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• W2068397766 abstract "The B block promoter element is the primary binding site for the RNA polymerase III transcription initiation factor TFIIIC. It is always located within the transcript coding region, except in the Saccharomyces cerevisiae U6 RNA gene (SNR6), in which the B block lies 120 base pairs downstream of the terminator. We have exploited the unique location of the SNR6 B block to examine the sequence specificity of its interaction with TFIIIC. The in vitro and in vivo effects of all possible single base pair substitutions in the 9-base pair core of the B block were determined. Five mutant alleles are recessive lethal when present at a low copy number; these alleles identify crucial contacts between TFIIIC and the B block promoter element. Transcript analysis reveals that lethal B block substitutions reduce U6 RNA synthesis at least 10-fold in vivo and 20-fold in vitro. One viable B block mutant strain has one-third the wild type amount of U6 RNA and exhibits reduced levels of the U4-U6 RNA complex required for spliceosome assembly. The locations of lethal single and double point mutations leads us to propose that two domains of TFIIIC contact overlapping sites on the B block element. The B block promoter element is the primary binding site for the RNA polymerase III transcription initiation factor TFIIIC. It is always located within the transcript coding region, except in the Saccharomyces cerevisiae U6 RNA gene (SNR6), in which the B block lies 120 base pairs downstream of the terminator. We have exploited the unique location of the SNR6 B block to examine the sequence specificity of its interaction with TFIIIC. The in vitro and in vivo effects of all possible single base pair substitutions in the 9-base pair core of the B block were determined. Five mutant alleles are recessive lethal when present at a low copy number; these alleles identify crucial contacts between TFIIIC and the B block promoter element. Transcript analysis reveals that lethal B block substitutions reduce U6 RNA synthesis at least 10-fold in vivo and 20-fold in vitro. One viable B block mutant strain has one-third the wild type amount of U6 RNA and exhibits reduced levels of the U4-U6 RNA complex required for spliceosome assembly. The locations of lethal single and double point mutations leads us to propose that two domains of TFIIIC contact overlapping sites on the B block element. Initiation of transcription by eukaryotic RNA polymerases requires the recognition of auxiliary factors bound stably to promoter elements adjacent to the start site. The most common class of genes transcribed by RNA polymerase III (RNAPIII), 1The abbreviations used are: RNAPIII, RNA polymerase III; TFIIIC, transcription factor-IIIC; 5-FOA, 5-fluoroorotic acid. which includes the tRNA genes and other cellular and viral small RNA genes, has a promoter that consists of two intragenic elements called the A and B blocks (or box A and box B; Ref. 1Galli G. Hofstetter H. Birnstiel M.L. Nature. 1981; 294: 626-631Crossref PubMed Scopus (281) Google Scholar). The A and B blocks are 11- or 12-base pair elements that bind the multisubunit transcription factor (TF) IIIC (reviewed in Refs. 2Geiduschek E.P. Tocchini-Valentini G.P. Annu. Rev. Biochem. 1988; 57: 873-914Crossref PubMed Scopus (448) Google Scholar, 3Willis I. Eur. J. Biochem. 1993; 212: 1-11Crossref PubMed Scopus (192) Google Scholar). The binding of TFIIIC to the A and B blocks directs the binding of another multisubunit factor, TFIIIB, immediately upstream of the transcription start site (4Kassavetis G.A. Riggs D.L. Negri R. Nguyen L.H. Geiduschek E.P. Mol. Cell. Biol. 1989; 9: 2551-2566Crossref PubMed Scopus (185) Google Scholar). Once TFIIIB is stably bound to DNA, RNAPIII binds and initiates transcription. The A and B block sequences apparently first functioned as RNA elements (the dihydroU and TΨC loops of tRNA, respectively) and were later recruited as promoter elements, since they are also present in prokaryotic tRNA genes. Indeed, prokaryotic tRNA genes are efficiently transcribed in HeLa cell extracts (5Gruissem W. Prescott D.M. Greenberg B.M. Hallick R.B. Cell. 1982; 30: 81-92Abstract Full Text PDF PubMed Scopus (37) Google Scholar), Xenopus oocytes (6Folk W.R. Hofstetter H. Birnstiel M.L. Nucleic Acids Res. 1982; 10: 7153-7162Crossref PubMed Scopus (12) Google Scholar, 7Bossi L. Ciampi M.S. Nucleic Acids Res. 1983; 11: 3207-3226Crossref PubMed Scopus (4) Google Scholar), and yeast cells (8Lee C.P. RajBhandary U.