scholarly journals High-Mobility-Group Proteins NHP6A and NHP6B Participate in Activation of the RNA Polymerase IIISNR6 Gene

2001 ◽  
Vol 21 (9) ◽  
pp. 3096-3104 ◽  
Author(s):  
Sébastien Lopez ◽  
Magda Livingstone-Zatchej ◽  
Sabine Jourdain ◽  
Fritz Thoma ◽  
André Sentenac ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
High Mobility ◽  
High Mobility Group ◽  
Wild Type ◽  
High Mobility Group Proteins ◽  
General Transcription Factors ◽  
Pol Iii

ABSTRACT Transcription of yeast class III genes involves the formation of a transcription initiation complex that comprises RNA polymerase III (Pol III) and the general transcription factors TFIIIB and TFIIIC. Using a genetic screen for positive regulators able to compensate for a deficiency in a promoter element of the SNR6 gene, we isolated the NHP6A and NHP6B genes. Here we show that the high-mobility-group proteins NHP6A and NHP6B are required for the efficient transcription of the SNR6 gene both in vivo and in vitro. The transcripts of wild-type and promoter-defectiveSNR6 genes decreased or became undetectable in annhp6AΔ nhp6BΔ double-mutant strain, and the protection over the TATA box of the wild-type SNR6 gene was lost innhp6AΔ nhp6BΔ cells at 37°C. In vitro, NHP6B specifically stimulated the transcription of SNR6 templates up to fivefold in transcription assays using either cell nuclear extracts from nhp6AΔ nhp6BΔ cells or reconstituted transcription systems. Finally, NHP6B activated SNR6transcription in a TFIIIC-independent assay. These results indicate that besides the general transcription factors TFIIIB and TFIIIC, additional auxilliary factors are required for the optimal transcription of at least some specific Pol III genes.

scholarly journals Novel Small-Molecule Inhibitors of RNA Polymerase III

2003 ◽  
Vol 2 (2) ◽  
pp. 256-264 ◽  
Author(s):  
Liping Wu ◽  
Jing Pan ◽  
Vala Thoroddsen ◽  
Deborah R. Wysong ◽  
Ronald K. Blackman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Rna Polymerase Iii ◽  
Wild Type ◽  
Biochemical Assays ◽  
Transcription In Vitro ◽  
Pol Iii

ABSTRACT A genetic approach utilizing the yeast Saccharomyces cerevisiae was used to identify the target of antifungal compounds. This analysis led to the identification of small molecule inhibitors of RNA polymerase (Pol) III from Saccharomyces cerevisiae. Three lines of evidence show that UK-118005 inhibits cell growth by targeting RNA Pol III in yeast. First, a dominant mutation in the g domain of Rpo31p, the largest subunit of RNA Pol III, confers resistance to the compound. Second, UK-118005 rapidly inhibits tRNA synthesis in wild-type cells but not in UK-118005 resistant mutants. Third, in biochemical assays, UK-118005 inhibits tRNA gene transcription in vitro by the wild-type but not the mutant Pol III enzyme. By testing analogs of UK-118005 in a template-specific RNA Pol III transcription assay, an inhibitor with significantly higher potency, ML-60218, was identified. Further examination showed that both compounds are broad-spectrum inhibitors, displaying activity against RNA Pol III transcription systems derived from Candida albicans and human cells. The identification of these inhibitors demonstrates that RNA Pol III can be targeted by small synthetic molecules.

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 The transcription reinitiation properties of RNA polymerase III in the absence of transcription factors

2008 ◽  
Vol 13 (1) ◽  
Author(s):  
Roberto Ferrari ◽  
Giorgio Dieci
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
Transcription Unit ◽  
Transcription System ◽  
Transcription Reinitiation ◽  
Pol Iii

AbstractTranscription reinitiation by RNA polymerase (Pol) III proceeds through facilitated recycling, a process by which the terminating Pol III, assisted by the transcription factors TFIIIB and TFIIIC, rapidly reloads onto the same transcription unit. To get further insight into the Pol III transcription mechanism, we analyzed the kinetics of transcription initiation and reinitiation of a simplified in vitro transcription system consisting only of Pol III and template DNA. The data indicates that, in the absence of transcription factors, first-round transcription initiation by Pol III proceeds at a normal rate, while facilitated reinitiation during subsequent cycles is compromised.

scholarly journals In vitro analysis of elongation and termination by mutant RNA polymerases with altered termination behavior.

