scholarly journals Assembly of SNAPc, Bdp1, and TBP on the U6 snRNA Gene Promoter in Drosophila melanogaster

2020 ◽  
Vol 40 (12) ◽  
Author(s):  
Mun Kyoung Kim ◽  
An Tranvo ◽  
Ann Marie Hurlburt ◽  
Neha Verma ◽  
Phuc Phan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Drosophila Melanogaster ◽  
Rna Polymerase Iii ◽  
Gene Promoter ◽  
Stable Complex ◽  
Sequence Element ◽  
Content Type ◽  
U6 Snrna ◽  
U6 Promoter ◽  
Pol Iii

ABSTRACT U6 snRNA is transcribed by RNA polymerase III (Pol III) and has an external upstream promoter that consists of a TATA sequence recognized by the TBP subunit of the Pol III basal transcription factor IIIB and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). Previously, we found that Drosophila melanogaster SNAPc (DmSNAPc) bound to the U6 PSE can recruit the Pol III general transcription factor Bdp1 to form a stable complex with the DNA. Here, we show that DmSNAPc-Bdp1 can recruit TBP to the U6 promoter, and we identify a region of Bdp1 that is sufficient for TBP recruitment. Moreover, we find that this same region of Bdp1 cross-links to nucleotides within the U6 PSE at positions that also cross-link to DmSNAPc. Finally, cross-linking mass spectrometry reveals likely interactions of specific DmSNAPc subunits with Bdp1 and TBP. These data, together with previous findings, have allowed us to build a more comprehensive model of the DmSNAPc-Bdp1-TBP complex on the U6 promoter that includes nearly all of DmSNAPc, a portion of Bdp1, and the conserved region of TBP.

scholarly journals High-Level Activation of Transcription of the Yeast U6 snRNA Gene in Chromatin by the Basal RNA Polymerase III Transcription Factor TFIIIC

2004 ◽  
Vol 24 (9) ◽  
pp. 3596-3606 ◽  
Author(s):  
Sushma Shivaswamy ◽  
George A. Kassavetis ◽  
Purnima Bhargava
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Direct Role ◽  
U6 Snrna ◽  
Snrna Gene ◽  
Polymerase Iii ◽  
Activation Of Transcription ◽  
Pol Iii

ABSTRACT Transcription of the U6 snRNA gene (SNR6) in Saccharomyces cerevisiae by RNA polymerase III (pol III) requires TFIIIC and its box A and B binding sites. In contrast, TFIIIC has little or no effect on SNR6 transcription with purified components in vitro due to direct recognition of the SNR6 TATA box by TFIIIB. When SNR6 was assembled into chromatin in vitro by use of the Drosophila melanogaster S-190 extract, transcription of these templates with highly purified yeast pol III, TFIIIC, and TFIIIB displayed a near-absolute requirement for TFIIIC but yielded a 5- to 15-fold-higher level of transcription relative to naked DNA (>100-fold activation over repressed chromatin). Analysis of chromatin structure demonstrated that TFIIIC binding leads to remodeling of U6 gene chromatin, resulting in positioning of a nucleosome between boxes A and B. The resulting folding of the intervening DNA into the nucleosome could bring the suboptimally spaced SNR6 box A and B elements into greater proximity and thus facilitate activation of transcription. In the absence of ATP, however, the binding of TFIIIC to box B in chromatin was not accompanied by remodeling and the transcription activation was ∼35% of that seen in its presence, implying that both TFIIIC binding and ATP-dependent chromatin remodeling were required for the full activation of the gene. Our results suggest that TFIIIC, which is a basal transcription factor of pol III, also plays a direct role in remodeling chromatin on the SNR6 gene.

scholarly journals Parvovirus Expresses a Small Noncoding RNA That Plays an Essential Role in Virus Replication

2017 ◽  
Vol 91 (8) ◽  
Author(s):  
Zekun Wang ◽  
Weiran Shen ◽  
Fang Cheng ◽  
Xuefeng Deng ◽  
John F. Engelhardt ◽  
...  
Keyword(s):  
Dna Replication ◽  
Noncoding Rna ◽  
Rna Polymerase Iii ◽  
Human Bocavirus ◽  
Nonstructural Proteins ◽  
Viral Dna ◽  
Content Type ◽  
Small Noncoding Rna ◽  
Pol Iii ◽  
Tract Infections