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11378-11382Crossref PubMed Scopus (41) Google Scholar). The U6 RNA gene from the yeast Saccharomyces cerevisiae, SNR6, is transcribed by RNAPIII (9Margottin F. Dujardin G. Gérard M. Egly J.-M. Huet J. Sentenac A. Science. 1991; 251: 424-426Crossref PubMed Scopus (116) Google Scholar). Like the tRNA genes, SNR6 contains A and B blocks, although the B block is located in a unique position, 120 base pairs downstream of the U6 RNA coding region (Fig. LA, Ref. 10Brow D. Guthrie C. Genes & Dev. 1990; 4: 1345-1356Crossref PubMed Scopus (109) Google Scholar). The A block is located 21 base pairs downstream from the transcription start site as compared to 19 ± 1 base pairs in yeast tRNA genes. Unlike tRNA genes, however, SNR6 contains a consensus TATA box 30 base pairs upstream of the transcription start site. The U6 RNA genes from vertebrates also have a TATA box at this location; however, the promoters of these genes contain two additional elements further upstream and no A or B blocks (reviewed in Ref. 11Hernandez N. McKnight S.L. Yamamoto K.R. Transcriptional Regulation. Vol. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992: 281-313Google Scholar). In vivo analysis of the promoter elements of SNR6 revealed that the A and B blocks are required for transcription, while the TATA box and sequences upstream of it are not, although complete substitution of the TATA box does alter start site selection in vivo (12Eschenlauer J.B. Kaiser M.W. Gerlach V.L. Brow D.A. Mol. Cell. Biol. 1993; 13: 3015-3026Crossref PubMed Scopus (80) Google Scholar). Thus the SNR6 transcription initiation complex is likely to be similar to that which assembles on a tRNA gene and unlike the vertebrate U6 RNA gene transcription complex. A highly purified in vitro transcription system exhibits different promoter requirements for SNR6 transcription: the B block is dispensable, as is TFIIIC (13Moenne A. Camier S. Anderson G. Margottin F. Beggs J. Sentenac A. EMBO J. 1990; 9: 271-277Crossref PubMed Scopus (67) Google Scholar, 14Joazeiro C.A.P. Kassavetis G.A. Geiduschek E.P. Mol. Cell. Biol. 1994; 14: 2798-2808Crossref PubMed Scopus (70) Google Scholar). Transcription can be reconstituted with TFIIIB and RNAPIII using an allele of SNR6 that contains the TATA box and A block but lacks the B block. However, if SNR6 is first assembled into nucleosomes or chromatin, transcription is repressed unless a TFIIIC fraction is added and the B block is present (15Burnol A.-F. Margottin F. Huet J. Almouzni G. Prioleau M-N. Méchali M. Sentenac A. Nature. 1993; 362: 475-477Crossref PubMed Scopus (87) Google Scholar). Furthermore, the B block can relieve nucleosomal inhibition in vitro even when its orientation is reversed (16Burnol A.-F. Margottin F. Schultz P. Marsolier M.-C. Oudet P. Sentenac A. J. Mol. Biol. 1993; 233: 644-658Crossref PubMed Scopus (57) Google Scholar). These results led Sentenac and co-workers (16Burnol A.-F. Margottin F. Schultz P. Marsolier M.