1996 ◽  
Vol 16 (11) ◽  
pp. 6468-6476 ◽  
Author(s):  
S A Shaaban ◽  
E V Bobkova ◽  
D M Chudzik ◽  
B D Hall
Keyword(s):  
Rna Polymerase Iii ◽  
Multiple Roles ◽  
Wild Type ◽  
Protein Motifs ◽  
Study Support ◽  
Vitro Analysis ◽  
Pol Iii ◽  
In Vitro Analysis

We have studied the in vitro elongation and termination properties of several yeast RNA polymerase III (pol III) mutant enzymes that have altered in vivo termination behavior (S. A. Shaaban, B. M. Krupp, and B. D. Hall, Mol. Cell. Biol. 15:1467-1478, 1995). The pattern of completed-transcript release was also characterized for three of the mutant enzymes. The mutations studied occupy amino acid regions 300 to 325, 455 to 521, and 1061 to 1082 of the RET1 protein (P. James, S. Whelen, and B. D. Hall, J. Biol. Chem. 266:5616-5624, 1991), the second largest subunit of yeast RNA pol III. In general, mutant enzymes which have increased termination require a longer time to traverse a template gene than does wild-type pol III; the converse holds true for most decreased-termination mutants. One increased-termination mutant (K310T I324K) was faster and two reduced termination mutants (K512N and T455I E478K) were slower than the wild-type enzyme. In most cases, these changes in overall elongation kinetics can be accounted for by a correspondingly longer or shorter dwell time at pause sites within the SUP4 tRNA(Tyr) gene. Of the three mutants analyzed for RNA release, one (T455I) was similar to the wild type while the two others (T455I E478K and E478K) bound the completed SUP4 pre-tRNA more avidly. The results of this study support the view that termination is a multistep pathway in which several different regions of the RET1 protein are actively involved. Region 300 to 325 likely affects a step involved in RNA release, while the Rif homology region, amino acids 455 to 521, interacts with the nascent RNA 3' end. The dual effects of several mutations on both elongation kinetics and RNA release suggest that the protein motifs affected by them have multiple roles in the steps leading to transcription termination.

scholarly journals TFIIIC1 acts through a downstream region to stabilize TFIIIC2 binding to RNA polymerase III promoters.

1996 ◽  
Vol 16 (12) ◽  
pp. 6841-6850 ◽  
Author(s):  
Z Wang ◽  
R G Roeder
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Weak Interaction ◽  
Strong Interaction ◽  
Rna Polymerase Iii ◽  
Cooperative Interaction ◽  
In Vitro System ◽  
Polymerase Iii ◽  
Footprint Analysis

An in vitro system reconstituted with highly purified RNA polymerase III, TFIIIC2, and TFIIIB has been used to identify two chromatographically distinct human RNA polymerase III transcription factors, TFIIIC1 and TFIIIC1', which are functionally equivalent to the previously defined TFIIIC1 (S. T. Yoshinaga, P. A. Boulanger, and A. J. Berk, Proc. Natl. Acad. Sci. USA 84:3585-3589, 1987). Interactions between TFIIIC2, TFIIIC1 (or TFIIIC1'), and the VA1 and tRNA1(Met) templates have been investigated by DNase I footprint analysis. Homogeneous TFIIIC2 alone shows only a weak footprint over the B-box region of the VA1 and tRNA1(Met) templates, whereas TFIIIC1 (or TFIIIC1') alone shows both a strong interaction over the downstream termination region and a very weak interaction near the A-box region. Importantly, when both factors are present simultaneously, TFIIIC1 (or TFIIIC1') dramatically enhances the level of TFIIIC2 binding and extends the footprint to a region that includes the A box. The downstream termination region is essential for this cooperative interaction between TFIIIC2 and TFIIIC1 (or TFIIIC1') on the VA1 and tRNA1(Met) templates and plays a role in the overall accuracy and efficiency of RNA polymerase III transcription.

scholarly journals RNA polymerase III transcription in synthetic nuclei assembled in vitro from defined DNA templates.

1995 ◽  
Vol 15 (9) ◽  
pp. 4873-4883 ◽  
Author(s):  
K S Ullman ◽  
D J Forbes
Keyword(s):  
Rna Polymerase ◽  
Nuclear Structure ◽  
De Novo ◽  
Rna Polymerase Iii ◽  
Transcriptional Analysis ◽  
Dna Encoding ◽  
Nuclear Assembly ◽  
Polymerase Iii ◽  
Pol Iii

Although much is known of the basic control of transcription, little is understood of the way in which the structural organization of the nucleus affects transcription. Synthetic nuclei, assembled de novo in extracts of Xenopus eggs, would be predicted to have a large potential for approaching the role of nuclear structure in RNA biogenesis. Synthetic nuclei provide a system in which the genetic content of the nuclei, as well as the structural and enzymatic proteins within the nuclei, can be manipulated. In this study, we have begun to examine transcription in such nuclei by using the most simple of templates, RNA polymerase III (pol III)-transcribed genes. DNA encoding tRNA or 5S genes was added to an assembly extract, and nuclei were formed entirely from the pol III templates. Conditions which allowed nuclear assembly and pol III transcription to take place efficiently and simultaneously in the assembly extract were found. To examine whether pol III transcription could initiate within synthetic nuclei, or instead was inhibited in nuclei and initiated only on rare unincorporated templates, we identified transcriptional inhibitors that were excluded from nuclei. We found that these inhibitors, heparin and dextran sulfate, blocked pol III transcription in the absence of assembly but did not do so following nuclear assembly. At the concentrations used, the inhibitors had no deleterious effect on nuclear structure itself or on nuclear import. We conclude that pol III transcription is active in synthetic nuclei, and this conclusion is further strengthened by the finding that pol III transcripts could be coisolated with synthetic nuclei. The rapid and direct transcriptional analysis possible with pol III templates, coupled with the simple experimental criteria developed in this study for distinguishing between nuclear and non-nuclear transcription, should now allow a molecular analysis of the effect of nuclear structure on transcriptional and posttranscriptional control.