ABSTRACT Human bocavirus 1 (HBoV1) belongs to the species Primate bocaparvovirus of the genus Bocaparvovirus of the Parvoviridae family. HBoV1 causes acute respiratory tract infections in young children and has a selective tropism for the apical surface of well-differentiated human airway epithelia (HAE). In this study, we identified an additional HBoV1 gene, bocavirus-transcribed small noncoding RNA (BocaSR), within the 3′ noncoding region (nucleotides [nt] 5199 to 5338) of the viral genome of positive sense. BocaSR is transcribed by RNA polymerase III (Pol III) from an intragenic promoter at levels similar to that of the capsid protein-coding mRNA and is essential for replication of the viral DNA in both transfected HEK293 and infected HAE cells. Mechanistically, we showed that BocaSR regulates the expression of HBoV1-encoded nonstructural proteins NS1, NS2, NS3, and NP1 but not NS4. BocaSR is similar to the adenovirus-associated type I (VAI) RNA in terms of both nucleotide sequence and secondary structure but differs from it in that its regulation of viral protein expression is independent of RNA-activated protein kinase (PKR) regulation. Notably, BocaSR accumulates in the viral DNA replication centers within the nucleus and likely plays a direct role in replication of the viral DNA. Our findings reveal BocaSR to be a novel viral noncoding RNA that coordinates the expression of viral proteins and regulates replication of viral DNA within the nucleus. Thus, BocaSR may be a target for antiviral therapies for HBoV and may also have utility in the production of recombinant HBoV vectors. IMPORTANCE Human bocavirus 1 (HBoV1) is pathogenic to humans, causing acute respiratory tract infections in young children. In this study, we identified a novel HBoV1 gene that lies in the 3′ noncoding region of the viral positive-sense genome and is transcribed by RNA polymerase III into a noncoding RNA of 140 nt. This bocavirus-transcribed small RNA (BocaSR) diverges from both adenovirus-associated (VA) RNAs and Epstein-Barr virus-encoded small RNAs (EBERs) with respect to RNA sequence, representing a third species of this kind of Pol III-dependent viral noncoding RNA and the first noncoding RNA identified in autonomous parvoviruses. Unlike the VA RNAs, BocaSR localizes to the viral DNA replication centers of the nucleus and is essential for expression of viral nonstructural proteins independent of RNA-activated protein kinase R and replication of HBoV1 genomes. The identification of BocaSR and its role in virus DNA replication reveals potential avenues for developing antiviral therapies.

scholarly journals Involvement of RNA Polymerase III in Immune Responses

2015 ◽  
Vol 35 (10) ◽  
pp. 1848-1859 ◽  
Author(s):  
Damian Graczyk ◽  
Robert J. White ◽  
Kevin M. Ryan
Keyword(s):  
Transcription Factor ◽  
Immune Responses ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Trna Genes ◽  
Malignant Cells ◽  
Polymerase Iii ◽  
Mouse Macrophages ◽  
Tightly Coupled ◽  
Pol Iii

Inflammation in the tumor microenvironment has many tumor-promoting effects. In particular, tumor-associated macrophages (TAMs) produce many cytokines which can support tumor growth by promoting survival of malignant cells, angiogenesis, and metastasis. Enhanced cytokine production by TAMs is tightly coupled with protein synthesis. In turn, translation of proteins depends on tRNAs, short abundant transcripts that are made by RNA polymerase III (Pol III). Here, we connect these facts by showing that stimulation of mouse macrophages with lipopolysaccharides (LPS) from the bacterial cell wall causes transcriptional upregulation of tRNA genes. The transcription factor NF-κB is a key transcription factor mediating inflammatory signals, and we report that LPS treatment causes an increased association of the NF-κB subunit p65 with tRNA genes. In addition, we show that p65 can directly associate with the Pol III transcription factor TFIIIB and that overexpression of p65 induces Pol III-dependent transcription. As a consequence of these effects, we show that inhibition of Pol III activity in macrophages restrains cytokine secretion and suppresses phagocytosis, two key functional characteristics of these cells. These findings therefore identify a radical new function for Pol III in the regulation of macrophage function which may be important for the immune responses associated with both normal and malignant cells.

scholarly journals Cell Cycle Kinase Polo Is Controlled by a Widespread 3′ Untranslated Region Regulatory Sequence inDrosophila melanogaster

2019 ◽  
Vol 39 (15) ◽  
Author(s):  
Marta S. Oliveira ◽  
Jaime Freitas ◽  
Pedro A. B. Pinto ◽  
Ana de Jesus ◽  
Joana Tavares ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Untranslated Region ◽  
Molecular Mechanisms ◽  
Alternative Polyadenylation ◽  
Upstream Sequence ◽  
Sequence Element ◽  
Content Type ◽  
Cell Cycle Kinase

