Specific initiation by RNA polymerase I in a whole-cell extract from yeast.

Schultz, Michael C.; Choe, Soo Young; Reeder, Ronald H.

doi:10.1073/pnas.88.3.1004

Cited by 100 publications

(82 citation statements)

References 29 publications

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“…Yeast Strain BJ1991 (prb1 pep4 gal2 leu2 trp1 ura3) was grown in yeast extract͞peptone͞dextrose (YEPD) to an A 600 of 1.5-2.0. Cells were harvested, and whole cell extracts were prepared by using a mortar and pestle (19). Protein concentration was determined by Bradford assay.…”

Section: Methodsmentioning

confidence: 99%

Probing TBP interactions in transcription initiation and reinitiation with RNA aptamers that act in distinct modes

Xiu

Shi

Adelman

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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The TATA-binding protein (TBP) is a critical general transcription factor that associates with the core promoter and acts as a nexus for gene regulation through its interactions with other factors. A large number of proteins recognize the relatively small yet highly conserved C-terminal domain of TBP. One subset of these proteins (general transcription factors) interacts with the TBP⅐TATA complex and RNA polymerase II to create the preinitiation complex. To study TBP functions in preinitiation complex and other complexes, we generated a set of RNA aptamers with high affinity to yeast TBP. These aptamers act on TBP in different ways: all of them bind TBP competitively with DNA bearing the TATA element, and some can actively disrupt the TBP⅐TATA interaction in preformed, higherorder complexes containing the additional general transcription factors TFIIB and TFIIA. In crude cell extracts, the aptamers inhibit transcription in ways that reveal the dynamic nature of TBP interactions during initiation and reinitiation. I nitiation of transcription by RNA polymerase II (Pol II) requires the assembly of a preinitiation complex (PIC) at the core promoter. The first, and often rate-limiting, step in this process is the binding of the TATA-binding protein (TBP) to the TATA element on DNA (1). The core domain of TBP has a saddle-shaped structure with a concave surface that binds and bends DNA severely (2). The PIC is largely built on this TBP⅐TATA foundation by an interlaced network of polypeptides that interact with each other and with the TBP⅐DNA complex (3). In vitro experiments suggest that the general transcription factor TFIIB binds to the promoter after TBP, providing a platform for the entry of Pol II͞TFIIF and playing a role in determining the transcription start sites (4, 5). TFIIA associates with the PIC through a distinct interaction surface on TBP and stimulates basal as well as activated transcription, presumably by counteracting the inhibitory effects of TBP-binding factors such as NC2 or Mot1 (reviewed in ref. 6). These inhibitory factors are thought to repress transcription by interfering with the TBP⅐TATA interaction and higher order complex formation (7,8).The critical role played by the TBP⅐TATA interaction in transcription makes it a frequently used target of regulation by numerous proteins that physically interact with TBP. Extensive investigations have been conducted to define the functions of discrete sites on the surface of TBP. Many genetic selections and screens have identified functionally distinct TBP mutants. Most mutants are defective in their interactions with the TATA element, TFIIA, or TFIIB, whereas others disrupt interactions with additional factors, including transcription activators and repressors (reviewed in ref. 9). Particularly informative was a systematic analysis, using alanine scanning mutagenesis of the surface of human TBP, which defined clusters of residues critical for its interactions with individual factors (10). Also, a particular surface of TBP has been shown to bind seve...

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Section: Methodsmentioning

confidence: 99%

Probing TBP interactions in transcription initiation and reinitiation with RNA aptamers that act in distinct modes

Xiu

Shi

Adelman

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…Whole-cell yeast extracts were produced according to the method described by Schultz et al (1991) with modifications. One and a half liters of yeast culture was grown in YPD to an OD 600 of 2.5-5.0 [v/v] glycerol, 1 mM EDTA, and 1 mM DTT), and frozen as drops in liquid nitrogen.…”

Section: Extract Preparationmentioning

confidence: 99%

Coupled RNA polymerase II transcription and 3′ end formation with yeast whole-cell extracts

Mariconti

Loll

Schlinkmann³

et al. 2010

RNA

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RNA polymerase II (RNAP II) transcription and pre-mRNA 39 end formation are linked through physical and functional interactions. We describe here a highly efficient yeast in vitro system that reproduces both transcription and 39 end formation in a single reaction. The system is based on simple whole-cell extracts that were supplemented with a hybrid Gal4-VP16 transcriptional activator and supercoiled plasmid DNA templates encoding G-less cassette reporters. We found that the coupling of transcription and processing in vitro enhanced pre-mRNA 39 end formation and reproduced requirements for poly(A) signals and polyadenylation factors. Unexpectedly, however, we show that in vitro transcripts lacked m 7 G-caps. Reconstitution experiments with CF IA factor assembled entirely from heterologous components suggested that the CTD interaction domain of the Pcf11 subunit was required for proper RNAP II termination but not 39 end formation. Moreover, we observed reduced termination activity associated with extracts prepared from cells carrying a mutation in the 59-39 exonuclease Rat1 or following chemical inhibition of exonuclease activity. Thus, in vitro transcription coupled to pre-mRNA processing recapitulates hallmarks of poly(A)-dependent RNAP II termination. The in vitro transcription/processing system presented here should provide a useful tool to further define the role of factors involved in coupling.

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“…Pol I transcription was performed using whole-cell extracts (Schultz et al 1991) under the conditions of Schultz et al {1992). Assays were also done with yeast nuclear extracts as the source of protein.…”

Section: Transcription Assaysmentioning

confidence: 99%

A yeast TFIIB-related factor involved in RNA polymerase III transcription.

Colbert

Hahn²

1992

Genes Dev.

175

127

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A suppressor gene was identified, which in high copy number rescues a temperature-sensitive mutation in yeast TATA-binding protein (TBP}. Suppression was allele specific because the suppressor did not rescue the temperature-sensitive phenotype of another TBP mutant. This suppressor gene encodes a 596-amino-acid protein of which the amino-terminal half is homologous to the Pol II-specific factor TFIIB. Disruption of this gene, termed BRF1, showed it to be essential for growth of yeast. Deletion of sequences at either the amino or carboxyl terminus of BRF1 gave both temperature-and cold-sensitive phenotypes. These temperature-and cold-sensitive strains were used to prepare extracts deficient in BRF1 activity and were tested for transcriptional activity by RNA polymerases I, II, and III in vitro. BRFl-deficient extracts are defective in Pol IlI transcription and can be reconstituted for Pol III transcription by the addition of recombinant BRF1. Western analysis shows that BRF1 is present in TFIIIB but not the TFIIIC fraction, suggesting that it is a component of TFIIIB. We propose that BRF1 plays a role in Pol III initiation analogous to the role played by TFIIB for Pol II in its interaction with TBP and polymerase. The identification of a Pol III-specific TFIIB-like factor extends the previously noted similarity of transcriptional initiation by the three nuclear polymerases.

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Specific initiation by RNA polymerase I in a whole-cell extract from yeast.

Cited by 100 publications

References 29 publications

Probing TBP interactions in transcription initiation and reinitiation with RNA aptamers that act in distinct modes

Probing TBP interactions in transcription initiation and reinitiation with RNA aptamers that act in distinct modes

Coupled RNA polymerase II transcription and 3′ end formation with yeast whole-cell extracts

A yeast TFIIB-related factor involved in RNA polymerase III transcription.

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