scholarly journals Different Functional Modes of p300 in Activation of RNA Polymerase III Transcription from Chromatin Templates

2008 ◽  
Vol 28 (18) ◽  
pp. 5764-5776 ◽  
Author(s):  
Claudia Mertens ◽  
Robert G. Roeder
Keyword(s):  
Rna Polymerase ◽  
Transcriptional Activation ◽  
Core Promoter ◽  
Rna Polymerase Iii ◽  
Initiation Factor ◽  
Trna Genes ◽  
U6 Snrna ◽  
Polymerase Iii

ABSTRACT Transcriptional coactivators that regulate the activity of human RNA polymerase III (Pol III) in the context of chromatin have not been reported. Here, we describe a completely defined in vitro system for transcription of a human tRNA gene assembled into a chromatin template. Transcriptional activation and histone acetylation in this system depend on recruitment of p300 by general initiation factor TFIIIC, thus providing a new paradigm for recruitment of histone-modifying coactivators. Beyond its role as a chromatin-modifying factor, p300 displays an acetyltransferase-independent function at the level of preinitiation complex assembly. Thus, direct interaction of p300 with TFIIIC stabilizes binding of TFIIIC to core promoter elements and results in enhanced transcriptional activity on histone-free templates. Additional studies show that p300 is recruited to the promoters of actively transcribed tRNA and U6 snRNA genes in vivo. These studies identify TFIIIC as a recruitment factor for p300 and thus may have important implications for the emerging concept that tRNA genes or TFIIIC binding sites act as chromatin barriers to prohibit spreading of silenced heterochromatin domains.

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.

scholarly journals Chromatin Structure and Expression of a Gene Transcribed by RNA Polymerase III Are Independent of H2A.Z Deposition

2008 ◽  
Vol 28 (8) ◽  
pp. 2598-2607 ◽  
Author(s):  
Aneeshkumar Gopalakrishnan Arimbasseri ◽  
Purnima Bhargava
Keyword(s):  
Rna Polymerase ◽  
Chromatin Structure ◽  
Rna Polymerase Iii ◽  
Nutritional Deprivation ◽  
U6 Snrna ◽  
Polymerase Iii ◽  
Downstream Box ◽  
Pol Iii

ABSTRACT The genes transcribed by RNA polymerase III (Pol III) generally have intragenic promoter elements. One of them, the yeast U6 snRNA (SNR6) gene is activated in vitro by a positioned nucleosome between its intragenic box A and extragenic, downstream box B separated by ∼200 bp. We demonstrate here that the in vivo chromatin structure of the gene region is characterized by the presence of an array of positioned nucleosomes, with only one of them in the 5′ end of the gene having a regulatory role. A positioned nucleosome present between boxes A and B in vivo does not move when the gene is repressed due to nutritional deprivation. In contrast, the upstream nucleosome which covers the TATA box under repressed conditions is shifted ∼50 bp further upstream by the ATP-dependent chromatin remodeler RSC upon activation. It is marked with the histone variant H2A.Z and H4K16 acetylation in active state. In the absence of H2A.Z, the chromatin structure of the gene does not change, suggesting that H2A.Z is not required for establishing the active chromatin structure. These results show that the chromatin structure directly participates in regulation of a Pol III-transcribed gene under different states of its activity in vivo.

scholarly journals The Retinoblastoma Tumor Suppressor Protein Targets Distinct General Transcription Factors To Regulate RNA Polymerase III Gene Expression

2000 ◽  
Vol 20 (24) ◽  
pp. 9182-9191 ◽  
Author(s):  
Heather A. Hirsch ◽  
Liping Gu ◽  
R. William Henry
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
U6 Snrna ◽  
General Transcription Factors ◽  
Retinoblastoma Tumor Suppressor ◽  
Polymerase Iii ◽  
Retinoblastoma Tumor

