No abstract

Transcription factor IIIC (TFIIIC) plays an important role in assembling the initiation factor TFIIIB on genes transcribed by RNA polymerase III (Pol III). In Saccharomyces cerevisiae, assembly of the TFIIIB complex by promoter-bound TFIIIC is thought to be initiated by its tetratricopeptide repeat (TPR)-containing subunit, TFIIIC 131 , which interacts directly with the TFIIB-related factor, TFIIIB 70 /Brf1. In this work, we have identified 10 dominant mutations in TFIIIC 131 that increase Pol III gene transcription. All of these mutations are found within a discrete 53-amino-acid region of the protein encompassing TPR2. Biochemical studies of one of the mutations (PCF1-2) show that the increase in transcription is due to an increase in the recruitment of TFIIIB 70 to TFIIIC-DNA. The PCF1-2 mutation does not affect the affinity of TFIIIC for DNA, and the differential in both transcription and TFIIIB complex assembly is observed at saturating levels of TFIIIB 70 . This indicates that mutant and wild-type TFIIIC-DNA complexes have the same affinity for TFIIIB 70 and suggests that the increased recruitment of this factor is achieved by a nonequilibrium binding mechanism. A novel mechanism of activation in which the TPR mutations facilitate a conformational change in TFIIIC that is required for TFIIIB 70 binding is proposed. The implications of this model for the regulation of processes involving TPR proteins are discussed.The multisubunit transcription factor IIIC (TFIIIC) is an assembly factor that directs the binding of the general initiation factor, TFIIIB, upstream of the transcription start site of RNA polymerase III (Pol III) genes (21, 37). On tRNA gene templates, TFIIIC interacts with intragenic A and B block promoter elements. High-affinity binding of TFIIIC to DNA is mediated by the B block. This element is positioned at variable distances from the start site and, in at least one example, can function, albeit less efficiently, in an inverted orientation (5). These properties, together with the requirement for TFIIIC in transcription from chromatin templates (antirepression), provide a conceptual view of TFIIIC as an enhancer binding factor (5, 6). TFIIIC, through interactions with the A block, also plays a role in start site selection. Typically, the start site is located about 20 bp upstream of the A block. However, its position can be shifted within a 20-to 30-bp window by altering the placement of TFIIIB on the DNA (7,10,12,15). Redirection of TFIIIB involves its TATA-binding protein (TBP) subunit, in addition to TFIIIC, and can be achieved by inserting, repositioning, or mutating an upstream TATA element. The conformational flexibility required for this variable placement of TFIIIB has been suggested to reside in the TFIIIB-assembling subunit of TFIIIC, TFIIIC 131 (15).Specific transcription by Pol III can be achieved in the absence of TFIIIC on nucleosome-free templates which contain a suitable TATA box and an initiation site. Transcription of the Saccharomyces cerevisiae U6 gene, which contains the ...