-C. Oudet P. Sentenac A. J. Mol. Biol. 1993; 233: 644-658Crossref PubMed Scopus (57) Google Scholar) to suggest that the SNR6 B block is an enhancer element, since it acts at a distance and functions in both orientations. Regardless of how the B block is viewed, as a basal promoter element or an enhancer element, the first event in transcription of SNR6 in vivo is likely to be the binding of TFIIIC via the A and B blocks. We have chosen SNR6 to study the interaction of TFIIIC with the B block for two reasons. First, SNR6 is single copy and essential, which allows us to assay the activity of any allele of SNR6 in vivo using a plasmid shuffle assay. Second, since the SNR6 B block is located outside of the RNA coding region, mutations in it are expected to affect only transcription and not RNA stability or function. Previous mutational studies of the B block have used tRNA genes and have primarily been done in vitro. In vivo analysis of tRNA gene transcription is problematical because most are present in multiple copies in the genome, and because intragenic promoter mutations can affect RNA stability as well as synthesis, confounding interpretation of the results. Furthermore, the analyses have not been very thorough; only one-third of the possible substitutions have been tested in yeast tRNA gene B blocks (17Allison D.S. Goh S.H. Hall B.D. Cell. 1983; 34: 655-664Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 18Nichols M. Bell J. Klekamp M.S. Weil P.A. Söll D. J. Biol. Chem. 1989; 264: 17084-17090Abstract Full Text PDF PubMed Google Scholar). Saturation mutagenesis of the Drosophila tRNAArg gene B block has been done (19Gaëta B.A. Sharp S.J. Stewart T.S. Nucleic Acids Res. 1990; 18: 1541-1548Crossref PubMed Scopus (6) Google Scholar), but many of the mutations had little effect on in vitro transcription, including substitutions which had previously been shown to substantially reduce transcription of yeast tRNA genes (17Allison D.S. Goh S.H. Hall B.D. Cell. 1983; 34: 655-664Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 18Nichols M. Bell J. Klekamp M.S. Weil P.A. Söll D. J. Biol. Chem. 1989; 264: 17084-17090Abstract Full Text PDF PubMed Google Scholar). Here we report a complete in vivo and in vitro analysis of the sequence requirements for B block function in RNAPIII transcription. We have created all possible substitutions in the 9-base pair core of the SNR6 B block. Four mutations decreased U6 RNA levels more than 20-fold in vivo and were recessive lethal when carried on a low copy number plasmid. Another mutation decreased in vivo U6 RNA levels 10-fold and resulted in low viability when on a centromere plasmid and lethality when present in the chromosomal copy of SNR6. At least one non-lethal B block mutation decreases U6 RNA synthesis to the point that it is limiting for U4-U6 RNA complex formation, a necessary step in spliceosome assembly. We have determined the quantitative effect of each substitution in vivo and in vitro and find that (i) the effect of mutations on transcription is more severe in vitro than in vivo, and (ii) the effect of previously studied substitutions on expression of tRNA genes and the U6 gene in vitro is quite similar. Based on the effects of mutations, we propose that the B block consists of two overlapping sites recognized by different domains of TFIIIC. The B block mutant alleles of SNR6 may allow the selection of change-of-specificity mutations in the TFC3 gene, which codes for the B block-binding subunit of TFIIIC. For the in vivo analysis, a library of B block substitutions was created by site-directed mutagenesis of the wild type -539H6 allele of SNR6 (10Brow D. Guthrie C. Genes & Dev. 1990; 4: 1345-1356Crossref PubMed Scopus (109) Google Scholar) in the yeast centromere plasmid pRS314 (TRP1 CEN6 ARSH4; Ref. 20Sikorski R.S. Heiter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). The plasmid was constructed by ligation of the EcoRI-PstI fragment from p-539H6 into EcoRI-PstI-digested pRS314. Mutagenesis was carried out as described (Ref. 12Eschenlauer J.B. Kaiser M.W. Gerlach V.L. Brow D.A. Mol. Cell. Biol. 1993; 13: 3015-3026Crossref PubMed Scopus (80) Google Scholar, except that a 1:1 DNA to primer ratio was used) using an oligonucleotide (“BBM,” see below) degenerate in the 11 base pair B block consensus sequence (2Geiduschek E.P. Tocchini-Valentini G.P. Annu. Rev. Biochem. 