Control of the Timing of Promoter Escape and RNA Catalysis by the Transcription Factor IIB Fingertip

Abstract

Transcription factor IIB (TFIIB) recruits RNA polymerase II to promoters and inserts a finger domain into its active site, with unknown consequences. Here we show that that the tip of this finger is important for two transcription initiation functions. First, TFIIB acts as a catalytic cofactor for initial RNA bond formation. It does so via a pair of fingertip aspartates that can bind magnesium, placing TFIIB within a family of proteins that insert finger domains to alter the catalytic functions of RNA polymerase. Second, the TFIIB fingertip mediates the timing of the release of TFIIB that is associated with appropriate promoter escape. These initiation requirements may assist in RNA quality control by minimizing functional synthesis when RNA polymerase becomes inappropriately associated with the genome without having been recruited there by TFIIB. TFIIB2 is a single-subunit protein that plays a central role in transcription by RNA polymerase II. It consists of three motifs with distinct structures and functions. The C terminus is composed of two cyclin repeats and is responsible for bringing TFIIB to promoters. It does so primarily by recognizing promoter-bound TATA-binding protein but also interacts with DNA sequences that flank the TATA box (1). A second motif is folded by association with a zinc atom (2). This binds a docking domain on RNA polymerase II (3) and recruits the enzyme to promoters to catalyze transcription initiation (4). A third motif is unstructured in solution but folds into a finger-like structure when bound to RNA polymerase (3). A number of functions have been suggested for this finger, mostly involving the escape by the recruited RNA polymerase (discussed below).TFIIB is unique among the general transcription factors in that it recycles during continuous transcription, i.e. TFIIF travels with the elongating RNA polymerase, and TATA-binding protein, TFIIH, and TFIIE largely remain bound to the promoter (5). Thus there can be a pioneer round of transcription that forms a scaffold upon which reinitiation can occur without requiring the reassembly of all factors from free solution. This allows transcription to occur at a facilitated rate (6). For each round of initiation, a new RNA polymerase must wait for the prior RNA polymerase to escape. This requires the recycling of TFIIB (7) by binding to the scaffold to recruit the next RNA polymerase. These initial steps of transcription initiation, involving initial bond formation, production of small RNAs, and disruption of contacts to the promoter, are collectively termed "escape" (8 -10).The finger domain of TFIIB has been proposed to play multiple roles in promoter escape. Both chemical probing (3) and structural studies (11) have shown that the finger penetrates the main channel of RNA polymerase II to approach the active site of the enzyme. From this location it could influence these early steps by interacting with the template DNA, the newly synthesized RNA, the catalytic center of the RNA polymerase, and perhaps TFIIF or other general transcr...

Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

“…B-reader loop function is required for transcription because deletion or mutation of B-reader loop residues 69-75 cause defects in vivo and in vitro (Supplementary Fig. 5), consistent with results for human TFIIB 23 and with in vitro data for yeast TFIIB 30 . B-reader loop function is conserved because replacing the loop for its Schizosaccharomyces pombe counterpart preserves function ( Supplementary Fig.…”

“…Consistent with this, the B-reader of the archaeal TFIIB homologue stimulates transcription 22 ; truncation of the B-reader loop in human TFIIB impairs extension of a RNA dinucleotide 23 ; and the presence of TFIIB and TBP stimulates transcription initiation from pre-opened DNA 15,24 . We find here that TFIIB alone stimulates de novo RNA synthesis by Pol II at low or near-physiological NTP concentrations ( Fig.…”

The TFIIB Tip Domain Couples Transcription Initiation to Events Involved in RNA Processing

“…TFIIB is known to both directly recruit RNA polymerase and to alter its enzymatic properties (2)(3)(4). The recruitment relies on several protein domains to bind components of the transcription preinitiation complex as well as to bind RNA polymerase.…”

“…One of these domains is the "B-finger" or "Breader loop," which has a tip that approaches the active site of RNA polymerase within the preinitiation complex (5)(6)(7). This tip contains a pair of aspartate residues that bind magnesium and are required for full catalysis by RNA polymerase during the initiation phase of transcription (3)(4). The assistance presumably provides a quality control function in that RNA polymerases that associate "non-specifically" with the genome, which is without the assistance of TFIIB and the associated preinitiation complex, will have low catalytic activity.…”

“…The timing of promoter escape is centrally related to the release of TFIIB, which is controlled by its tip domain (3). That is, small deletions within the tip do not prevent TFIIB from properly joining the preinitiation transcription complex but do cause TFIIB to be released prematurely.…”