The N-terminal Region of Yeast TFIIB Contains Two Adjacent Functional Domains Involved in Stable RNA Polymerase II Binding and Transcription Start Site Selection

Timothy S Pardee,Chaitanya S Bangur,Alfred S Ponticelli

doi:10.1074/jbc.273.28.17859

Abstract

The general transcription factor IIB (TFIIB) is required for accurate and efficient transcription of protein-coding genes by RNA polymerase II (RNAPII). To define functional domains in the highly conserved N-terminal region of TFIIB, we have analyzed 14 site-directed substitution mutants of yeast TFIIB for their ability to support cell viability, transcription in vitro, accurate start site selection in vitro and in vivo, and to form stable complexes with purified RNAPII in vitro. Mutations impairing the formation of stable TFIIB.RNAPII complexes mapped to the zinc ribbon fold, whereas mutations conferring downstream shifts in transcription start site selection were identified at multiple positions within a highly conserved homology block adjacent and C-terminal to the zinc ribbon. These results demonstrate that the N-terminal region of yeast TFIIB contains two separable and adjacent functional domains involved in stable RNAPII binding and transcription start site selection, suggesting that downstream shifts in transcription start site selection do not result from impairment of stable TFIIB.RNAPII binding. We discuss models for yeast start site selection in which TFIIB may affect the ability of preinitiation complexes to interact with downstream DNA or to affect start site recognition by a scanning polymerase.

Highlights

Eukaryotic RNA polymerase II (RNAPII)1 requires the action of at least six accessory proteins to accurately initiate transcription
Mutations impairing the formation of stable TFIIB1⁄7RNAPII complexes mapped to the zinc ribbon fold, whereas mutations conferring downstream shifts in transcription start site selection were identified at multiple positions within a highly conserved homology block adjacent and C-terminal to the zinc ribbon. These results demonstrate that the N-terminal region of yeast transcription factor IIB (TFIIB) contains two separable and adjacent functional domains involved in stable RNAPII binding and transcription start site selection, suggesting that downstream shifts in transcription start site selection do not result from impairment of stable TFIIB1⁄7RNAPII binding
The core domain of TFIIB did not detectably bind RNAPII in these assays. These results demonstrate that the second rubredoxin knuckle and the C-terminal ␤-strand of the zinc ribbon in the N-terminal region of TFIIB are involved in the direct interaction between TFIIB and RNAPII

Summary

Introduction

Eukaryotic RNA polymerase II (RNAPII)1 requires the action of at least six accessory proteins to accurately initiate transcription. To define functional domains in the highly conserved Nterminal region of TFIIB, we have analyzed 14 site-directed substitution mutants of yeast TFIIB for their ability to support cell viability, transcription in vitro, accurate start site selection in vitro and in vivo, and to form stable complexes with purified RNAPII in vitro.

Results

Conclusion