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Molecular and Cellular Biology, May 2004, p. 3983-3991, Vol. 24, No. 9
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.9.3983-3991.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Functional Interaction between TFIIB and the Rpb2 Subunit of RNA Polymerase II: Implications for the Mechanism of Transcription Initiation

Bo-Shiun Chen{dagger} and Michael Hampsey*

Division of Nucleic Acids Enzymology, Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854

Received 5 August 2003/ Returned for modification 31 October 2003/ Accepted 2 February 2004

The general transcription factor TFIIB is required for accurate initiation, although the mechanism by which RNA polymerase II (RNAP II) identifies initiation sites is not well understood. Here we describe results from genetic and biochemical analyses of an altered form of yeast TFIIB containing an arginine-78 -> cysteine (R78C) replacement in the "B-finger" domain. TFIIB R78C shifts start site selection downstream of normal and confers a cold-sensitive growth defect (Csm–). Suppression of the R78C Csm– phenotype identified a functional interaction between TFIIB and the Rpb2 subunit of RNAP II and defined a novel role for Rpb2 in start site selection. The rpb2 suppressor encodes a glycine-369 -> serine (G369S) replacement, located in the "lobe" domain of Rpb2 and near the Rpb9 subunit, which was identified previously as an effector of start site selection. The Rpb2-Rpb9 "lobe-jaw" region of RNAP II is downstream of the catalytic center and distal to the site of RNAP II-TFIIB interaction. A TFIIB R78C mutant extract was defective for promoter-specific run-on transcription but yielded an altered pattern of abortive initiation products, indicating that the R78C defect does not preclude initiation. The sua7-3 rpb2-101 double mutant was sensitive to 6-azauracil in vivo and to nucleoside triphosphate substrate depletion in vitro. In the context of the recent X-ray structure of the yeast RNAP II-TFIIB complex, these results define a functional interaction between the B-finger domain of TFIIB and the distal lobe-jaw region of RNAP II and provide insight into the mechanism of start site selection.

* Corresponding author. Mailing address: Department of Biochemistry, Robert Wood Johnson Medical School, 683 Hoes Ln., Piscataway, NJ 08854-0009. Phone: (732) 235-5888. Fax: (732) 235-5889. E-mail: michael.hampsey{at}umdnj.edu.

{dagger} Present address: Receptor Biology Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.

Molecular and Cellular Biology, May 2004, p. 3983-3991, Vol. 24, No. 9
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.9.3983-3991.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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