Abstract
Gene looping, defined as the interaction of the promoter and the terminator regions of a gene during transcription, requires transcription factor IIB (TFIIB). We have earlier demonstrated association of TFIIB with the distal ends of a gene in an activator-dependent manner (El Kaderi, B., Medler, S., Raghunayakula, S., and Ansari, A. (2009) J. Biol. Chem. 284, 25015-25025). The presence of TFIIB at the 3' end of a gene required its interaction with cleavage factor 1 (CF1) 3' end processing complex subunit Rna15. Here, employing affinity chromatography and glycerol gradient centrifugation, we show that TFIIB associates with poly(A) polymerase and the entire CF1 complex in yeast cells. The factors required for general transcription such as TATA-binding protein, RNA polymerase II, and TFIIH are not a component of the TFIIB complex. This holo-TFIIB complex was resistant to MNase digestion. The complex was observed only in the looping-competent strains, but not in the looping-defective sua7-1 strain. The requirement of Rna15 in gene looping has been demonstrated earlier. Here we provide evidence that poly(A) polymerase (Pap1) as well as CF1 subunits Rna14 and Pcf11 are also required for loop formation of MET16 and INO1 genes. Accordingly, cross-linking of TFIIB to the 3' end of genes was abolished in the mutants of Pap1, Rna14, and Pcf11. We further show that in sua7-1 cells, where holo-TFIIB complex is not formed, the kinetics of activated transcription is altered. These results suggest that a complex of TFIIB, CF1 subunits, and Pap1 exists in yeast cells. Furthermore, TFIIB interaction with the CF1 complex and Pap1 is crucial for gene looping and transcriptional regulation.
Highlights
MCB1020911. □S The on-line version of this article contains supplemental Tables S1 and S2 and Figs
Recombinant transcription factor IIB (TFIIB), with a molecular mass in the range of 32–38 kDa, could complement all functions of native, biochemically purified TFIIB in an in vitro transcription assay (16 –19). These results suggested that TFIIB is a single polypeptide protein that exists as a monomer in solution
In the second analysis, poly(A)-binding protein (Pab1), which interacts with the poly(A) tail of mRNA, was identified as the only 3Ј end processing factor associated with TFIIB [20]
Summary
MCB1020911. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Figs. We provide evidence that poly(A) polymerase (Pap1) as well as CF1 subunits Rna14 and Pcf11 are required for loop formation of MET16 and INO1 genes. The absolute requirement of TFIIB in gene looping, cross-linking of TFIIB to both the promoter and the terminator regions of a looped gene, and its functional interaction with several 3Ј end processing/termination factors including CF1 subunit Rna15 [1, 11] suggested that a complex of TFIIB and termination factors exists in the cell.
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