Gene transcription by RNA polymerase III (pol III) 1 in Saccharomyces cerevisiae requires four chromatographically separable and functionally distinct transcription factors (TFIIIA, TFIIIB, TFIIIC, and TFIIIE) that bind to the promoters of pol III genes and/or facilitate their transcription (1-4). TFIIIA plays a unique gene-specific role in the transcription of the 5 S RNA genes, whereas the other factors play important functions in the transcription of all pol III genes in yeast. For TFIIIC, these functions include promoter recognition and the recruitment of TFIIIB, which is directed to a region upstream of the transcription start site (2, 5). Promoter-bound TFIIIB can, by itself, recruit pol III for multiple rounds of transcription (6).TFIIIB, therefore, plays a role in pol III transcription that is analogous to the general transcription factors required for initiation of transcription by RNA pol II.The limiting steps in the transcription of pol III genes in wild-type yeast cells have been defined by mutations or gene dosage effects that increase the synthesis of pol III gene products. Thus far, only missense mutations in one of the six subunits of TFIIIC (TFIIIC 131 ) (7, 8) and increased levels of one of the three subunits of TFIIIB (TFIIIB 70 ) (9) have been found to illicit a stimulation of pol III gene transcription. For TFIIIB 70 , this and other data demonstrate that the factor is stoichiometrically limiting for transcription in vivo (9) and in whole-cell extracts (10). Because of the limiting nature of TFIIIB 70 , global control of pol III gene expression can potentially be achieved by regulating the amount of this factor (1). Indeed, this appears to be the case. The reduced transcription observed in extracts derived from cells that are approaching stationary phase or whose growth has been inhibited by cycloheximide can be accounted for, in part, by a reduction in the amount of TFIIIB 70 (10, 11).The individual polypeptides that comprise yeast TFIIIB have been identified and cloned (4, 9, 12-21). In addition to TFIIIB 70 noted above, the components of yeast TFIIIB include the TATA-binding protein (TBP) and TFIIIB 90 . These polypeptides bind to TFIIIC⅐DNA complexes in a stepwise manner in vitro beginning with TFIIIB 70 . The subsequent binding of TBP followed by TFIIIB 90 leads to progressive changes in: (i) the size of the upstream region protected from digestion by DNase I; (ii) the degree of protection conferred by these proteins at specific sites; and (iii) the efficiency of cross-linking of various TFIIIB and TFIIIC subunits by photoprobes positioned at specific locations in the DNA (2). Each of the components of yeast TFIIIB has been expressed in bacteria, and collectively, they suffice to support TATA box-mediated transcription in the presence of highly purified pol III. Additionally, when provided with highly purified TFIIIC, the recombinant TFIIIB components reconstitute the transcription of tRNA genes (4, 21).TFIIIB 70 is a protein of 596 amino acids, the amino-terminal half of which is homol...

High levels of RNA polymerase III gene transcription are achieved by facilitated recycling of the polymerase on transcription factor IIIB (TFIIIB)-DNA complexes that are stable through multiple rounds of initiation. TFIIIB-DNA complexes in yeast comprise the TATAbinding protein (TBP), the TFIIB-related factor TFIIIB70, and TFIIIB90. The high stability of the TFIIIB-DNA complex is conferred by TFIIIB90 binding to TFIIIB70-TBP-DNA complexes. This stability is thought to result from compound bends introduced in the DNA by TBP and TFIIIB90 and by protein-protein interactions that obstruct DNA dissociation. Here we present biochemical evidence that the high stability of TFIIIB-DNA complexes results from kinetic trapping of the DNA. Thermodynamic analysis shows that the free energies of formation of TFIIIB70-TBP-DNA (⌬G°‫؍‬ ؊12.10 ؎ 0.12 kcal͞mol) and TFIIIB-DNA (⌬G°‫؍‬ ؊11.90 ؎ 0.14 kcal͞mol) complexes are equivalent whereas a kinetic analysis shows that the half-lives of these complexes (46 ؎ 3 min and 95 ؎ 6 min, respectively) differ significantly. The differential stability of these isoenergetic complexes demonstrates that TFIIIB90 binding energy is used to drive conformational changes and increase the barrier to complex dissociation. RNA polymerase (pol) III transcribes a variety of nontranslated RNA genes encoding transfer RNAs, 5S ribosomal RNA, U6 snRNA, and other small cellular RNAs (1). In Saccharomyces cerevisiae, transcription of these genes is directed by the initiation factor TFIIIB, which is assembled upstream of the start site by other factors (TFIIIA and͞or TFIIIC), bound to downstream promoter elements. Yeast TFIIIB is a heterotrimeric complex comprising the TATA-binding protein (TBP), a TFIIB-related component, TFIIIB70 (Brf1), and a SANT domain protein, TFIIIB90 (BЈЈ). Structural and functional homologs of these proteins have been identified in human cells and confer TFIIIB activity (termed TFIIIB-␣ in ref.2) for the transcription of tRNA and related pol III genes having internal promoter elements (2-4). Additionally, human cells contain a second TFIIIB activity (TFIIIB-␤) that is used by pol III genes whose promoter elements are located upstream of the start site (e.g., U6 snRNA and 7SK RNA). TFIIIB-␤ differs from TFIIIB-␣ in that it contains a different TFIIB-related component (termed BRFU or hTFIIIB50) and associated proteins (2, 3). Further complexity among TFIIIB complexes is suggested by the identification of three splice variants of human Brf1 (4). One of these variants, Brf2, appears to be active in the transcription U6 snRNA.Pol III genes are among the most actively transcribed genes in eukaryotic cells. High rates of pol III gene transcription are achieved through the facilitated recycling of pol molecules (5-8) on TFIIIB complexes that remain bound to the DNA for multiple rounds of initiation (9,10). The stability of TFIIIB-DNA complexes is therefore a key property that enables rapid reinitiation by eliminating rate-limiting steps in transcription complex assembly. Yeast TFIIIB-DNA compl...