1988; 57: 873-914Crossref PubMed Scopus (448) Google Scholar). The mutagenized plasmid was transformed into Escherichia coli (all E. coli transformations were by electroporation), transformants were grown up in liquid culture, and plasmid DNA was prepared by CsCl gradient purification as described (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) except for the following changes. After the isopropyl alcohol precipitation, the crude nucleic acid pellet was resuspended in 10 ml of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (TE) and extracted three times with 10 ml of 1:1 phenol/chloroform, followed by ethanol precipitation. The nucleic acid pellet was resuspended in TE and subjected to CsCl gradient centrifugation. For the isolation of individual B block substitutions, library DNA was digested with BstBI, which cuts within the wild type B block (TTCGAA, positions 2–7) and thus enriches for mutant alleles when the DNA was retransformed into E. coli. Plasmid DNA was prepared from individual transformants and sequenced with oligonucleotide 6seq4 to identify the B block substitution. To isolate substitutions not obtained by this method and for positions outside the BstBI site, additional oligonucleotides degenerated at individual positions were synthesized and mutagenesis was carried out. All mutant alleles were sequenced from position –50 to +260 of SNR6 to identify the B block mutations and to verify the lack of mutations in the rest of the gene. Plasmids were sequenced by the dideoxy method using Sequenase (United States Biochemical Corp., Cleveland, OH). For in vitro transcription, B block mutant alleles of SNR6 were introduced into pUC118 (22Vieira J. Messing J. Methods Enzymol. 1987; 153: 3-11Crossref PubMed Scopus (2009) Google Scholar) since the pRS314-based clones exhibited a high level of nonspecific transcription. The pUC118-based clones were constructed by mutagenesis of p-539H6 with the degenerate oligonucleotides described above or by ligation of the EcoRI-PstI fragment from the corresponding pRS314 clone into EcoRI-Ps(I-digested pUC118. The pABE6 internal control plasmid has been described previously (12Eschenlauer J.B. Kaiser M.W. Gerlach V.L. Brow D.A. Mol. Cell. Biol. 1993; 13: 3015-3026Crossref PubMed Scopus (80) Google Scholar). Yeast strain MWK023 has been described previously (12Eschenlauer J.B. Kaiser M.W. Gerlach V.L. Brow D.A. Mol. Cell. Biol. 1993; 13: 3015-3026Crossref PubMed Scopus (80) Google Scholar). Briefly, the strain contains a deletion of the chromosomal SNR6 gene and carries the –39D6 allele of SNR6 (12Eschenlauer J.B. Kaiser M.W. Gerlach V.L. Brow D.A. Mol. Cell. Biol. 1993; 13: 3015-3026Crossref PubMed Scopus (80) Google Scholar) on YCp50 (URA3 CEN4 ARS1). MVVK027 is identical to MWK023 except it carries the pseudo-wild type allele of SNR6 (23Madhani H.D. Bordonné R. Guthrie C. Genes & Dev. 1990; 4: 2264-2277Crossref PubMed Scopus (131) Google Scholar) on YCp50. MWK035 is MWK023 carrying the pRS314–539H6 plasmid (see above). The genotype of strain PJ43–2b is MATα trp1–1 ura3–52 can1–100 leu2–3,112 his3-11,15 ade2–1 met2-Δ1 lys2-Δ2. 2P. James, personal communication. BBM, 5′-GCAAAGGCTTXXXXXXXXXXXCCGAGAC (X = 96% wild type, 1.3% each of the non-wild type nucleotide); G235H, 5′-GGCTTAGGTTCGAADGCGAGACAATT (D = equal amounts of A, G, and T); C242D, 5′-GCAAAGGCTTAGHTTTCGAACGCG (H = equal amounts of A, C, and T); C243D 5′-GCAAAGGCTTAHGTTC-GAACGCG; T236V, 5′-GGCTTAGGTTCGABCGCGAGACAAT (B = equal amounts of C, G, and T); A241B, 5′-GCAAAGGCTTAGGVTC-GAACGCG (V = equal amounts of A, C, and G); G239H, 5′-GGCTTAG-GTTDGAACGCGAGAC; 6D, 5′-AAAACGAAATAAATCTCTTG; 14C, 5′-CACAATCTCGGACGAATCCTC; 14B, 5′-AGGTATTCCAAAAAT-TCCC; 6seq4, 5′-GCAGTGTATCTTTATCTTCC; U5B, 5′-AAGTTC-CAAAAAATATGGCAAGC. To identify lethal mutations, the SNR6 B block library was transformed into MWK023 by the lithium acetate procedure (24Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). The transformation plates were replica plated to medium containing 5-fluoroorotic acid (5-FOA) and scored for lethality after 4 days of growth at 30 °C. For strains which appeared to be 5-FOA inviable, the original transformants were streaked on 5-FOA-containing plates and rescored for lethality after 3 days growth at 30 °C. For 5-FOA inviable strains, the plasmid carrying the mutant B block was isolated from the original transformant by transformation of genomic DNA (25Hoffman C. Winston F. Gene (Amst.). 