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

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 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. Mutations impairing the formation of stable TFIIB⅐RNAPII 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 TFIIB⅐RNAPII 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. Eukaryotic RNA polymerase II (RNAPII)1 requires the action of at least six accessory proteins to accurately initiate transcription. These accessory proteins, termed the general transcription factors (GTFs), have been the focus of much investigation and include TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. The GTFs and RNAPII assemble in an ordered stepwise fashion on a class II promoter in vitro to form a functional preinitiation complex (PIC) (reviewed in Ref. 1). Assembly is initiated by the binding of TFIID to the TATA element via the TATA-binding protein (TBP) subunit, in some cases assisted by TFIIA. This complex is recognized by TFIIB, which binds and recruits RNAPII and TFIIF. PIC formation is completed by the association of TFIIE and then TFIIH, and the resulting complex can hydrolyze ATP and initiate mRNA synthesis. In contrast to this ordered-assembly model for PIC formation, it has been proposed that a preassembled holoenzyme, consisting of RNAPII, most of the GTFs, and additional factors, is recruited in one step to promoter-bound TFIID in vivo (reviewed in Ref. 1).The general transcription factor TFIIB has an essential role in RNAPII transcription, and together with RNAPII and TBP, defines the minimal set of factors necessary for promoter-dependent transcription of a supercoiled DNA template in vitro (1). In both the ordered-assembly and holoenzyme-recruitment models of PIC formation, TFIIB recognizes promoter-bound TFIID and facilitates association of the remaining GTFs and RNAPII. Consistent with this role, TFIIB interacts with DNA adjacent to the TATA box (2) and binds to TBP (3-5), the TBP-associated factor TAF40 (6), RNAPII (4,7,8), and both subunits of TFIIF (4, 9). TFIIB may also play a r...

Transcription Factor B Contacts Promoter DNA Near the Transcription Start Site of the Archaeal Transcription Initiation Complex

“…2 and 3), and on documented roles of TFB and TFIIB N-terminal domain CSBs in transcription-initiation NTP concentration dependence, transcription-initiation efficiency, and start-site selection (33,(37)(38)(39)(40)(41)(42), we suggest that the TFB and TFIIB N-terminal domains in the archaeal and eukaryal transcription initiation complexes occupy positions analogous to those of R3.2 in the bacterial transcription initiation complex. Specifically, we suggest that the TFB or TFIIB N-terminal-domain zinc ribbon binds in or near the RNAP RNA exit channel (as documented by protein-protein photocrosslinking and protein affinity cleaving (64)) and that the TFB and TFIIB N-terminal domain CSBs bind in the RNAP active-center cleft, between the two strands of the melted transcription bubble, near the transcription start site.…”

“…The proposed interaction of residues of the TFB N-terminal domain and/or interdomain linker with the transcription-bubble region, near the transcription start site, immediately suggests a structural and mechanistic explanation for roles of the TFB and TFIIB N-terminal domains in post-recruitment reactions involving the transcription start site: transcription-initiation NTP concentration dependence (33), transcription-initiation efficiency (33, 38 -40), and start-site selection (33,(37)(38)(39)(40)(41)(42). We propose that residues of the TFB and TFIIB N-terminal domains contact or closely approach the transcription-bubble region near the transcription start site and mediate, through direct interactions near the transcription start site, roles in these post-recruitment reactions.…”

“…Available evidence suggests that the zinc ribbon is responsible for effects of TFB and TFIIB on recruitment of RNAP to initiation complexes (31,34,38,39) and that the CSB is responsible for effects of TFB and TFIIB on transcription-initiation NTP concentration dependence, transcription-initiation efficiency, and start-site selection (33,(37)(38)(39)(40)(41)(42). Thus, deletions or substitutions within the zinc ribbon prevent recruitment of RNAP (31,34,38,39), whereas deletions or substitutions within the CSB do not affect recruitment of RNAP (33,39,40,42) but do affect transcription-initiation NTP concentration dependence (33), transcription-initiation efficiency (33, 38 -40), and start-site selection (37)(38)(39)(40)(41)(42). We propose that residues of the TFB and TFIIB N-terminal-domain CSBs contact or closely approach the transcription-bubble region near the transcription start site and mediate, through direct interactions near the transcription start site, roles in transcrip- 4 N. Naryshkin and R. H. Ebright, unpublished data.…”

“…33). Substitutions in the conserved sequence block in archaeal TFB indicate a role in transcription-initiation NTP concentration dependence (33), and substitutions in eukaryal TFIIB indicate roles in transcription-initiation efficiency and start-site selection (37)(38)(39)(40)(41)(42). Although the roles of the TFB and TFIIB N-terminal domains in transcription initiation and start-site selection have been well characterized, the structural and mechanistic basis of these roles has remained undefined.…”

Nuclear Import of TFIIB Is Mediated by Kap114p, a Karyopherin with Multiple Cargo-binding Domains

“…Western blotting was performed by transferring the proteins onto polyvinylidene difluoride membrane and probing with antibodies as noted. The Sua7p antibody used in Western blots (Pardee et al, 1998) was a kind gift from Dr. Alfred Ponticelli (University of Buffalo, NY). Kap114-Myc was probed with monoclonal antic-myc antibody (Sigma, St. Louis, MO).…”