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G A Kassavetis

Publications and source records attributed to G A Kassavetis.

At least 19 recordsLinked to original sources
PubMed2006

Transcription factor TFIIIB and transcription by RNA polymerase III.

pol (RNA polymerase) III is charged with the task of transcribing nuclear genes encoding diverse small structural and catalytic RNAs. We present a brief review of the current understanding of several aspects of the pol III transcription apparatus. The focus is on yeast and, more specifically, on Saccharomyces cerevisiae; preponderant attention is given to the TFs (transcription initiation factors) and especially to TFIIIB, which is the core pol III initiation factor by virtue of its role in recruiting pol III to the transcriptional start site and its essential roles in forming the transcription-ready open promoter complex. Certain relatively recent developments are also selected for brief comment: (i) the genome-wide analysis of occupancy of pol III-transcribed genes (and other loci) by the transcription apparatus and the location of pol III transcription in the cell; (ii) progress toward a mechanistic and molecular understanding of the regulation of transcription by pol III in yeast; and (iii) recent experiments identifying a high mobility group protein as a fidelity factor that assures selection of the precise transcriptional start site at certain pol III promoters.

Gene Expression Regulation↗
PubMed2001

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.

Base Sequence↗
PubMed1999

Alignment of the B" subunit of RNA polymerase III transcription factor IIIB in its promoter complex.

TFIIIB, the central transcription initiation factor of the eukaryotic nuclear RNA polymerase (pol) III is composed of three subunits: the TATA-binding protein; Brf, the TFIIB-related subunit; and B", the Saccharomyces cerevisiae, TFC5 gene product. The orientation of the B" subunit within the TFIIIB-DNA complex has been analyzed at two promoters by two approaches that involve site-specific photochemical protein-DNA cross-linking: a collection of B" internal and external deletion proteins has been surveyed for those deletions that alter the interaction of B" with DNA or change the orientation of B" relative to DNA; a method for regionally mapping cross-links between specific DNA sites and (32)P-end-labeled protein has also been applied. The results map an N-proximal segment of B" to the upstream end of the TFIIIB-DNA complex and amino acids 299-315 to the principal DNA-contact site, approximately 8 base pairs upstream of the TATA box. The analysis also indicates that a segment comprising amino acids 316-434 loops away from DNA, and locates the C-proximal 170 amino acids of B" downstream of the TATA box. Examination of two-cross-link products formed by DNA with adjacent and nearby photoactive nucleotides supports the conclusion that Brf and B" share an extended interface along the length of the TFIIIB-DNA complex.

Base Sequence↗
PubMed1999

A minimal RNA polymerase III transcription system.

Transcription factor (TF) IIIB recruits RNA polymerase (pol) III for specific initiation of transcription. All three subunits of TFIIIB, TBP, Brf (the TFIIB-related subunit) and B", are required for transcription of supercoiled and linear duplex DNA, but we show here that B" is non-essential on a promoter that has been partly pre-opened by unpairing a short segment of the transcription bubble. These findings expose a striking similarity between transcriptional initiation by pol II, pol III and bacterial RNA polymerases: a preformed single-stranded DNA bubble upstream of the transcriptional start removes the dependence of pol II on TFIIE, TFIIH and ATP hydrolysis, and the dependence of pol III on B"; the favored placement of the transcription bubble for B"-independent transcription by pol III overlaps a DNA segment that interacts sequence specifically as single-stranded DNA with the sigma(70 )initiation subunit of Escherichia coli RNA polymerase holoenzyme.

Adenosine Triphosphate↗
PubMed1999

Abortive initiation by Saccharomyces cerevisiae RNA polymerase III.

Promoter escape can be rate-limiting for transcription by bacterial RNA polymerases and RNA polymerase II of higher eukaryotes. Formation of a productive elongation complex requires disengagement of RNA polymerase from promoter-bound eukaryotic transcription factors or bacterial sigma factors. RNA polymerase III (pol III) stably associates with the TFIIIB-DNA complex even in the absence of localized DNA unwinding associated with the open promoter complex. To explore the role that release of pol III from the TFIIIB-DNA complex plays in limiting the overall rate of transcription, we have examined the early steps of RNA synthesis. We find that, on average, only three rounds of abortive initiation precede the formation of each elongation complex and that nearly all pol III molecules escape the abortive initiation phase of transcription without significant pausing or arrest. However, when elongation is limited to 5 nucleotides, the intrinsic exoribonuclease activity of pol III cleaves 5-mer RNA at a rate considerably faster than product release or reinitiation. This cleavage also occurs in the normal process of forming a productive elongation complex. The possible role of nucleolytic retraction in disengaging pol III from TFIIIB is discussed.

