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

1998 ◽  
Vol 18 (9) ◽  
pp. 5587-5599 ◽  
Author(s):  
George A. Kassavetis ◽  
Ashok Kumar ◽  
Enrique Ramirez ◽  
E. Peter Geiduschek
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Mutational Analysis ◽  
Rna Polymerase Iii ◽  
Initiation Factor ◽  
Highly Active ◽  
Polymerase Iii ◽  
Dna 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.

scholarly journals Inhibition of TATA Binding Protein Dimerization by RNA Polymerase III Transcription Initiation Factor Brf1

2004 ◽  
Vol 279 (31) ◽  
pp. 32401-32406 ◽  
Author(s):  
Diane E. Alexander ◽  
David J. Kaczorowski ◽  
Amy J. Jackson-Fisher ◽  
Drew M. Lowery ◽  
Sara J. Zanton ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Binding Protein ◽  
Rna Polymerase Iii ◽  
Tata Binding Protein ◽  
Initiation Factor ◽  
Protein Dimerization ◽  
Polymerase Iii ◽  
Transcription Initiation Factor

scholarly journals Termination-altering mutations in the second-largest subunit of yeast RNA polymerase III.

1995 ◽  
Vol 15 (3) ◽  
pp. 1467-1478 ◽  
Author(s):  
S A Shaaban ◽  
B M Krupp ◽  
B D Hall
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
The Other ◽  
Polymerase Iii

In order to identify catalytically important amino acid changes within the second-largest subunit of yeast RNA polymerase III, we mutagenized selected regions of its gene (RET1) and devised in vivo assays for both increased and decreased transcription termination by this enzyme. Using as the reporter gene a mutant SUP4-o tRNA gene that in one case terminates prematurely and in the other case fails to terminate, we screened mutagenized RET1 libraries for reduced and increased transcription termination, respectively. The gain in suppression phenotype was in both cases scored as a reduction in the accumulation of red pigment in yeast strains harboring the ade2-1 ochre mutation. Termination-altering mutations were obtained in regions of the RET1 gene encoding amino acids 300 to 325, 455 to 486, 487 to 521, and 1061 to 1082 of the protein. In degree of amino acid sequence conservation, these range from highly variable in the first to highly conserved in the last two regions. Residues 300 to 325 yielded mainly reduced-termination mutants, while in region 1061 to 1082, increased-termination mutants were obtained exclusively. All mutants recovered, while causing gain of suppression with one SUP4 allele, brought about a reduction in suppression with the other allele, thus confirming that the phenotype is due to altered termination rather than an elevated level of transcription initiation. In vitro transcription reactions performed with extracts from several strong mutants demonstrated that the mutant polymerases respond to RNA terminator sequences in a manner that matches their in vivo termination phenotypes.

scholarly journals Essential Roles of Bdp1, a Subunit of RNA Polymerase III Initiation Factor TFIIIB, in Transcription and tRNA Processing

2002 ◽  
Vol 22 (10) ◽  
pp. 3264-3275 ◽  
Author(s):  
Akira Ishiguro ◽  
George A. Kassavetis ◽  
E. Peter Geiduschek
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Genomic Library ◽  
Rna Polymerase Iii ◽  
Rnase P ◽  
Initiation Factor ◽  
Physical Interaction ◽  
Polymerase Iii ◽  
A Subunit ◽  
Pol Iii

ABSTRACT The essential Saccharomyces cerevisiae gene BDP1 encodes a subunit of RNA polymerase III (Pol III) transcription factor (TFIIIB); TATA box binding protein (TBP) and Brf1 are the other subunits of this three-protein complex. Deletion analysis defined three segments of Bdp1 that are essential for viability. A central segment, comprising amino acids 327 to 353, was found to be dispensable, and cells making Bdp1 that was split within this segment, at amino acid 352, are viable. Suppression of bdp1 conditional viability by overexpressing SPT15 and BRF1 identified functional interactions of specific Bdp1 segments with TBP and Brf1, respectively. A Bdp1 deletion near essential segment I was synthetically lethal with overexpression of PCF1-1, a dominant gain-of-function mutation in the second tetracopeptide repeat motif (out of 11) of the Tfc4 (τ131) subunit of TFIIIC. The analysis also identifies a connection between Bdp1 and posttranscriptional processing of Pol III transcripts. Yeast genomic library screening identified RPR1 as the specific overexpression suppressor of very slow growth at 37°C due to deletion of Bdp1 amino acids 253 to 269. RPR1 RNA, a Pol III transcript, is the RNA subunit of RNase P, which trims pre-tRNA transcript 5′ ends. Maturation of tRNA was found to be aberrant in bdp1-Δ253-269 cells, and RPR1 transcription with the highly resolved Pol III transcription system in vitro was also diminished when recombinant Bdp1Δ253-269 replaced wild-type Bdp1. Physical interaction of RNase P with Bdp1 was demonstrated by coimmunoprecipitation and pull-down assays.

scholarly journals A Novel Upstream RNA Polymerase III Promoter Element Becomes Essential When the Chromatin Structure of the Yeast U6 RNA Gene Is Altered

2001 ◽  
Vol 21 (19) ◽  
pp. 6429-6439 ◽  
Author(s):  
Michael P. Martin ◽  
Valerie L. Gerlach ◽  
David A. Brow
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Point Mutations ◽  
Tata Box ◽  
Initiation Factor ◽  
Base Pairs ◽  
Polymerase Iii ◽  
U6 Rna

ABSTRACT The Saccharomyces cerevisiae U6 RNA gene,SNR6, possesses upstream sequences that allow productive binding in vitro of the RNA polymerase III (Pol III) transcription initiation factor IIIB (TFIIIB) in the absence of TFIIIC or other assembly factors. TFIIIC-independent transcription ofSNR6 in vitro is highly sensitive to point mutations in a consensus TATA box at position −30. In contrast, the TATA box is dispensable for SNR6 transcription in vivo, apparently because TFIIIC bound to the intragenic A block and downstream B block can recruit TFIIIB via protein-protein interactions. A mutant allele ofSNR6 with decreased spacing between the A and B blocks,snr6-Δ42, exhibits increased dependence on the upstream sequences in vivo. Unexpectedly, we find that in vivo expression of snr6-Δ42 is much more sensitive to mutations in a (dT-dA)7 tract between the TATA box and transcription start site than to mutations in the TATA box itself. Inversion of single base pairs in the center of the dT-dA tract nearly abolishes transcription of snr6-Δ42, yet inversion of all 7 base pairs has little effect on expression, indicating that the dA-dT tract is relatively orientation independent. Although it is within the TFIIIB footprint, point mutations in the dT-dA tract do not inhibit TFIIIB binding or TFIIIC-independent transcription ofSNR6 in vitro. In the absence of the chromatin architectural protein Nhp6, dT-dA tract mutations are lethal even when A-to-B block spacing is wild type. We conclude that the (dT-dA)7 tract and Nhp6 cooperate to direct productive transcription complex assembly on SNR6 in vivo.

Transcription factor TFIIIB and transcription by RNA polymerase III

2006 ◽  
Vol 34 (6) ◽  
pp. 1082-1087 ◽  
Author(s):  
G.A. Kassavetis ◽  
E.P. Geiduschek
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Transcriptional Start Site ◽  
Rna Polymerase Iii ◽  
Initiation Factor ◽  
Regulation Of Transcription ◽  
Start Site ◽  
High Mobility Group Protein ◽  
Polymerase Iii ◽  
Pol 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.

Sign in / Sign up

Export Citation Format

Share Document