Functional Dissection of the B″ Component of RNA Polymerase III Transcription Factor IIIB: a Scaffolding Protein with Multiple Roles in Assembly and Initiation of Transcription

Kumar, Ashok; Kassavetis, George A.; Geiduschek, E. Peter; Hambalko, M; Brent, C J

doi:10.1128/mcb.17.4.1868

Cited by 52 publications

(112 citation statements)

References 55 publications

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“…Bdp1 is essential for transcription by pol III in vitro, but no single region of Bdp1 is required for TFIIIC-independent transcription of SNR6 as supercoiled DNA (13). Much of Bdp1 can be deleted with retention of yeast viability (the N-terminal 240 amino acids, the C-terminal 108 amino acids, 68 amino acids between residues 312 and 372, and 17 amino acids between 253 and 269), leaving just 161 Bdp1 residues that are essential (26,28).…”

Section: Discussionmentioning

confidence: 99%

Mapping the Principal Interaction Site of the Brf1 and Bdp1 Subunits of Saccharomyces cerevisiae TFIIIB

Kassavetis

Driscoll²,

Geiduschek

2006

Journal of Biological Chemistry

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The Brf1 subunit of the central RNA polymerase (pol) III transcription initiation factor TFIIIB is bipartite; its N-terminal TFIIBrelated half is principally responsible for recruiting pol III to the promoter and for promoter opening near the transcriptional start site, whereas its pol III-specific C-terminal half contributes most of the affinities that hold the three subunits of TFIIIB together. Here, the principal attachment site of Brf1 for the Bdp1 subunit of TFIIIB has been mapped by a combination of structure-informed, site-directed mutagenesis and photochemical protein-DNA cross-linking. A 66-amino acid segment of Brf1 is shown to serve as a two-sided adhesive surface, with the side chains projecting away from its extended interface with TATA-binding protein anchoring Bdp1 binding. An extensive collection of N-terminal, C-terminal, and internal deletion proteins has been used to demarcate the interacting Bdp1 domain to a 66-amino acid segment that includes the SANT domain of this subunit and is phylogenetically the most conserved region of Bdp1.The RNA polymerase (pol) 3 III transcription apparatus of yeast comprises, in addition to the polymerase itself, three transcription initiation factors, TFIIIA, -B, and -C.The three-subunit TFIIIB is the core initiation factor required and sufficient for recruiting pol III to the promoter and accurately initiating transcription. The large six-subunit TFIIIC places TFIIIB on DNA upstream of the transcriptional start site and protects the generally very small pol III transcription units from encroachment by repressive chromatin (1, 2). TFIIIA is the 5 S rRNA gene-specific factor that places TFIIIC on the transcription unit-internal promoter of these genes (reviewed in Refs. 3-5).The work that is presented here deals with the structure of TFIIIB, which is composed of three subunits: the TATA-binding protein (TBP), Brf1, and Bdp1. TBP and Brf1 bind tightly and co-purify, whereas Bdp1 dissociates during later steps of conventional column chromatography. (The two separated fractions of TFIIIB have been designated BЈ and BЉ, and B double prime has been adopted as the eponym of the Bdp1 subunit.) The 596-amino acid Brf1 is a fusion protein, with an N-terminal half that is related to the pol II transcription initiation factor TFIIB and a pol III-specific C-terminal half. A conserved segment of the C-terminal half (homology domain II; amino acids 439 -515) constitutes the Brf1 high affinity binding site for the evolutionarily highly conserved core of TBP (comprising all but the N-terminal 60 amino acids of the Saccharomyces cerevisiae protein); the N-terminal half of Brf1 engages TBP through a separate lower affinity interaction.The N-and C-terminal halves of Brf1 also provide separate Bdp1-docking sites, with the C-proximal homology domain II contributing the higher Bdp1 affinity. TBP makes only a minor direct contribution to bringing Bdp1 into TFIIIB. Thus, the pol III-specific half of Brf1 appears to contribute the interactions that principally hold TFIIIB together. Ind...