ABSTRACTAlternative polyadenylation generates transcriptomic diversity, although the physiological impact and regulatory mechanisms involved are still poorly understood. The cell cycle kinase Polo is controlled by alternative polyadenylation in the 3′ untranslated region (3′UTR), with critical physiological consequences. Here, we characterized the molecular mechanisms required forpoloalternative polyadenylation. We identified a conserved upstream sequence element (USE) close to thepoloproximal poly(A) signal. Transgenic flies without this sequence show incorrect selection ofpolopoly(A) signals with consequent downregulation of Polo expression levels and insufficient/defective activation of Polo kinetochore targets Mps1 and Aurora B. Deletion of the USE results in abnormal mitoses in neuroblasts, revealing a role for this sequencein vivo. We found that Hephaestus binds to the USE RNA and thathephaestusmutants display defects inpoloalternative polyadenylation concomitant with a striking reduction in Polo protein levels, leading to mitotic errors and aneuploidy. Bioinformatic analyses show that the USE is preferentially localized upstream of noncanonical polyadenylation signals inDrosophila melanogastergenes. Taken together, our results revealed the molecular mechanisms involved inpoloalternative polyadenylation, with remarkable physiological functions in Polo expression and activity at the kinetochores, and disclosed a newin vivofunction for USEs inDrosophila melanogaster.

scholarly journals A carboxy-terminal basic region controls RNA polymerase III transcription factor activity of human La protein.

1997 ◽  
Vol 17 (10) ◽  
pp. 5823-5832 ◽  
Author(s):  
J L Goodier ◽  
H Fan ◽  
R J Maraia
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Binding ◽  
Rna Polymerase Iii ◽  
Transcription Factor Activity ◽  
Factor Activity ◽  
Terminal Domain ◽  
La Protein ◽  
Pol Iii ◽  
Basic Region

Human La protein has been shown to serve as a transcription factor for RNA polymerase III (pol III) by facilitating transcription termination and recycling of transcription complexes. In addition, La binds to the 3' oligo(U) ends common to all nascent pol III transcripts, and in the case of B1-Alu RNA, protects it from 3'-end processing (R. J. Maraia, D. J. Kenan, and J. D. Keene, Mol. Cell. Biol. 14:2147-2158, 1994). Others have previously dissected the La protein into an N-terminal domain that binds RNA and a C-terminal domain that does not. Here, deletion and substitution mutants of La were examined for general RNA binding, RNA 3'-end protection, and transcription factor activity. Although some La mutants altered in a C-terminal basic region bind RNA in mobility shift assays, they are defective in RNA 3'-end protection and do not support transcription, while one C-terminal substitution mutant is defective only in transcription. Moreover, a C-terminal fragment lacking RNA binding activity appears able to support low levels of transcription by pol III. While efficient multiround transcription is supported only by mutants that bind RNA and contain a C-terminal basic region. These analyses indicate that RNA binding contributes to but is not sufficient for La transcription factor activity and that the C-terminal domain plays a role in transcription that is distinguishable from simple RNA binding. The transcription factor activity of La can be reversibly inhibited by RNA, suggesting the potential for feedback inhibition of pol III transcription.

scholarly journals Distinct Mechanisms for Repression of RNA Polymerase III Transcription by the Retinoblastoma Tumor Suppressor Protein

2004 ◽  
Vol 24 (13) ◽  
pp. 5989-5999 ◽  
Author(s):  
Heather A. Hirsch ◽  
Gauri W. Jawdekar ◽  
Kang-Ae Lee ◽  
Liping Gu ◽  
R. William Henry
Keyword(s):  
Rna Polymerase ◽  
Gene Transcription ◽  
Transcriptional Repression ◽  
Rna Polymerase Iii ◽  
Control Cell ◽  
U6 Snrna ◽  
Retinoblastoma Tumor Suppressor ◽  
Polymerase Iii ◽  
U6 Promoter ◽  
Retinoblastoma Tumor

ABSTRACT The retinoblastoma (RB) protein represses global RNA polymerase III transcription of genes that encode nontranslated RNAs, potentially to control cell growth. However, RNA polymerase III-transcribed genes exhibit diverse promoter structures and factor requirements for transcription, and a universal mechanism explaining global repression is uncertain. We show that RB represses different classes of RNA polymerase III-transcribed genes via distinct mechanisms. Repression of human U6 snRNA (class 3) gene transcription occurs through stable promoter occupancy by RB, whereas repression of adenovirus VAI (class 2) gene transcription occurs in the absence of detectable RB-promoter association. Endogenous RB binds to a human U6 snRNA gene in both normal and cancer cells that maintain functional RB but not in HeLa cells whose RB function is disrupted by the papillomavirus E7 protein. Both U6 promoter association and transcriptional repression require the A/B pocket domain and C region of RB. These regions of RB contribute to U6 promoter targeting through numerous interactions with components of the U6 general transcription machinery, including SNAPC and TFIIIB. Importantly, RB also concurrently occupies a U6 promoter with RNA polymerase III during repression. These observations suggest a novel mechanism for RB function wherein RB can repress U6 transcription at critical steps subsequent to RNA polymerase III recruitment.