scholarly journals MAF1 is a Chronic Repressor of RNA Polymerase III Transcription in the Mouse

2019 ◽  
Author(s):  
Nicolas Bonhoure ◽  
Viviane Praz ◽  
Robyn D. Moir ◽  
Gilles Willemin ◽  
François Mange ◽  
...  
Keyword(s):  
Energy Expenditure ◽  
Rna Polymerase ◽  
Ribosomal Proteins ◽  
Rna Polymerase Iii ◽  
Mrna Levels ◽  
Wild Type ◽  
Pol Iii ◽  
The Impact ◽  
Global Translation ◽  
Modest Effect

AbstractMaf1-/- mice are lean, obesity-resistant and metabolically inefficient. Their increased energy expenditure is thought to be driven by a futile RNA cycle that reprograms metabolism to meet an increased demand for nucleotides stemming from the deregulation of RNA polymerase (pol) III transcription. Metabolic changes consistent with this model have been reported in both fasted and refed mice, however the impact of the fasting-refeeding-cycle on pol III function has not been examined. Here we show that changes in pol III occupancy in the liver of fasted versus refed wild-type mice are largely confined to low and intermediate occupancy genes; high occupancy genes are unchanged. However, in Maf1-/- mice, pol III occupancy of the vast majority of active loci in liver and the levels of specific precursor tRNAs in this tissue and other organs are higher than wild-type in both fasted and refed conditions. Thus, MAF1 functions as a chronic repressor of active pol III loci and can modulate transcription under different conditions. Our findings support the futile RNA cycle hypothesis, elaborate the mechanism of pol III repression by MAF1 and demonstrate a modest effect of MAF1 on global translation via reduced mRNA levels and translation efficiencies for several ribosomal proteins.

scholarly journals Functional characterization of Polr3a hypomyelinating leukodystrophy mutations in the S. cerevisiae homolog, RPC160

2020 ◽  
Author(s):  
Robyn D. Moir ◽  
Christian Lavados ◽  
JaeHoon Lee ◽  
Ian M. Willis
Keyword(s):  
Functional Characterization ◽  
Rna Polymerase Iii ◽  
State Level ◽  
Temperature Sensitive ◽  
Missense Mutations ◽  
Wild Type ◽  
Functional Deficits ◽  
Pol Iii ◽  
Transcriptional Output

AbstractMutations in RNA polymerase III (Pol III) cause hypomeylinating leukodystrophy (HLD) and neurodegeneration in humans. POLR3A and POLR3B, the two largest Pol III subunits, together form the catalytic center and carry the majority of disease alleles. Disease-causing mutations include invariant and highly conserved residues that are predicted to negatively affect Pol III activity and decrease transcriptional output. A subset of HLD missense mutations in POLR3A cluster in the pore region that provides nucleotide access to the Pol III active site. These mutations were engineered at the corresponding positions in the Saccharomyces cerevisiae homolog, Rpc160, to evaluate their functional deficits. None of the mutations caused a growth or transcription phenotype in yeast. Each mutation was combined with a frequently occurring pore mutation, POLR3A G672E, which was also wild-type for growth and transcription. The double mutants showed a spectrum of phenotypes from wild-type to lethal, with only the least fit combinations showing an effect on Pol III transcription. In one slow-growing temperature-sensitive mutant the steady-state level of tRNAs was unaffected, however global tRNA synthesis was compromised, as was the synthesis of RPR1 and SNR52 RNAs. Affinity-purified mutant Pol III was broadly defective in both factor-independent and factor-dependent transcription in vitro across genes that represent the yeast Pol III transcriptome. Thus, the robustness of yeast to Pol III leukodystrophy mutations in RPC160 can be overcome by a combinatorial strategy.

TFIIIR is an isoleucine tRNA

1994 ◽  
Vol 14 (6) ◽  
pp. 3588-3595
Author(s):  
H M Dunstan ◽  
L S Young ◽  
K U Sprague
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Silk Gland ◽  
Class Iii ◽  
Nuclear Extracts ◽  
Hybrid Selection ◽  
Class Iii Genes ◽  
Selection Of

Promoter-specific transcription by silkworm RNA polymerase III is dependent on several transcription factors (TFs) in addition to the polymerase itself. The activities present in silk gland nuclear extracts that are necessary to reconstitute transcription from class III genes in vitro have been resolved into several partially purified components. These include TFIIIR, which is unusual because it is composed of RNA. Here, we identify the RNA that provides TFIIIR activity as silkworm tRNA(IleIAU). This conclusion is based on copurification of tRNA(IleIAU) with TFIIIR activity, TFIIIR activity in synthetic tRNA(Ile), and hybrid selection of TFIIIR activity by antisense tRNA(IleIAU). We have tested the ability of a variety of other tRNAs to stimulate transcription and find that TFIIIR activity is highly specific to silkworm tRNA(IleIAU).

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