A selection for mutants of the RNA polymerase III transcription apparatus: PCF1 stimulates transcription of tRNA and 5S RNA genes.

Willis, Ian M.; Schmidt, Petra; Söll, Dieter

doi:10.1002/j.1460-2075.1989.tb08614.x

Cited by 32 publications

(26 citation statements)

References 36 publications

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“…Multicopy plasmids pRS423TBP, pRS423B70 (BRF1), and pRS423B90 (BDP1) were gifts from I. Willis (54). pRS423 PCF1 and pRS423 PCF1-1 were derived from pRS313 PCF1 and pRS313 PCF1-1 (50,66), also from I. Willis, by transfer between the two PvuI sites of pRS423.…”

Section: Methodsmentioning

confidence: 99%

Essential Roles of Bdp1, a Subunit of RNA Polymerase III Initiation Factor TFIIIB, in Transcription and tRNA Processing

Ishiguro

Kassavetis

Geiduschek

2002

Molecular and Cellular Biology

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The essential Saccharomyces cerevisiae gene BDP1 encodes a subunit of RNA polymerase III (Pol III) transcription factor (TFIIIB); TATA box binding protein (TBP) and Brf1 are the other subunits of this three-protein complex. Deletion analysis defined three segments of Bdp1 that are essential for viability. A central segment, comprising amino acids 327 to 353, was found to be dispensable, and cells making Bdp1 that was split within this segment, at amino acid 352, are viable. Suppression of bdp1 conditional viability by overexpressing SPT15 and BRF1 identified functional interactions of specific Bdp1 segments with TBP and Brf1, respectively. A Bdp1 deletion near essential segment I was synthetically lethal with overexpression of PCF1-1, a dominant gain-of-function mutation in the second tetracopeptide repeat motif (out of 11) of the Tfc4 ( 131 ) subunit of TFIIIC. The analysis also identifies a connection between Bdp1 and posttranscriptional processing of Pol III transcripts. Yeast genomic library screening identified RPR1 as the specific overexpression suppressor of very slow growth at 37°C due to deletion of Bdp1 amino acids 253 to 269. RPR1 RNA, a Pol III transcript, is the RNA subunit of RNase P, which trims pre-tRNA transcript 5 ends. Maturation of tRNA was found to be aberrant in bdp1-⌬253-269 cells, and RPR1 transcription with the highly resolved Pol III transcription system in vitro was also diminished when recombinant Bdp1⌬253-269 replaced wild-type Bdp1. Physical interaction of RNase P with Bdp1 was demonstrated by coimmunoprecipitation and pull-down assays.RNA polymerase III (Pol III) transcribes genes encoding tRNAs, 5S rRNA, U6 snRNA, and other small RNAs. Accurate initiation of this transcription requires basal transcription factor IIIA (TFIIIA), TFIIIB, and TFIIIC. In the yeast Saccharomyces cerevisiae, TFIIIB is required for all Pol III transcription in vitro and in vivo, TFIIIC is required for all Pol III transcription in vivo, and TFIIIA is required only for 5S rRNA gene transcription. TFIIIB is composed of three subunits, TATA-binding protein (TBP), Brf1 (the TFIIB-related factor), and Bdp1; all three are also essential for Pol III transcription in vitro (7,12,14,21,27,28,33,40,51,52,65). In vivo analysis defines multiple interactions of TFIIIB with the rest of the S. cerevisiae Pol III transcription machinery: Brf1 and Bdp1 interact with Tfc4 (the second largest subunit of TFIIIC), and Brf1 also interacts with the RPC34 and RPC17 subunits of Pol III (3,5,19,20,39,47,52,64).In human cells, two TFIIIB-related assemblies have been identified (46,60). TFIIIB␣, which contains TBP, Bdp1 (previously called hTFIIIB150), and Brf2 (a hBrf1 paralogue previously called hTFIIIB50), is required for transcription of Pol III genes with upstream promoter elements, such as 7SK and U6 (53, 61). TFIIIB␤, containing TBP, Bdp1, and Brf1, is required for transcription of genes with internal promoters (53). Alternatively spliced variants of hBrf1 have also been noted (44). Human TFIIIB interacts with a subcomplex of P...