BRF1, a subunit of RNA polymerase III transcription factor TFIIIB, is essential for cell growth of Trypanosoma brucei

Parasitology ◽  
2015 ◽  
Vol 142 (13) ◽  
pp. 1563-1573 ◽  
Author(s):  
D. E. VÉLEZ-RAMÍREZ ◽  
L. E. FLORENCIO-MARTÍNEZ ◽  
G. ROMERO-MEZA ◽  
S. ROJAS-SÁNCHEZ ◽  
R. MORENO-CAMPOS ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Homology Modelling ◽  
Rna Polymerase Iii ◽  
Related Factor ◽  
Characteristic Structure ◽  
Rna Molecules ◽  
Polymerase Iii ◽  
Pol Iii

SUMMARYRNA polymerase III (Pol III) synthesizes small RNA molecules that are essential for cell viability. Accurate initiation of transcription by Pol III requires general transcription factor TFIIIB, which is composed of three subunits: TFIIB-related factor BRF1, TATA-binding protein and BDP1. Here we report the molecular characterization of BRF1 in Trypanosoma brucei (TbBRF1), a parasitic protozoa that shows distinctive transcription characteristics. In silico analysis allowed the detection in TbBRF1 of the three conserved domains located in the N-terminal region of all BRF1 orthologues, namely a zinc ribbon motif and two cyclin repeats. Homology modelling suggested that, similarly to other BRF1 and TFIIB proteins, the TbBRF1 cyclin repeats show the characteristic structure of five α-helices per repeat, connected by a short random-coiled linker. As expected for a transcription factor, TbBRF1 was localized in the nucleus. Knock-down of TbBRF1 by RNA interference (RNAi) showed that this protein is essential for the viability of procyclic forms of T. brucei, since ablation of TbBRF1 led to growth arrest of the parasites. Nuclear run-on and quantitative real-time PCR analyses demonstrated that transcription of all the Pol III-dependent genes analysed was reduced, at different levels, after RNAi induction.

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.

Analysis of transcription factors binding to the human 7SL RNA gene promoter

1999 ◽  
Vol 77 (5) ◽  
pp. 431-438 ◽  
Author(s):  
Jürgen Müller ◽  
Bernd-Joachim Benecke
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Gene Promoter ◽  
7Sl Rna ◽  
Polymerase Iii ◽  
Responsive Element

Transcription of the human 7SL RNA gene by RNA polymerase III depends on the concerted action of transcription factors binding to the gene-internal and gene-external parts of its promoter. Here, we investigated which transcription factors interact with the human 7SL RNA gene promoter and which are required for transcription of the human 7SL RNA gene. A-box/B-box elements were previously identified in 5S RNA, tRNA, and virus associated RNA genes and are recognized by transcription factor IIIC (TFIIIC). The gene-internal promoter region of the human 7SL RNA gene shows only limited similarity to those elements. Nevertheless, competition experiments and the use of highly enriched factor preparations demonstrate that TFIIIC is required for human 7SL transcription. The gene-external part of the promoter includes an authentic cAMP-responsive element previously identified in various RNA polymerase II promoters. Here we demonstrate that members of the activating transcription factor/cyclic AMP-responsive element binding protein (ATF/CREB) transcription factor family bind specifically to this element in vitro. However, the human 7SL RNA gene is not regulated by cAMP in vivo. Furthermore, in vitro transcription of the gene does not depend on ATF/CREB transcription factors. It rather appears that a transcription factor with DNA-binding characteristics like ATF/CREB proteins but otherwise different properties is required for human 7SL RNA transcription.Key words: 7SL RNA, ATF, CRE, TFIIIC, RNA polymerase III.

scholarly journals A post-recruitment function for the RNA polymerase III transcription–initiation factor IIIB

1998 ◽  
Vol 95 (16) ◽  
pp. 9196-9201 ◽  
Author(s):  
George A. Kassavetis ◽  
Ashok Kumar ◽  
Garth A. Letts ◽  
E. Peter Geiduschek
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Transcriptional Start Site ◽  
Rna Polymerase Iii ◽  
Active Role ◽  
Normal Function ◽  
Initiation Factor ◽  
Transcriptional Initiation ◽  
Pol Iii

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.

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