Base Sequence↗
PubMed1999

The RNA polymerase III-recruiting factor TFIIIB induces a DNA bend between the TATA box and the transcriptional start site.

TFIIIB, the RNA polymerase III-recruiting factor of Saccharomyces cerevisiae, may be assembled upstream of the transcriptional start site, either through the interaction of its constituent TATA-binding protein (TBP) with a strong TATA-box, or by means of the multisubunit assembly factor, TFIIIC. Missing nucleoside interference analysis of TFIIIC-dependent TFIIIB-DNA complex formation revealed enhanced complex formation at 0 degreesC when the DNA is missing nucleosides in two broad 7-10 bp regions centered around base-pairs -17 and -3 relative to the transcriptional start site; no effect of missing nucleosides was evident at 20 degreesC. The implication of these results for required DNA flexure in TFIIIC-mediated TFIIIB-DNA complex formation was pursued in a TFIIIC-independent context, using DNA with a suboptimal 6 bp TATA box (TATAAA). A unique missing nucleoside at the downstream end of the TATA box, corresponding to the position of one of two TBP-mediated DNA kinks, significantly enhances TBP-DNA complex formation. In contrast, TFIIIB displays a broad preference for missing nucleosides within an approximately 15 bp region immediately downstream of the TATA box. Consecutive mismatches (4-nt loops), either at the sites of TBP-mediated DNA kinking at both ends of the TATA box or within the identified region where missing nucleosides promote TFIIIB-DNA complex formation, also result in enhanced and specific TFIIIB assembly; 4-nt loops further downstream do not lead to preferential placement of TFIIIB. We conclude that TFIIIB induces an additional DNA deformation between the TATA box and the start site of transcription that is likely to be more extended than the sharp kinks generated by TBP.

Base Sequence↗
PubMed1998

Abortive initiation of transcription at a hybrid promoter. An analysis of the sliding clamp activator of bacteriophage T4 late transcription, and a comparison of the sigma70 and T4 gp55 promoter recognition proteins.

Bacteriophage T4 late promoters are transcribed by an RNA polymerase holoenzyme comprising the Escherichia coli core, E, the phage gene 55-encoded promoter recognition subunit, gp55, and the gene 33-encoded co-activator, gp33. Transcriptional initiation is activated by the T4 gene 45-encoded sliding clamp, which is loaded on to DNA at enhancer-like sites by its clamp-loader. Correct initiation of transcription at late promoters in basal mode requires only RNA polymerase core and gp55 (E.gp55). Dinucleotide-primed abortive initiation of basal and activated T4 late transcription has been compared. Only the trinucleotide non-productive transcript is made at a high rate; all other short transcripts are made at rates of less than one molecule per productive transcript. Gp45 increases abortive trinucleotide synthesis along with productive transcription, although the proportion of productive transcripts is also elevated. Nevertheless, this increase accounts for only a small part of the activation of T4 late transcription that is generated by its activator and co-activator. The pattern of production of short transcripts differs subtly between basal and enhanced transcription, indicating that linking the RNA polymerase with its sliding clamp activator only generates minor changes in the transition from abortive to productive RNA chain elongation. The T4 late promoter is converted to a strong sigma70 promoter by inserting an appropriate -35 promoter element. A direct comparison at such a hybrid promoter shows sigma70 and gp55 generating qualitatively and quantitative different patterns of abortive initiation at the same start site.

Bacteriophage T4↗
PubMed1998

A post-recruitment function for the RNA polymerase III transcription-initiation factor IIIB.

Transcription factor (TF) IIIB, which directs RNA polymerase (pol) III to its promoters, is made up of three components: the TATA box-binding protein, the TFIIB-related Brf, and the pol III-specific B". Certain mutations in Saccharomyces cerevisiae Brf and B" retain TFIIIB transcription factor activity with supercoiled DNA but are inactive with linear duplex DNA. Further analysis shows that these inactive TFIIIB-DNA complexes bind pol III and position it appropriately over the transcriptional start site but do not form DNA strand-separated open promoter complexes. It is proposed that the normal function of TFIIIB combines pol III recruitment with an active role in a subsequent step of transcriptional initiation leading to promoter opening.

Base Sequence↗
PubMed1998

Polymerase (Pol) III TATA box-binding protein (TBP)-associated factor Brf binds to a surface on TBP also required for activated Pol II transcription.