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Section: Discussionmentioning

confidence: 99%

Mapping the Principal Interaction Site of the Brf1 and Bdp1 Subunits of Saccharomyces cerevisiae TFIIIB

Kassavetis

Driscoll²,

Geiduschek

2006

Journal of Biological Chemistry

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“…Addition of Bdp1p to the TFIIIB complex increases complex stability (Kassavetis et al 1990) and extends its footprint on DNA, likely due to Bdp1p's ability to remodel the complex by changing the configuration of Brf1p on DNA (Kumar et al 1997;Shah et al 1999). The Bdp1p itself is reorganized upon DNA binding, with subsequent increase in accessibility of residues 190-210 to hydroxy radical cleavage (Kumar et al 1997). Binding of Bdp1p is essential to promoter opening, which is likely to coordinate with its induction of a major bend (135°) in the DNA (Kassavetis et al 1998;Grove et al 1999).…”

Section: Ty1 Is a Long Terminal Repeat (Ltr) Retrotransposon Inmentioning

confidence: 99%

“…Binding of Bdp1p is essential to promoter opening, which is likely to coordinate with its induction of a major bend (135°) in the DNA (Kassavetis et al 1998;Grove et al 1999). More than half of Bdp1p can be removed by deletion without obvious adverse consequences on viability, transcription activity in vitro, or the DNase I protection footprint of the TFIIIB complex (Kumar et al 1997).…”

Section: Ty1 Is a Long Terminal Repeat (Ltr) Retrotransposon Inmentioning

confidence: 99%

“…Transcription initiation begins with stable binding of TFIIIC to the internal promoter B box, followed by binding of the subunits of TFIIIB: Brf1p, TBP, and Bdp1p (for review, see Geiduschek and Kassavetis 2001). Addition of Bdp1p to the TFIIIB complex increases complex stability (Kassavetis et al 1990) and extends its footprint on DNA, likely due to Bdp1p's ability to remodel the complex by changing the configuration of Brf1p on DNA (Kumar et al 1997;Shah et al 1999). The Bdp1p itself is reorganized upon DNA binding, with subsequent increase in accessibility of residues 190-210 to hydroxy radical cleavage (Kumar et al 1997).…”

Section: Ty1 Is a Long Terminal Repeat (Ltr) Retrotransposon Inmentioning

confidence: 99%

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TFIIIB subunit Bdp1p is required for periodic integration of the Ty1 retrotransposon and targeting of Isw2p to S. cerevisiae tDNAs

Bachman¹,

Gelbart²,

Tsukiyama³

et al. 2005

Genes Dev.

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Retrotransposons are RNA elements that reverse transcribe their RNA genomes and make a cDNA copy that is inserted back into a new genomic location by the element-encoded integrase protein. Ty1 is a long terminal repeat (LTR) retrotransposon in Saccharomyces cerevisiae that inserts into an ∼700-bp integration window upstream of tRNA genes with a periodicity of ∼80 bp. ATP-dependent chromatin remodeling by Isw2 upstream of tRNA genes leads to changes in chromatin structure and Ty1 integration site selection. We show that the N terminus of Bdp1p, a component of the RNA polymerase III transcription factor TFIIIB, is required for periodic integration of Ty1 into the integration window. Deletion of the Bdp1p N terminus and mutation of ISW2 result in similar disruption of nucleosome positioning upstream of some tRNA genes, and the N-terminal domain of Bdp1p is required for targeting of Isw2 complex to tRNA genes. This study provides the first example for recruitment of an ATP-dependent chromatin-remodeling factor by a general transcription factor in vivo.[Keywords: RNA polymerase III; chromatin; integrase; retrotransposon; transcription factor; tRNA gene] Supplemental material is available at http://www.genesdev.org.

