A Class III Transcription Factor Composed of RNA

Young, Lisa; Dunstan, Heather M.; Witte, Pamela R.; Smith, Timothy P. L.; Ottonello, Simone; Sprague, Karen U.

doi:10.1126/science.1708526

Cited by 55 publications

(23 citation statements)

References 34 publications

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“…Alternatively, as has been found for some other proteins involved in macromolecular nucleic acid metabolism (Kole and Altman 1981;Greider and Blackburn 1987;Young et al 1991), Mts3 might be composed of RNA. RNA has been found in the 40,000-fold purified fraction VI, but the available evidence suggests that it is not a functional subunit of the Mts 1/Mts2 protein-DNA binding activity (Wahls 1994).…”

Section: Evidence For a Third Factormentioning

confidence: 99%

A heteromeric protein that binds to a meiotic homologous recombination hot spot: correlation of binding and hot spot activity.

Wahls¹,

Smith²

1994

Genes Dev.

107

179

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Homologous recombination hot spots are DNA sites that increase the frequency of recombination in their vicinity. The M26 allele of the ade6 gene in Schizosaccharomyces pombe is the first meiotic hot spot with an identified unique nucleotide sequence. We have purified 40,000-fold a heteromeric protein, containing polypeptides Mtsl (70 kD) and Mts2 (28 kD1, that binds to the M26 site. Binding in vitro strictly correlates with hot spot activity in vivo for numerous single base pair substitutions in the vicinity of the M26 site, indicating that Mtsl/Mts2 activates the M26 site and promotes a rate-limiting step of meiotic recombination. These and other data suggest that homologous recombination may be regulated primarily by discrete DNA sites and proteins that interact with those sites.

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Section: Evidence For a Third Factormentioning

confidence: 99%

A heteromeric protein that binds to a meiotic homologous recombination hot spot: correlation of binding and hot spot activity.

Wahls¹,

Smith²

1994

Genes Dev.

107

179

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“…TFIIIB then directs initiation by RNAPIII (28). Transcription of a silkworm tRNA gene in a homologous cell extract requires an additional factor composed of RNA, called TFIIIR (60). It is not known whether TFIIIR is required for tRNA gene transcription in other organisms. U6 RNA, the smallest of five small nuclear RNAs found in the spliceosome, is synthesized by RNAPIII (32,47), while the other four spliceosomal small nuclear RNAs are made by RNAPII.…”

mentioning

confidence: 99%

Architecture of a yeast U6 RNA gene promoter.

Eschenlauer¹,

Kaiser²,

Gerlach³

et al. 1993

Mol. Cell. Biol.

131

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actions responsible for the presumptive positioning of TF-IIIB upstream of vertebrate U6 RNA genes are unknown. The absence of A-and B-block promoter elements argues against the involvement of TFIIIC. The surprising discovery that in vitro transcription of vertebrate U6 genes requires the TATA-binding protein (TBP) subunit of RNAPII initiation factor TFIID (38, 51) and an RNAPII factor that stabilizes TBP binding to the TATA box, TFIIA (55), raises the possibility that TFIIIB is positioned by TBP and associated proteins bound to the vertebrate U6 gene TATA box.We have previously isolated the U6 RNA gene, SNR6, from the yeast Saccharomyces cerevisiae and found that unlike vertebrate U6 genes, it contains an essential B-block promoter element (7). The SNR6 B block is in a unique position, downstream of the coding region (see Fig. 1). A candidate A-block element in the conventional intragenic position was also identified, and it was suggested that the yeast U6 RNA gene binds TFIIIB by the same TFIIICdependent mechanism used by tRNA genes (7). However, the yeast U6 gene has upstream sequences similar to the vertebrate U6 gene TATA box and PSE (6)

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“…The finding that tRNA'U (TFIIIR) acts to prevent transcription inhibition is surprising, given our earlier results (29).…”