The TATA box-binding protein (TBP) plays an essential role in transcription by all three eukaryotic nuclear RNA polymerases, polymerases (Pol) I, II, and III. In each case, TBP interacts with class-specific TBP-associated factors (TAFs) to form class-specific transcription initiation factors. For yeast Pol III transcription, TBP associates with Brf (from TFIIB-related factor) and B", two Pol III TAFs, to form Pol III transcription factor TFIIIB. Here, we identify TBP surface residues that are required for interaction with yeast Pol III TAFs. Ninety-one human TBP surface residue mutants with radical substitutions were analyzed for the ability to form stable gel shift complexes with purified Brf and B" and for their activities for in vitro synthesis of yeast U6 snRNA. Mutations in a large positively charged epitope extending from the top (that is, on the surface opposite the DNA-facing "saddle" of TBP) and onto the side of the first TBP repeat inhibited binding to Brf (residues K181, L185, R186, E206, R231, L232, R235, K236, R239, Q242, K243, K249, and F250). A triple-mutant TBP (R231E + R235E + R239S) had greatly reduced activity for yeast U6 snRNA gene transcription while remaining active for Pol II basal transcription. Similar results were observed when selected mutations were introduced into yeast TBP at equivalent positions. A C-terminal fragment of Brf lacking the region of homology with TFIIB retains the ability to bind TBP-DNA complexes (G. Kassavetis, C. Bardeleben, A. Kumar, E. Ramirez, and E. P. Geiduschek, Mol. Cell. Biol. 17:5299-5306, 1997); the same TBP mutations reduced binding by this fragment. Mutations in TBP residues that interact with TFIIB did not affect Brf binding or U6 gene transcription. These results indicate that Brf and TFIIB interact differently with TBP. An extensively overlapping epitope on the top surface of TBP was found previously to be required for activated Pol II transcription and has been hypothesized to interact with Pol II TAFs. Our results map the surface of TBP that interacts with Brf and suggest that Pol II and Pol III TAFs interact with the same surface of TBP.

DNA-Binding Proteins↗
PubMed1998

Functional and structural organization of Brf, the TFIIB-related component of the RNA polymerase III transcription initiation complex.

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.

Base Sequence↗
PubMed1997

Mechanism of transcription through the nucleosome by eukaryotic RNA polymerase.

Nucleosomes, the nucleohistone subunits of chromatin, are present on transcribed eukaryotic genes but do not prevent transcription. It is shown here that the large yeast RNA polymerase III transcribes through a single nucleosome. This takes place through a direct internal nucleosome transfer in which histones never leave the DNA template. During this process, the polymerase pauses with a pronounced periodicity of 10 to 11 base pairs, which is consistent with restricted rotation in the DNA loop formed during transfer. Transcription through nucleosomes by the eukaryotic enzyme and by much smaller prokaryotic RNA polymerases thus shares many features, reflecting an important property of nucleosomes.

Base Sequence↗
PubMed1997

Dual targets of a transcriptional activator that tracks on DNA.

The sliding clamp of the bacteriophage T4 DNA polymerase, gp45, is also the proximal effector for activation of transcription of T4 late genes. We have identified the phage T4-encoded sigma factor gp55 and the co-activator gp33 as targets of gp45 in promoter complexes, and have shown that a conserved carboxy-terminal amino acid sequence of gp55 and gp33 is required for interaction with gp45. The respective contribution of each target-gp45 interaction to activation of transcription has been assessed by measuring promoter opening rates. The opening rate supported by interaction with both targets is far greater than the arithmetical sum of the separate contributions of each target, implying a synergistic activation of transcription through at least two separate interactions of the trimeric gp45.

Bacteriophage T4↗
PubMed1997

Functional dissection of the B" component of RNA polymerase III transcription factor IIIB: a scaffolding protein with multiple roles in assembly and initiation of transcription.

Transcription factor IIIB (TFIIIB), the central transcription factor of Saccharomyces cerevisiae RNA polymerase III, is composed of TATA-binding protein, the TFIIB-related protein Brf, and B". B", the last component to enter the TFIIIB-DNA complex, confers extremely tight DNA binding on TFIIIB. Terminally and internally deleted B" derivatives were tested for competence to form TFIIIB-DNA complexes by TFIIIC-dependent and -independent pathways on the SUP4 tRNA(Tyr) and U6 snRNA (SNR6) genes, respectively, and for transcription. Selected TFIIIB-TFIIIC-DNA complexes assembled with truncated B" were analyzed by DNase I footprinting, and the surface topography of B" in the TFIIIB-DNA complex was also analyzed by hydroxyl radical protein footprinting. These analyses define functional domains of B" and also reveal roles in start site selection by RNA polymerase III and in clearing TFIIIC from the transcriptional start. Although absolutely required for transcription, B" can be extensively truncated. Core proteins retaining as few as 176 (of 594) amino acids remain competent to transcribe the SNR6 gene in vitro. TFIIIC-dependent assembly on DNA and transcription requires a larger core of B": two domains (I and II) that are required for SNR6 transcription on an either-or basis are simultaneously required for TFIIIC-dependent assembly of DNA complexes and transcription. Domains I and II of B" are buried upon assembly of the TFIIIB-DNA complex, as determined by protein footprinting. The picture of the TFIIIB-DNA complex that emerges is that B" serves as its scaffold and is folded over in the complex so that domains I and II are near one another.

