scholarly journals A New Strategy of RNA Interference That Targets Heterologous Sequences Reveals CITFA1 as an Essential Component of Class I Transcription Factor A in Trypanosoma brucei

2014 ◽  
Vol 13 (6) ◽  
pp. 785-795 ◽  
Author(s):  
Sung Hee Park ◽  
Bao N. Nguyen ◽  
Justin K. Kirkham ◽  
Tu N. Nguyen ◽  
Arthur Günzl
Keyword(s):  
Transcription Factor ◽  
Rna Interference ◽  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Transcription Initiation ◽  
Single Copy ◽  
Rna Polymerase I ◽  
Class I ◽  
Content Type ◽  
Polymerase I

ABSTRACTConditional gene silencing by RNA interference inTrypanosoma bruceican be inconclusive if knockdowns are inefficient or have off-target effects. To enable efficient, specific silencing of single-copy genes in mammalian-infective, bloodstream form trypanosomes, we developed a system that targets the heterologous and functionalTrypanosoma cruziU2AF353′ untranslated region (UTR) (Tc3) or, alternatively, the sequence of the PTP tag, which can be fused to any mRNA of interest. Two cell lines were created, single-marker Tc3 (smTc3) and smPTP, which conditionally express Tc3 and PTP double-stranded RNA (dsRNA), respectively. The system depends on manipulating both alleles of the gene of interest so that cells exclusively express the target mRNA as a fusion to one of these heterologous sequences. We generated allele integration vectors in which the C-terminal part of a gene's coding sequence can be fused to either heterologous sequence in a single cloning step. We first tested this system withCITFA7, which encodes a well-characterized subunit of the class I transcription factor A (CITFA), an essential factor for transcription initiation by RNA polymerase I. Targeting either Tc3 or PTP fused to theCITFA7mRNA resulted in gene knockdowns that were as efficient and specific as targeting the endogenousCITFA7mRNA. Moreover, application of this system toCITFA1, which could not be silenced by established methods, demonstrated that the gene encodes an essential CITFA subunit that mediates binding of the transcription factor complex to RNA polymerase I promoters.

scholarly journals Characterization of a Novel Class I Transcription Factor A (CITFA) Subunit That Is Indispensable for Transcription by the Multifunctional RNA Polymerase I of Trypanosoma brucei

2012 ◽  
Vol 11 (12) ◽  
pp. 1573-1581 ◽  
Author(s):  
Tu N. Nguyen ◽  
Bao N. Nguyen ◽  
Ju Huck Lee ◽  
Aswini K. Panigrahi ◽  
Arthur Günzl
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Rna Polymerase I ◽  
Class I ◽  
Mammalian Host ◽  
Subunit Structure ◽  
Content Type ◽  
Pol I ◽  
Polymerase I

ABSTRACT Trypanosoma brucei is the only organism known to have evolved a multifunctional RNA polymerase I (pol I) system that is used to express the parasite's ribosomal RNAs, as well as its major cell surface antigens, namely, the variant surface glycoprotein (VSG) and procyclin, which are vital for establishing successful infections in the mammalian host and the tsetse vector, respectively. Thus far, biochemical analyses of the T. brucei RNA pol I transcription machinery have elucidated the subunit structure of the enzyme and identified the class I transcription factor A (CITFA). CITFA binds to RNA pol I promoters, and its CITFA-2 subunit was shown to be absolutely essential for RNA pol I transcription in the parasite. Tandem affinity purification (TAP) of CITFA revealed the subunits CITFA-1 to -6, which are conserved only among kinetoplastid organisms, plus the dynein light chain DYNLL1. Here, by tagging CITFA-6 instead of CITFA-2, a complex was purified that contained all known CITFA subunits, as well as a novel proline-rich protein. Functional studies carried out in vivo and in vitro , as well as a colocalization study, unequivocally demonstrated that this protein is a bona fide CITFA subunit, essential for parasite viability and indispensable for RNA pol I transcription of ribosomal gene units and the active VSG expression site in the mammalian-infective life cycle stage of the parasite. Interestingly, CITFA-7 function appears to be species specific, because expression of an RNA interference (RNAi)-resistant CITFA-7 transgene from Trypanosoma cruzi could not rescue the lethal phenotype of silencing endogenous CITFA-7 .

scholarly journals Promoter occupancy of the basal class I transcription factor A differs strongly between active and silent VSG expression sites in Trypanosoma brucei

2013 ◽  
Vol 42 (5) ◽  
pp. 3164-3176 ◽  
Author(s):  
Tu N. Nguyen ◽  
Laura S. M. Müller ◽  
Sung Hee Park ◽  
T. Nicolai Siegel ◽  
Arthur Günzl
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Transcription Initiation ◽  
Rna Polymerase I ◽  
Surface Glycoprotein ◽  
Class I ◽  
Monoallelic Expression ◽  
Promoter Occupancy ◽  
Polymerase I

