George A. Kassavetis

S. cerevisiae TFIIIB is the transcription initiation factor proper of RNA polymerase III, while TFIIIA and TFIIIC are assembly factors

The S. cerevisiae RNA polymerase III (pol III) transcription factor TFIIIB binds to DNA upstream of the transcription start site of the SUP4 tRNA(Tyr) gene in a TFIIIC-dependent reaction and to the major 5S rRNA gene in a reaction requiring TFIIIC and TFIIIA. It is shown here that TFIIIB alone correctly positions pol III for repeated cycles of transcription on both genes, with the same efficiency as fully assembled transcription complexes. Thus, TFIIIB is the sole transcription initiation factor of S. cerevisiae pol III; TFIIIC and TFIIIA are assembly factors for TFIIIB. The TFIIIB-dependent binding of pol III to the SUP4 tRNA and 5S rRNA genes has been analyzed in binary (protein and DNA only) and precisely arrested ternary (protein, DNA, and RNA) transcription complexes. Pol III unwinds at least 14 bp of DNA at the SUP4 transcription start in a temperature-dependent process. The unwound DNA segment moves downstream with nascent RNA as a transcription bubble of approximately the same size.

The subunit structure of Saccharomyces cerevisiae transcription factor IIIC probed with a novel photocrosslinking reagent.

A photocrosslinking nucleotide, 5‐[N‐(p‐azidobenzoyl)‐3‐aminoallyl]‐deoxyuridine monophosphate (N3Rd‐UMP), has been used to identify four polypeptides that are associated with the large Saccharomyces cerevisiae RNA polymerase III transcription factor TFIIIC, and to map the locations of these subunits along DNA when TFIIIC binds to the S.cerevisiae SUP4 tRNA(Tyr) gene. The 145 kd subunit of TFIIIC is primarily accessible to photocrosslinking from the vicinity of the box B + internal promoter element; 95 and 55 kd subunits are located on opposite sides of the DNA helix in the vicinity of the box A internal promoter element; a 135 kd subunit is less strongly crosslinked to the box A region and to a DNA segment between boxes B and A. DNA probes containing more than one N3RdUMP residue can form crosslinks between polypeptide chains. The specific circumstances of formation and the apparent mol. wts of two of these products lead to the tentative suggestion that a protomer of TFIIIC may contain two 95 kd subunits.

Two components of Saccharomyces cerevisiae transcription factor IIIB (TFIIIB) are stereospecifically located upstream of a tRNA gene and interact with the second-largest subunit of TFIIIC.

A novel photocrosslinking method has been used to identify the components of transcription factor IIIB (TFIIIB) and TFIIIC that associate with DNA upstream of the Saccharomyces cerevisiae SUP4 tRNATyr gene and to map these components to specific positions in DNA. When TFIIIC binds to the tRNA gene, only its second-largest subunit (135 kDa) is accessible for reaction with a photoactive nucleotide, 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP, inserted into DNA upstream of the transcriptional start. Formation of TFIII(C + B)-tRNA gene complexes specifically brings two additional polypeptides (90 and 70 kDa) within reach of upstream photoprobes. A collection of 13 probes has been used to map the locations of these three proteins along a 45-bp segment of DNA upstream of the transcriptional start site. Evidence is presented that the 90- and 70-kDa polypeptides are separate and distinct components of yeast TFIIIB, that they are accessible to crosslinking on opposite sides of the DNA helix in a 6-bp segment centered 35 bp upstream of the tRNATyr gene transcriptional start, and that they interact with the second-largest subunit of TFIIIC.

