yeast
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