Abstract Monoallelic expression within a gene family is found in pathogens exhibiting antigenic variation and in mammalian olfactory neurons. Trypanosoma brucei, a lethal parasite living in the human bloodstream, expresses variant surface glycoprotein (VSG) from 1 of 15 bloodstream expression sites (BESs) by virtue of a multifunctional RNA polymerase I. The active BES is transcribed in an extranucleolar compartment termed the expression site body (ESB), whereas silent BESs, located elsewhere within the nucleus, are repressed epigenetically. The regulatory mechanisms, however, are poorly understood. Here we show that two essential subunits of the basal class I transcription factor A (CITFA) predominantly occupied the promoter of the active BES relative to that of a silent BES, a phenotype that was maintained after switching BESs in situ. In these experiments, high promoter occupancy of CITFA was coupled to high levels of both promoter-proximal RNA abundance and RNA polymerase I occupancy. Accordingly, fluorescently tagged CITFA-7 was concentrated in the nucleolus and the ESB. Because a ChIP-seq analysis found that along the entire BES, CITFA-7 is specifically enriched only at the promoter, our data strongly indicate that monoallelic BES transcription is activated by a mechanism that functions at the level of transcription initiation.

scholarly journals The Transcription Factor THO Promotes Transcription Initiation and Elongation by RNA Polymerase I

2015 ◽  
Vol 291 (6) ◽  
pp. 3010-3018 ◽  
Author(s):  
Yinfeng Zhang ◽  
Sarah L. French ◽  
Ann L. Beyer ◽  
David A. Schneider
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Rna Polymerase I ◽  
Polymerase I

Events during eucaryotic rRNA transcription initiation and elongation: conversion from the closed to the open promoter complex requires nucleotide substrates

1988 ◽  
Vol 8 (5) ◽  
pp. 1940-1946
Author(s):  
E Bateman ◽  
M R Paule
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Topological Analysis ◽  
Acanthamoeba Castellanii ◽  
Rna Polymerase I ◽  
Initiation Factor ◽  
Rrna Transcription ◽  
Promoter Complex ◽  
Polymerase I

Chemical footprinting and topological analysis were carried out on the Acanthamoeba castellanii rRNA transcription initiation factor (TIF) and RNA polymerase I complexes with DNA during transcription initiation and elongation. The results show that the binding of TIF and polymerase to the promoter does not alter the supercoiling of the DNA template and the template does not become sensitive to modification by diethylpyrocarbonate, which can identify melted DNA regions. Thus, in contrast to bacterial RNA polymerase, the eucaryotic RNA polymerase I-promoter complex is in a closed configuration preceding addition of nucleotides in vitro. Initiation and 3'-O-methyl CTP-limited translocation by RNA polymerase I results in separation of the polymerase-TIF footprints, leaving the TIF footprint unaltered. In contrast, initiation and translocation result in a significant change in the conformation of the polymerase-DNA complex, culminating in an unwound DNA region of at least 10 base pairs.

scholarly journals Molecular Topology of RNA Polymerase I Upstream Activation Factor

2020 ◽  
Vol 40 (13) ◽  
Author(s):  
Bruce A. Knutson ◽  
Marissa L. Smith ◽  
Alana E. Belkevich ◽  
Aula M. Fakhouri
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Dual Function ◽  
Molecular Topology ◽  
Content Type ◽  
Histone Fold ◽  
Topological Features ◽  
Pol I ◽  
Activation Factor ◽  
Polymerase I

ABSTRACT Upstream activation factor (UAF) is a multifunctional transcription factor in Saccharomyces cerevisiae that plays dual roles in activating RNA polymerase I (Pol I) transcription and repression of Pol II. For Pol I, UAF binds to a specific upstream element in the ribosomal DNA (rDNA) promoter and interacts with two other Pol I initiation factors, the TATA-binding protein (TBP) and core factor (CF). We used an integrated combination of chemical cross-linking mass spectrometry (CXMS), molecular genetics, protein biochemistry, and structural modeling to understand the topological framework responsible for UAF complex formation. Here, we report the molecular topology of the UAF complex, describe new structural and functional domains that play roles in UAF complex integrity, assembly, and biological function, and provide roles for previously identified UAF domains that include the Rrn5 SANT and histone fold domains. We highlight the role of new domains in Uaf30 that include an N-terminal winged helix domain and a disordered tethering domain as well as a BORCS6-like domain found in Rrn9. Together, our results reveal a unique network of topological features that coalesce around a histone tetramer-like core to form the dual-function UAF complex.

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