Molecular basis of RNA polymerase III transcription repression by Maf1

RNA polymerase III (Pol III) transcribes short RNAs required for cell growth. Under stress conditions, the conserved protein Maf1 rapidly represses Pol III transcription. We report the crystal structure of Maf1 and cryo-electron microscopic structures of Pol III, an active Pol III-DNA-RNA complex, and a repressive Pol III-Maf1 complex. Binding of DNA and RNA causes ordering of the Pol III-specific subcomplex C82/34/31 that is required for transcription initiation. Maf1 binds the Pol III clamp and rearranges C82/34/31 at the rim of the active center cleft. This impairs recruitment of Pol III to a complex of promoter DNA with the initiation factors Brf1 and TBP and thus prevents closed complex formation. Maf1 does however not impair binding of a DNA-RNA scaffold and RNA synthesis. These results explain how Maf1 specifically represses transcription initiation from Pol III promoters and indicate that Maf1 also prevents reinitiation by binding Pol III during transcription elongation.

Human TFIID Binds to Core Promoter DNA in a Reorganized Structural State

A mechanistic description of metazoan transcription is essential for understanding the molecular processes that govern cellular decisions. To provide structural insights into the DNA recognition step of transcription initiation, we used single-particle electron microscopy (EM) to visualize human TFIID with promoter DNA. This analysis revealed that TFIID coexists in two predominant and distinct structural states that differ by a 100 Å translocation of TFIIDs lobe A. The transition between these structural states is modulated by TFIIA, as the presence of TFIIA and promoter DNA facilitates the formation of a rearranged state of TFIID that enables promoter recognition and binding. DNA labeling and footprinting, together with cryo-EM studies, were used to map the locations of TATA, Initiator (Inr), motif ten element (MTE), and downstream core promoter element (DPE) promoter motifs within the TFIID-TFIIA-DNA structure. The existence of two structurally and functionally distinct forms of TFIID suggests that the different conformers may serve as specific targets for the action of regulatory factors.

Functional and Structural Organization of Brf, the TFIIB-Related Component of the RNA Polymerase III Transcription Initiation Complex

ABSTRACT Brf is the TFIIB-related component of Saccharomyces cerevisiae RNA polymerase III transcription initiation factor IIIB (TFIIIB). An extensive set of Brf fragments has been examined for the abilities to assemble the TFIIIB-DNA complex and recruit RNA polymerase III to accurately initiate transcription. The principal TFIIIB-assembly function of Brf was found to be contributed by a C-proximal segment spanning amino acids 435 to 545, while the principal transcription-directing function was contributed by a segment of its N-proximal, TFIIB-homologous half. The diverse activities of Brf were also reconstituted from combined fragments. The fragments spanning amino acids 1 to 282 and 284 to 596 were found to assemble into TFIIIB-DNA and TFIIIC-TFIIIB-DNA complexes that were very stable, transcriptionally highly active, and indistinguishable (by in vitro footprinting) from complexes formed with intact Brf. The proximities of the individual halves of split Brf to DNA were extensively mapped by photochemical cross-linking of the TFIIIB-DNA complex. We also identified sites of interaction of Brf fragments with TATA-binding protein (TBP), taking advantage of a recently completed mutational analysis of the TBP surface. The constraints established by these analyses specify a global model of the functional segments of Brf and how they fit into the structure of the TFIIIB-DNA complex.

Identical components of yeast transcription factor IIIB are required and sufficient for transcription of TATA box-containing and TATA-less genes

Specific transcription by RNA polymerase III requires recognition of the promoter-bound transcription factor IIIB (TFIIIB), of which the TATA-binding protein (TBP) is a subunit. The recruitment of TFIIIB to TATA-less genes is mediated by protein-protein interactions with transcription factor IIIC (TFIIIC) bound to the box A and box B elements. Here we examine interactions involved in the recruitment of TFIIIB to the TATA element-containing yeast U6 small nuclear RNA gene SNR6. TFIIIC is not required for the formation of TFIIIB-SNR6 gene complexes with purified components. The same three components of TFIIIB that are necessary for TFIIIC-dependent transcription of tRNA genes (recombinant TBP and Brf and the denaturing-gel-purified 90-kDa subunit) are required and sufficient for TATA box-directed U6 transcription. Despite its TFIIIC-independent, DNA sequence-dependent assembly, the TFIIIB-SNR6 complex shares important features with tDNA- and 5S rDNA-TFIIIB complexes, such as extent and location of footprint, stability, and resistance to heparin. These properties are clearly distinct from those of a TBP-SNR6 complex. In the SNR6 gene, box B, the primary binding site for TFIIIC, is suboptimally spaced relative to box A. At limiting TBP concentrations and on bare DNA, TFIIIC stimulates the formation of TFIIIB complexes with SNR6 but contributes poorly, at best, to the formation of properly placed complexes.