Amino Acid Sequence↗
PubMed1997

Domains of the Brf component of RNA polymerase III transcription factor IIIB (TFIIIB): functions in assembly of TFIIIB-DNA complexes and recruitment of RNA polymerase to the promoter.

Saccharomyces cerevisiae transcription factor IIIB (TFIIIB) is composed of three subunits: the TATA-binding protein, the TFIIB-related protein Brf, and B". TFIIIB, which is brought to RNA polymerase III-transcribed genes indirectly through interaction with DNA-bound TFIIIC or directly through DNA recognition by the TATA-binding protein, in turn recruits RNA polymerase III to the promoter. N-terminally deleted derivatives of Brf have been examined for their ability to interact with DNA-bound TFIIIC and with the other components of TFIIIB and for participation in transcription. Brf(165-596), lacking 164 N-proximal TFIIB-homologous amino acids, is competent to participate in the assembly of TFIIIB-DNA complexes and in TFIIIC-independent transcription. Even deletion of the entire TFIIB-homologous half of the protein, as in Brf(317-596) and Brf(352-596), allows some interaction with DNA-bound TBP and with the B" component of TFIIIB to be retained. The function of Brf(165-596) in transcription has also been examined in the context of B" with small internal deletions. The ability of Brf with this sizable N-terminal segment deleted to function in TFIIIC-independent transcription requires segments of B" that are individually indispensable although required on an either/or basis, in the context of complete Brf. These findings suggest a functional complementarity and reciprocity between the Brf and B" components of TFIIIB.

Base Sequence↗
PubMed1996

A direct interaction between a DNA-tracking protein and a promoter recognition protein: implications for searching DNA sequence.

Bacteriophage T4 gene 45 protein, gp45, serves as the sliding clamp of viral DNA replication and as the activator of T4 late gene transcription. In the latter context, DNA tracking is an essential feature of the unique mechanism of action. T4 late promoters, which consist of a simple TATA box, TATAAATA, are recognized by the small sigma-family gene 55 protein, gp55, which binds to Escherichia coli RNA polymerase core. A direct and RNA polymerase-independent interaction of gp45 with gp55 has been demonstrated in two ways. (i) gp45 tracks along DNA; co-tracking of gp55 requires the previously documented DNA-loading process of gp45, and can be detected by photochemical crosslinking. (ii) The dynamics of DNA tracking by gp45 can be followed by footprinting; the catenated DNA-tracking state of gp45 is short-lived, but is stabilized by gp55. The ability of this topologically linked DNA-tracking transcriptional activator to interact directly with a promoter recognition protein suggests the existence of multiple pathways of promoter location, which are discussed.

Bacterial Proteins↗
PubMed1996

Alternative outcomes in assembly of promoter complexes: the roles of TBP and a flexible linker in placing TFIIIB on tRNA genes.

Saccharomyces cerevisiae transcription factor (TF) IIIB, a TATA-binding protein (TBP)-containing multisubunit factor, recruits RNA polymerase (Pol) III for multiple rounds of transcription. TFIIIC is an assembly factor for TFIIIB on TATA-less tRNA gene promoters. To investigate the role of TBP-DNA interactions in tRNA gene transcription, we generated sequence substitutions in the SUP4 tRNATyr gene TFIIIB binding site. Purified transcription proteins were used to analyze the selection of transcription initiation sites and the physical structures of the protein complexes formed on these mutant genes. We show that the association of TFIIIB with tRNA genes proceeds through an initial step of binding-site selection that is codirected by its TBP subunit and by TFIIIC. TFIIIB is assembled in a predominantly metric manner with regard to box A, the start site-proximal binding site of TFIIIC, but TFIIIC opens a window within which wild-type TBP can select the TFIIIB-binding site. Despite its clear preference for AT-rich sequences, TBP can mediate TFIIIB assembly at diverse DNA sequences, including stretches containing only G and C. However, a mutant TBP, m3, which recognizes TATAAA and TGTAAA and is active for Pol III transcription, utilizes other sequences only poorly. We also show that alternative alignments between DNA-bound TFIIIB and TFIIIC are possible, implying a remarkably flexible linkage, and suggest that Tfc4, the TFIIIB-assembling subunit of TFIIIC, could be responsible for such elasticity. The relevance of these findings to alternative initiation of Pol II- and other Pol III-transcribed genes is discussed.

Base Sequence↗