Mutational Analysis of the D1/E1 Core Helices and the Conserved N-Terminal Region of Yeast Transcription Factor IIB (TFIIB): Identification of an N-terminal Mutant That Stabilizes TATA-Binding Protein-TFIIB-DNA Complexes

Abstract

The general transcription factor IIB (TFIIB) plays an essential role in transcription of protein-coding genes by RNA polymerase II. We have used site-directed mutagenesis to assess the role of conserved amino acids in several important regions of yeast TFIIB. These include residues in the highly conserved amino-terminal region and basic residues in the D1 and E1 core domain ␣-helices. Acidic substitutions of residues K190 (D1) and K201 (E1) resulted in growth impairments in vivo, reduced basal transcriptional activity in vitro, and an inability to form stable TFIIB-TATA-binding protein-DNA (DB) complexes. Significantly, these mutants retained the ability to respond to acidic activators in vivo and to the Gal4-VP16 activator in vitro, supporting the view that these basic residues play a role in basal transcription. In addition, 14 single-amino-acid substitutions were introduced in the conserved amino-terminal region. Three of these mutants, the L50D, R64E, and R78L mutants, displayed altered growth properties in vivo and were compromised for supporting transcription in vitro. The L50D mutant was impaired for RNA polymerase II interaction, while the R64E mutant exhibited altered transcription start site selection both in vitro and in vivo and, surprisingly, was more active than the wild type in the formation of stable DB complexes. These results support the view that the amino-terminal domain is involved in the direct interaction between yeast TFIIB and RNA polymerase II and suggest that this domain may interact with DNA and/or modulate the formation of a DB complex.To specifically initiate transcription, eukaryotic RNA polymerase II (RNAPII) requires the assistance of at least six distinct auxiliary factors, commonly known as the general transcription factors (GTFs). The formation of an active preinitiation complex on the promoter of a protein-coding gene is thought to occur by the initial binding of transcription factor IID (TFIID) to the TATA element, followed either by an ordered assembly of the remaining GTFs and RNAPII or by the direct recruitment of a preassembled RNAPII holoenzyme (reviewed in reference 26). Some fundamental goals regarding the investigation of RNAPII transcription are to determine the structure and functions of the GTFs, elucidate the interactions between them and their mode of association, and determine the precise molecular mechanisms by which transcription complex assembly or recruitment and the ensuing processes of initiation and elongation are regulated.The general transcription factor TFIIB plays an essential role in RNAPII transcription (reviewed in reference 26). It has been proposed that the primary function of TFIIB is to act as a physical bridge between TFIID bound at the TATA element and the remainder of the preinitiation complex. Consistent with this view, biochemical studies have shown that TFIIB interacts with several components of the general transcription machinery, including TFIIF (13, 16), RNAPII (8, 16), TATAbinding protein (TBP) (6,16,24), and the TBP-associated facto...

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.…”

“…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.…”

“…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.…”

“…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.…”

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

“…Interaction of an Inr thymine with the B-reader residue R64 (Fig. 3c) apparently occurs in the open DNA complex, because mutation of R64 causes TSS shifts that are sensitive to changes in Inr sequence 27,28 . The B-reader function in Inr recognition is supported by mutational analysis of the invariant TFIIB residue R78, which buttresses the B-reader loop, including the DNAbinding residue D69 (Fig.…”