Transcriptional activation by a DNA-tracking protein: Structural consequences of enhancement at the T4 late promoter

Transcriptional initiation at bacteriophage T4 late promoters is activated from enhancer-like distal sites by the T4 gene 44, 62, and 45 DNA polymerase accessory proteins (gp44, gp62, and gp45, respectively). Enhancement is ATP hydrolysis-dependent and requires protein tracking along DNA. The structural analysis of the enhanced transcription initiation complex shows gp45 located at the upstream end of this promoter complex in the vicinity of its transcriptional coactivator, the T4 gene 33 protein. The ATP-cleaving gene 44 protein-gene 62 protein complex serves as the assembly factor for gp45, but does not stably associate with the enhanced promoter complex. Transcriptional enhancement quantitatively favors, but does not qualitatively change, DNA strand separation in the transcription bubble. A model of the transcriptional activation that rationalizes its DNA-tracking and activation-polarity properties is presented.

10 RNA Polymerase III Transcription Complexes

OVERVIEW RNA polymerase III (pol III) synthesizes tRNA, 5S ribosomal RNA, and other small RNAs in conjunction with a group of transcription factors (TFIII) that is not yet completely enumerated and characterized. In yeast, TFIIIB serves as the central transcription factor, necessary and sufficient for correctly positioning RNA pol III over the transcriptional start. TFIIIB does not recognize specific DNA sequence, nor is it able to bind to DNA by itself. Instead, it must be directed to its transcription start-proximal upstream DNA-binding site by other TFIIIs acting as assembly factors. The assembly factors of tRNA and 5S RNA genes, TFIIIC and TFIIIA, bind within the transcription unit, with TFIIIA serving as an adapter for TFIIIC in 5S RNA genes. In a U6 snRNA gene, other transcription factors, including the RNA polymerase II transcription factor TFIID, probably assemble TFIIIB from upstream of the transcription start site. It is widely anticipated that these functional principles are generalizable to transcription by RNA pol III in all eukaryotes, but that remains to be proven. INTRODUCTION It is 15 years since the publication of the first clear and reliable demonstrations of specifically initiated eukaryotic transcription in vitro by RNA pol III (Parker and Roeder 1977; Wu et al. 1977). The fact that about three-fourths of the chapters of this book deal with eukaryotic transcription reflects (beyond fashion and finance) the enormous development that our understanding of the subject has subsequently undergone. This chapter deals with transcriptional initiation by RNA pol III, which synthesizes small RNAs,...

The RNA polymerase III transcription initiation factor TFIIIB participates in two steps of promoter opening

Evidence for post‐recruitment functions of yeast transcription factor (TF)IIIB in initiation of transcription was first provided by the properties of TFIIIB–RNA polymerase III–promoter complexes assembled with deletion mutants of its Brf and B″ subunits that are transcriptionally inactive because they fail to open the promoter. The experiments presented here show that these defects can be repaired by unpairing short (3 or 5 bp) DNA segments spanning the transcription bubble of the open promoter complex. Analysis of this suppression phenomenon indicates that TFIIIB participates in two steps of promoter opening by RNA polymerase III that are comparable to the successive steps of promoter opening by bacterial RNA polymerase holoenzyme. B″ deletions between amino acids 355 and 421 interfere with the initiating step of DNA strand separation at the upstream end of the transcription bubble. Removing an N‐terminal domain of Brf interferes with downstream propagation of the transcription bubble to and beyond the transcriptional start site.