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Section: Methodsmentioning

confidence: 99%

Essential Roles of Bdp1, a Subunit of RNA Polymerase III Initiation Factor TFIIIB, in Transcription and tRNA Processing

Ishiguro

Kassavetis

Geiduschek

2002

Molecular and Cellular Biology

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“…PCF1-1, PCF1-2, and PCF1-23 were cloned from genomic libraries of mutant strains which showed increased expression of the Pol III reporter gene sup9-e A19-supS1 (32,39). Unique dominant mutations were confirmed by sequencing the entire gene.…”

Section: Methodsmentioning

confidence: 99%

“…This limiting step is defined by a dominant mutation, PCF1-1, which increases Pol III transcription in vivo and in vitro (39). The PCF1-1 mutation causes a histidine-totyrosine change in the second of 11 tetratricopeptide repeats (TPRs) found in TFIIIC 131 (32) (Fig.…”

mentioning

confidence: 99%

A Tetratricopeptide Repeat Mutation in Yeast Transcription Factor IIIC₁₃₁ (TFIIIC₁₃₁) Facilitates Recruitment of Tfiib-Related Factor TFIIIB₇₀

Moir

Sethy-Coraci

Puglia

et al. 1997

Molecular and Cellular Biology

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Transcription factor IIIC (TFIIIC) plays an important role in assembling the initiation factor TFIIIB on genes transcribed by RNA polymerase III (Pol III). In Saccharomyces cerevisiae, assembly of the TFIIIB complex by promoter-bound TFIIIC is thought to be initiated by its tetratricopeptide repeat (TPR)-containing subunit, TFIIIC 131 , which interacts directly with the TFIIB-related factor, TFIIIB 70 /Brf1. In this work, we have identified 10 dominant mutations in TFIIIC 131 that increase Pol III gene transcription. All of these mutations are found within a discrete 53-amino-acid region of the protein encompassing TPR2. Biochemical studies of one of the mutations (PCF1-2) show that the increase in transcription is due to an increase in the recruitment of TFIIIB 70 to TFIIIC-DNA. The PCF1-2 mutation does not affect the affinity of TFIIIC for DNA, and the differential in both transcription and TFIIIB complex assembly is observed at saturating levels of TFIIIB 70 . This indicates that mutant and wild-type TFIIIC-DNA complexes have the same affinity for TFIIIB 70 and suggests that the increased recruitment of this factor is achieved by a nonequilibrium binding mechanism. A novel mechanism of activation in which the TPR mutations facilitate a conformational change in TFIIIC that is required for TFIIIB 70 binding is proposed. The implications of this model for the regulation of processes involving TPR proteins are discussed.The multisubunit transcription factor IIIC (TFIIIC) is an assembly factor that directs the binding of the general initiation factor, TFIIIB, upstream of the transcription start site of RNA polymerase III (Pol III) genes (21, 37). On tRNA gene templates, TFIIIC interacts with intragenic A and B block promoter elements. High-affinity binding of TFIIIC to DNA is mediated by the B block. This element is positioned at variable distances from the start site and, in at least one example, can function, albeit less efficiently, in an inverted orientation (5). These properties, together with the requirement for TFIIIC in transcription from chromatin templates (antirepression), provide a conceptual view of TFIIIC as an enhancer binding factor (5, 6). TFIIIC, through interactions with the A block, also plays a role in start site selection. Typically, the start site is located about 20 bp upstream of the A block. However, its position can be shifted within a 20-to 30-bp window by altering the placement of TFIIIB on the DNA (7,10,12,15). Redirection of TFIIIB involves its TATA-binding protein (TBP) subunit, in addition to TFIIIC, and can be achieved by inserting, repositioning, or mutating an upstream TATA element. The conformational flexibility required for this variable placement of TFIIIB has been suggested to reside in the TFIIIB-assembling subunit of TFIIIC, TFIIIC 131 (15).Specific transcription by Pol III can be achieved in the absence of TFIIIC on nucleosome-free templates which contain a suitable TATA box and an initiation site. Transcription of the Saccharomyces cerevisiae U6 gene, which contains the ...

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“…Expression of the sup9-e A19-supS1 reporter gene was monitored at 30°C by the suppression of auxotrophies in tryptophan and methionine biosynthesis. Since growth differences were not apparent under non-selective conditions at this temperature, differences observed on Trp-Met-medium reflect changes in sup9-e A19-supS1 transcription (34). One mutation, I515K at position 14 in TPR8, conferred a modest increase in suppressor activity implying either the loss of an inhibitory interaction or the acquisition of a positive interaction.…”

Section: Amino Acid Substitutions At Phylogenetically Conserved Positmentioning

confidence: 90%

“…The resulting mutant genes were plasmid shuffled into S. cerevisiae (strain supAC1ϩ, Ref. 14) and evaluated for growth at several temperatures on synthetic complete medium and for their ability to express the pol III reporter gene, sup9-e A19-supS1 (34). None of the mutations were lethal at 30°C since the URA3-marked wild-type rescuing plasmid could be successfully evicted on 5-FOA-containing medium.…”

Section: Amino Acid Substitutions At Phylogenetically Conserved Positmentioning

confidence: 99%

The Brf1 and Bdp1 Subunits of Transcription Factor TFIIIB Bind to Overlapping Sites in the Tetratricopeptide Repeats of Tfc4