We have conducted a quantitative thermodynamic study of the effects of the TATA element and TATAflanking sequences on the assembly of complexes containing TATA-binding protein (TBP) and the TFIIB-related factor, TFIIIB 70 . TBP binds to the sequence TATAAAAG in the context of the yeast U6 gene (yU6 hybrid TATA) or the adenovirus major late promoter (AdMLP) with different affinities demonstrating that the sequence context of a TATA element contributes to TBP binding. We also determined the cooperative free energies of formation of TBP⅐TFIIIB 70 ⅐DNA complexes on the yU6 TATA element, the yU6 hybrid TATA element and a nonconsensus TATA element. The yU6 hybrid TATA displayed a moderate, less than 5-fold, increase in TBP affinity similar to the 3-fold increase observed for the AdMLP. In contrast, the nonconsensus and yU6 TATAs increased the affinity of TBP for DNA 12-and 17-fold, respectively. Since the TBP-TFIIIB 70 cooperativity is greater on lower affinity TATA boxes and most polymerase III genes contain low affinity "TATA boxes," we conclude that the cooperative binding of TFIIIB 70 and TBP to DNA represents an important driving force in the assembly of polymerase III-specific transcription complexes. An effect of the sequences surrounding the TATA box was also observed on TBP-TFIIIB 70 cooperativity. The mechanistic implications of the thermodynamic linkage between DNA sequence and binding cooperativity are discussed.The TATA-binding protein (TBP) 1 is required for transcription by all three nuclear RNA polymerases (pols I, II, and III) (1). This universal role of TBP is achieved, in most organisms, through its sequestration into polymerase-specific complexes (e.g. SL1, TFIID, and TFIIIB). Although sequence-specific binding of TBP to DNA is observed in two of these complexes (TFIID and TFIIIB), the efficient recruitment of each complex to the appropriate promoters in vivo is dependent upon additional interactions with other proteins termed "activating factors," "coactivators," and/or "initiators" (2-6). The recruitment of SL1, TFIID, and TFIIIB to DNA has been studied extensively because of the fundamental importance of these steps in transcription by the respective polymerase and because of their regulatory significance. DNA binding by TBP or its complexes appears to be a rate-limiting step for transcription initiation at many promoters (7-9). Accordingly, many regulatory factors have been described that interact directly with TBP or its associated components (4, 5).The interaction of TBP with the TATA element has been the subject of extensive genetic, biochemical and biophysical studies. These studies have shown that TBP binds to a wide variety of sequences and that it exhibits the highest affinities for sequences of the general form TATAa/tAa/t (10). The crystal structures that have been solved of Arabidopsis thaliana TBP2 and the conserved C-terminal cores of Saccharomyces cerevisiae and Homo sapiens TBP complexed with DNA containing different TATA boxes are strikingly similar and revealed a remarkable p...