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“…Ref. 10); 2) replacement of TFIIIB with recombinant subunits elevates bp ϩ4 and ϩ8 initiation events to the level at bp ϩ1 (13,14). A crude Bdp1 fraction was also found to contain an excess of a factor or factors that re-establishes the fidelity of initiation at bp ϩ1 on the SUP4 gene with recombinant TFIIIB.…”

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confidence: 99%

Nhp6 Is a Transcriptional Initiation Fidelity Factor for RNA Polymerase III Transcription in Vitro and in Vivo

Kassavetis

Steiner

2006

Journal of Biological Chemistry

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The binding of the RNA polymerase III (pol III) transcription factor TFIIIC to the box A intragenic promoter element of tRNA genes specifies the placement of TFIIIB on upstream-lying DNA. In turn, TFIIIB recruits pol III to the promoter and specifies transcription initiating 17-19 base pairs upstream of box A. The resolution of the pol III transcription apparatus into recombinant TFIIIB, highly purified TFIIIC, and pol III is accompanied by a loss of precision in specifying where transcription initiation occurs due to heterogeneous placement of TFIIIB. In this paper we show that Nhp6a, an abundant high mobility group B (HMGB) family, non-sequencespecific DNA-binding protein in Saccharomyces cerevisiae restores transcriptional initiation fidelity to this highly purified in vitro system. Restoration of initiation fidelity requires the presence of Nhp6a prior to TFIIIB-DNA complex formation. Chemical nuclease footprinting of TFIIIC-and TFIIIB-TFIIIC-DNA complexes reveals that Nhp6a markedly alters the TFIIIC footprint over box A and reduces the size of the TFIIIB footprint on upstream DNA sequence. Analyses of unprocessed tRNAs from yeast lacking Nhp6a and its closely related paralogue Nhp6b demonstrate that Nhp6 is required for transcriptional initiation fidelity of some but not all tRNA genes, in vivo.The sites at which RNA polymerase III (pol III) 2 initiates tRNA gene transcription are specified by sequence-specific interactions of its two transcription factors, TFIIIC and TFIIIB, and by pol III itself. TFIIIC is a 520-kDa protein, containing six distinct subunits arranged into two globular subdomains, A (subunits Tfc1, Tfc4, and Tfc7) and B (subunits Tfc3, Tfc6, and Tfc8), which are connected by a flexible linker (Tfc8; see Refs. 1-3 for reviews). TFIIIC binds to two intragenic sequence elements, the start site-proximal box A (binding A) and the distal box B (binding B), which are optimally separated by 30 -60 bp. The B-box B interaction contributes nearly all of the affinity of TFIIIC for tRNA genes (K D Ͻ1 nM) (4). Nevertheless, it is the weak A-box A interaction that primarily specifies initiation 17-19 bp upstream of box A (5, 6). Specification is indirect; the A-box A interaction determines the placement of TFIIIB, which, in turn, recruits pol III. TFIIIB placement is mediated through an interaction between Tfc4 and the Brf1 subunit of TFIIIB. Brf1 forms a stable complex with TBP, and the major DNA contacts during TFIIIC-dependent assembly of TFIIIB upstream of the start site of transcription are mediated by TBP. Consistent with this role of TBP, the DNA segments occupied by TFIIIB (ϳbp Ϫ40 to bp Ϫ10, relative to predicted start sites of transcription) are uniformly AT-rich (Ͼ70% (7-9)).The notion that preferential sequence selection by TBP can occur during TFIIIC-dependent assembly of TFIIIB comes from in vivo and in vitro studies in which a shift in placement of a TATA box or partial TATA box shifts the start site accordingly but retains dependence on TFIIIC for transcription (10, 11). Pol III also cont...

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Functional Dissection of the B″ Component of RNA Polymerase III Transcription Factor IIIB: a Scaffolding Protein with Multiple Roles in Assembly and Initiation of Transcription

Cited by 52 publications

References 55 publications

Mapping the Principal Interaction Site of the Brf1 and Bdp1 Subunits of Saccharomyces cerevisiae TFIIIB

Mapping the Principal Interaction Site of the Brf1 and Bdp1 Subunits of Saccharomyces cerevisiae TFIIIB

TFIIIB subunit Bdp1p is required for periodic integration of the Ty1 retrotransposon and targeting of Isw2p to S. cerevisiae tDNAs

Nhp6 Is a Transcriptional Initiation Fidelity Factor for RNA Polymerase III Transcription in Vitro and in Vivo

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