1987; 57: 267-272Crossref PubMed Scopus (2043) Google Scholar) into E. coli. Plasmids were sequenced to identify the B block mutation. For the in vivo screening of individual mutants, snr6 alleles were transformed into MWK023, grown up overnight at 30 °C and then streaked onto medium containing 5-FOA. Viability was determined after 3 days of growth at 30 °C. For quantitative analysis of transcript levels in vivo, all snr6 B block point mutant alleles were transformed into MWK027 (contains pseudo-wild type U6 gene), and RNA was prepared as described previously (10Brow D. Guthrie C. Genes & Dev. 1990; 4: 1345-1356Crossref PubMed Scopus (109) Google Scholar). Primer extension analysis (12Eschenlauer J.B. Kaiser M.W. Gerlach V.L. Brow D.A. Mol. Cell. Biol. 1993; 13: 3015-3026Crossref PubMed Scopus (80) Google Scholar) of 2 µg of a single preparation of total RNA from each strain was done twice, once using 32P-labeled oligonucleotides 6D and 14C (complementary to U6, and U4 RNAs, respectively), and once using oligonucleotides 6D and U5B (complementary to U6 and U5 RNAs). The cDNA products were resolved on a 6% acrylamide, 8.3 m urea sequencing gel and were quantitated either with an AMBIS betaimager or with a PhosphorImager (Molecular Dynamics model SI). Total RNA was prepared from the wild type strain three times, and each RNA preparation was analyzed by primer extension three times. The yield of U6 RNA in the three wild type RNA samples varied less than ±8% from the mean value. The U4 or U5 bands were used to normalize the amount of RNA/lane. The solution hybridization analysis was carried out as described (26Li Z. Brow D.A. Nucleic Acids Res. 1993; 21: 4645-4646Crossref PubMed Scopus (35) Google Scholar) using 32P-labeled oligonucleotides 6D and 14B. RNA for the solution hybridization analysis was prepared as described (27Treco D.A. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New York, NY1989: 13.12.1-13.12.3Google Scholar) under conditions that leave U4-U6 base pairing intact. The gel was quantitated by PhosphorImager analysis. The G5C and G5T substitutions were introduced into the chromosomal copy of SNR6 by the “pop-in/pop-out” procedure (28Scherer S. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4951-4955Crossref PubMed Scopus (485) Google Scholar). The KpnI-SacI fragment of pRS314 containing the G5C and G5T alleles of SNR6 was ligated into KpnI-SacI-digested pRS306, a URA3-marked integrating plasmid (20Sikorski R.S. Heiter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). Eight µg of pRS306 plasmid containing the G5C or G5T alleles was digested with NruI and transformed into PJ43–2b by the lithium acetate procedure. After integration, the strains were transformed with a wild type copy of SNR6 carried on pRS317 (LYS2 CEN6 ARSH4, Ref. 29Sikorski R.S. Boeke J.D. Methods Enzymol. 1991; 194: 302-329Crossref PubMed Scopus (495) Google Scholar) followed by plating on medium containing 5-FOA to force loss of one of the two copies of chromosomal SNR6. After the pop-out, the presence of the B block mutation was confirmed by polymerase chain reaction amplification of the genomic locus and direct sequencing of the polymerase chain reaction product. The strains were transformed with YCp50 containing the -39D6 allele (12Eschenlauer J.B. Kaiser M.W. Gerlach V.L. Brow D.A. Mol. Cell. Biol. 1993; 13: 3015-3026Crossref PubMed Scopus (80) Google Scholar) of SNR6 followed by plating on medium containing α-aminoadipate to select for loss of the LYS2 plasmid (30Chattoo B.B. Sherman F. Fjellstedt T.A. Mehnert D. Ogur M. Genetics. 