Section: Methodsmentioning

confidence: 51%

“…The activities necessary for in vitro transcription of tRNA genes have been chromatographically separated into five partially purified fractions (17,29). These consist of the RNA polymerase III fraction and factor-containing fractions TFIIIB, TFIIIC, TFIIID, and TFIIIR, a fraction in which the transcription activity is provided by RNA (29). In an accompanying report (6), we demonstrate that transcriptional activity of the TFIIIR fraction is due to silkworm tRNA"eu and that transcriptional activity is highly specific for this RNA. Silkworm tRNA gene transcription is initiated by the association of the transcription factors TFIIIB, TFIIIC, and TFIIID with the promoter to form a highly stable complex that can support multiple rounds of transcription in vitro (17).…”

mentioning

confidence: 99%

“…In the silkworm, Bombyx mori, RNA polymerase III (pol III) and the auxiliary transcription factors involved in transcription of class III genes can be provided by nuclear extracts that support promoter-specific transcription in vitro (22). The activities necessary for in vitro transcription of tRNA genes have been chromatographically separated into five partially purified fractions (17,29). These consist of the RNA polymerase III fraction and factor-containing fractions TFIIIB, TFIIIC, TFIIID, and TFIIIR, a fraction in which the transcription activity is provided by RNA (29).…”

mentioning

confidence: 99%

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tRNA(IleIAU) (TFIIIR) plays an indirect role in silkworm class III transcription in vitro and inhibits low-frequency DNA cleavage.

1994

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tRNAleU provides an activity, originally called TFIIIR, necessary to reconstitute transcription by silkworm RNA polymerase III in vitro from partially purified components. Here we report studies on the role of tRNA'IAU in in vitro transcription. We show that tRNA['eU does not act positively but, rather, is required to prevent the action of a transcriptional inhibitor. We also show that the presence of tRNA'U in transcription reaction mixtures prevents low-frequency DNA cleavage by the TFIIIB fraction. Studies on the mechanism of transcriptional inhibition suggest that this DNA cleavage could cause transcriptional inhibition through trans-inactivation of transcription machinery. The ability to block DNA cleavage, like the ability to facilitate transcription, is highly specific to silkworm tRNA"'1U.Promoter-specific transcription by eukaryotic RNA polymerases requires the action of transcription factors in addition to the polymerases themselves (reviewed in references 7, 8, and 21). In the silkworm, Bombyx mori, RNA polymerase III (pol III) and the auxiliary transcription factors involved in transcription of class III genes can be provided by nuclear extracts that support promoter-specific transcription in vitro (22). The activities necessary for in vitro transcription of tRNA genes have been chromatographically separated into five partially purified fractions (17,29). These consist of the RNA polymerase III fraction and factor-containing fractions TFIIIB, TFIIIC, TFIIID, and TFIIIR, a fraction in which the transcription activity is provided by RNA (29). In an accompanying report (6), we demonstrate that transcriptional activity of the TFIIIR fraction is due to silkworm tRNA"eu and that transcriptional activity is highly specific for this RNA. Silkworm tRNA gene transcription is initiated by the association of the transcription factors TFIIIB, TFIIIC, and TFIIID with the promoter to form a highly stable complex that can support multiple rounds of transcription in vitro (17). We report here that, in contrast to these transcription factors, tRNA"U is not necessary for active transcription complex formation. Instead, tRNAI"u is necessary to protect transcription complexes by preventing the generation of a transcriptional inhibitor. The transcription inhibition that occurs in the absence of tRNA'U involves the TFIIIB fraction and DNA and correlates with low-level DNA cleavage. Our results suggest that the DNA cleavage that occurs in the absence of tRNA"'u is responsible for transcription inhibition. MATERIALS AND METHODSCloned genes used in this work The template DNA for all experiments except that whose results are shown in Fig. 4 used for the experiments in Fig. 4 was a 2.5-kb deletion derivative of this plasmid that includes the tRNACIa gene and sequences from -221 to +89 inserted between the EcoRI and PvuII sites of pBR322 (13).RNA preparation. Silkworm tRNA'U was purified from total silk gland nucleic acids by gel filtration chromatography and polyacrylamide gel fractionation, as described previously (6). Silk...

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A Class III Transcription Factor Composed of RNA

Cited by 55 publications

References 34 publications

A heteromeric protein that binds to a meiotic homologous recombination hot spot: correlation of binding and hot spot activity.

A heteromeric protein that binds to a meiotic homologous recombination hot spot: correlation of binding and hot spot activity.

Architecture of a yeast U6 RNA gene promoter.

tRNA(IleIAU) (TFIIIR) plays an indirect role in silkworm class III transcription in vitro and inhibits low-frequency DNA cleavage.

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