ABSTRACT The retinoblastoma protein (RB) represses RNA polymerase III transcription effectively both in vivo and in vitro. Here we demonstrate that the general transcription factors snRNA-activating protein complex (SNAPc) and TATA binding protein (TBP) are important for RB repression of human U6 snRNA gene transcription by RNA polymerase III. RB is associated with SNAPc as detected by both coimmunoprecipitation of endogenous RB with SNAPc and cofractionation of RB and SNAPc during chromatographic purification. RB also interacts with two SNAPc subunits, SNAP43 and SNAP50. TBP or a combination of TBP and SNAPcrestores efficient U6 transcription from RB-treated extracts, indicating that TBP is also involved in RB regulation. In contrast, the TBP-containing complex TFIIIB restores adenovirus VAI but not human U6 transcription in RB-treated extracts, suggesting that TFIIIB is important for RB regulation of tRNA-like genes. These results suggest that different classes of RNA polymerase III-transcribed genes have distinct general transcription factor requirements for repression by RB.

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 Stable transcription complex on a class III gene in a minichromosome.

1985 ◽  
Vol 5 (1) ◽  
pp. 40-45 ◽  
Author(s):  
A B Lassar ◽  
D H Hamer ◽  
R G Roeder
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Stable Complex ◽  
Trna Genes ◽  
Class Iii ◽  
5S Rna ◽  
Polymerase Iii

We have constructed recombinant simian virus 40 molecules containing Xenopus 5S RNA and tRNA genes. Recombinant minichromosomes containing these genes were isolated to study the interaction of RNA polymerase III transcription factors with these model chromatin templates. Minichromosomes containing a tRNAMet gene can be isolated in a stable complex with transcription factors (IIIB and IIIC) and are active in vitro templates for purified RNA polymerase III. In contrast, minichromosomes containing a 5S RNA gene are refractory to transcription by purified RNA polymerase III in either the absence or the presence of other factors.

scholarly journals Differential Utilization of TATA Box-binding Protein (TBP) and TBP-related Factor 1 (TRF1) at Different Classes of RNA Polymerase III Promoters

2013 ◽  
Vol 288 (38) ◽  
pp. 27564-27570 ◽  
Author(s):  
Neha Verma ◽  
Ko-Hsuan Hung ◽  
Jin Joo Kang ◽  
Nermeen H. Barakat ◽  
William E. Stumph
Keyword(s):  
Rna Polymerase ◽  
Binding Protein ◽  
Rna Polymerase Iii ◽  
Tata Box ◽  
Related Factor ◽  
U6 Snrna ◽  
Polymerase Iii ◽  
Tata Box Binding Protein ◽  
In Cells

In the fruit fly Drosophila melanogaster, RNA polymerase III transcription was found to be dependent not upon the canonical TATA box-binding protein (TBP) but instead upon the TBP-related factor 1 (TRF1) (Takada, S., Lis, J. T., Zhou, S., and Tjian, R. (2000) Cell 101, 459–469). Here we confirm that transcription of fly tRNA genes requires TRF1. However, we unexpectedly find that U6 snRNA gene promoters are occupied primarily by TBP in cells and that knockdown of TBP, but not TRF1, inhibits U6 transcription in cells. Moreover, U6 transcription in vitro effectively utilizes TBP, whereas TBP cannot substitute for TRF1 to promote tRNA transcription in vitro. Thus, in fruit flies, different classes of RNA polymerase III promoters differentially utilize TBP and TRF1 for the initiation of transcription.

A factor with Sp1 DNA-binding specificity stimulates Xenopus U6 snRNA in vivo transcription by RNA polymerase III

1992 ◽  
Vol 228 (2) ◽  
pp. 387-394 ◽  
Author(s):  
Alain Lescure ◽  
Graham Tebb ◽  
Iain W. Mattaj ◽  
Alain Krol ◽  
Philippe Carbon
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Binding Specificity ◽  
U6 Snrna ◽  
Dna Binding Specificity ◽  
Polymerase Iii
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