Liao

Willis

Moir

2003

Journal of Biological Chemistry

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The RNA polymerase III initiation factor TFIIIB is assembled onto DNA through interactions involving the Tfc4 subunit of the assembly factor TFIIIC and two subunits of TFIIIB, Brf1 and Bdp1. Tfc4 contains two arrays of tetratricopeptide repeats (TPRs), each of which provides a binding site for Brf1. Dominant mutations in the ligand binding channel of the first TPR array, TPRs1-5, and on the back side of this array, increase Brf1 binding by Tfc4. Here we examine the biological importance of the second TPR array, TPRs6 -9. Radical mutations at phylogenetically conserved residues in the ligand binding channel of TPRs6 -9 impair pol III reporter gene transcription. Biochemical studies on one such mutation, L469K in TPR7, revealed a defect in the recruitment of Brf1 into TFIIIB-TFIIIC-DNA complexes and diminished the direct interaction between Tfc4 and Brf1. Multicopy suppression analysis implicates TPR9 in Brf1 binding and TPRs7 and 8 in binding to more than one ligand. Indeed, the L469K mutation also decreased the binding affinity for Bdp1 incorporation into TFIIIB-TFIIIC-DNA complexes and inhibited binary interactions between Bdp1 and Tfc4. The Bdp1 binding domain in Tfc4 was mapped to TPRs1-9, a domain that contains both TPR arrays and thus overlaps two of the known binding sites for Brf1. The properties of the L469K mutation identify both Brf1 and Bdp1 as ligands for the second TPR array.The assembly of the initiation factor TFIIIB by TFIIIC is a limiting step in RNA polymerase III (pol III) 1 transcription. Although multiple subunits of both TFIIIB and TFIIIC interact (1-4) TFIIIB assembly is mediated initially by protein-protein interactions between the TFIIIC subunit, Tfc4, and the TFIIIB subunit, Brf1. Biochemical and genetic studies in yeast indicate that the recruitment of Brf1 by TFIIIC is a major limiting step in preinitiation complex assembly (5-7). Subsequent binding of the TBP and Bdp1 subunits of TFIIIB generates a series of structural changes in Tfc4, Brf1 and DNA that leads to progressively increased accessibility of Brf1 to DNA (8). The protein-protein interactions between TFIIIB subunits, together with DNA bending, generate a highly stable TFIIIB complex, a structure that surrounds and kinetically traps the DNA (9 -12).Tfc4 contains eleven copies of a ubiquitous protein-protein interaction motif known as a tetratricopeptide repeat (13,14).TPR motifs, as the name implies, are typically 34 amino acids in length and fold into two antiparallel ␣-helices (designated A and B, Ref. 15). Although no position within the motif is invariant, a pattern of small and large hydrophobic residues defines the loose TPR consensus and provides stacking interactions between adjacent repeats (15-17). Multiple adjacent repeats form a right-handed superhelix in which the inner channel (formed by the A-helices) provides a ligand binding interface (18 -20). The TPRs in Tfc4 are organized into two arrays in the N terminus, TPR1-5 and TPR6 -9 (with five and four repeats, respectively) that are separated by a region of mini...

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A selection for mutants of the RNA polymerase III transcription apparatus: PCF1 stimulates transcription of tRNA and 5S RNA genes.

Cited by 32 publications

References 36 publications

Essential Roles of Bdp1, a Subunit of RNA Polymerase III Initiation Factor TFIIIB, in Transcription and tRNA Processing

Essential Roles of Bdp1, a Subunit of RNA Polymerase III Initiation Factor TFIIIB, in Transcription and tRNA Processing

A Tetratricopeptide Repeat Mutation in Yeast Transcription Factor IIIC₁₃₁ (TFIIIC₁₃₁) Facilitates Recruitment of Tfiib-Related Factor TFIIIB₇₀

The Brf1 and Bdp1 Subunits of Transcription Factor TFIIIB Bind to Overlapping Sites in the Tetratricopeptide Repeats of Tfc4

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A selection for mutants of the RNA polymerase III transcription apparatus: PCF1 stimulates transcription of tRNA and 5S RNA genes.

Cited by 32 publications

References 36 publications

Essential Roles of Bdp1, a Subunit of RNA Polymerase III Initiation Factor TFIIIB, in Transcription and tRNA Processing

Essential Roles of Bdp1, a Subunit of RNA Polymerase III Initiation Factor TFIIIB, in Transcription and tRNA Processing

A Tetratricopeptide Repeat Mutation in Yeast Transcription Factor IIIC131 (TFIIIC131) Facilitates Recruitment of Tfiib-Related Factor TFIIIB70

The Brf1 and Bdp1 Subunits of Transcription Factor TFIIIB Bind to Overlapping Sites in the Tetratricopeptide Repeats of Tfc4

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A Tetratricopeptide Repeat Mutation in Yeast Transcription Factor IIIC₁₃₁ (TFIIIC₁₃₁) Facilitates Recruitment of Tfiib-Related Factor TFIIIB₇₀