1979; 93: 51-65Crossref PubMed Google Scholar). These final strains which contained either the G5C or G5T substitutions in the chromosomal copy of SNR6 and the URA3-marked plasmid YCp50 containing wild type SNR6 were then streaked on medium containing 5-FOA and scored for lethality after 3 days growth at 30 °C. Transcription reactions using subcellular extract (31Evans C.F. Engelke D.R. Methods Enzymol. 1990; 181: 439-450Crossref PubMed Scopus (23) Google Scholar) were carried out as described (12Eschenlauer J.B. Kaiser M.W. Gerlach V.L. Brow D.A. Mol. Cell. Biol. 1993; 13: 3015-3026Crossref PubMed Scopus (80) Google Scholar) except for the following changes. For the first set of experiments, reactions contained 60 ng of mutant plasmid and 20 ng of pΔBE6 internal control plasmid. For the second and third set of experiments, reactions contained 75 ng of mutant plasmid only. After reactions were completed, 6 µl of stop buffer (0.4 m Tris-Cl (pH 6.8), 0.5 m dithiothreitol, 10% SDS, 50% glycerol, 20 mm EDTA, 0.1% bromphenol blue) were added, samples were heated at 90 °C for 3 min, and then loaded directly onto a discontinuous 0.1% SDS, 7 m urea polyacrylamide gel without further manipulation (32Brow D.A. Geiduschek E.P. J. Biol Chem. 1987; 262: 13953-13958Abstract Full Text PDF PubMed Google Scholar). Since the entire sample is loaded on the gel without phenol extraction and ethanol precipitation, there is no need to control for loss of RNA product. The 19 cm high, 14 cm wide, 1.5 mm thick gel was run at 60 mA for 3.5 h until the bromphenol blue was 2.5 cm from the bottom of the gel. The portion of the gel containing the unincorporated [α-32P]GTP and the stacking gel were then removed, and the gel was fixed for 30 min each in 45% methanol, 10% acetic acid, then 10% methanol, 7.5% acetic acid, and dried. The dried gel was exposed to Kodak X-Omat AR5 film with or without a Cronex Lightning-Plus intensifying screen (DuPont). U6 RNA bands were quantitated by excising bands out of the dried gels and counting in a scintillation counter. To normalize for variations in template concentration, a 100-ng aliquot of each mutant plasmid (the same DNA stocks that were used for the in vitro transcriptions) was digested with EcoRI and electrophoresed on a 0.8% agarose gel. A photograph was taken of the gel after ethidium bromide staining, and the negative was scanned with an Applescanner using the Applescan program. The scan was quantitated using the Scan Analysis program (version 2.21, Specom Research). The linearity of the scan analysis was verified for 50–150 ng of DNA. The data from the in vitro transcription reactions was normalized to the amount of DNA (transcript yield increases linearly with increasing DNA concentration in this range; data not shown). The concentration of DNA in the stock solutions varied less than ± 10% from the mean value. In order to identify essential base pairs in the conserved core of the B block promoter element, a library of snr6 alleles was created by site-directed mutagenesis using a degenerate oligonucleotide that randomly substitutes the central 9 base pairs of the B block. These will be referred to as positions 1–9 and corresponds to positions 235–243 of SNR6 and positions 53–61 in the standard tRNA numbering system (Fig. 1B). Mutagenesis was carried out on SNR6 cloned into the yeast centromere vector pRS314. The library of mutants was then transformed into a yeast strain containing a deletion of the chromosomal U6 gene and a URA3-marked plasmid carrying wild type SNR6. Transformants were replica plated to medium containing 5-FOA, which selects for loss of the URA3/SNR6 plasmid. Colonies that do not survive on the 5-FOA plates must therefore have a lethal snr6 allele on pRS314. The pRS314-snr6 plasmid was then isolated from the 5-FOA inviable transformants and sequenced to identify the B block mutation. From approximately 1000 colonies screened, three lethal point mutations were obtained: T3C, C4A, and C4G (Fig. 1, B and C). In addition, two lethal double mutations, T3G-C9T and T2G-C9T were isolated. Because we could not be sure that the library contained the full range of mutations, B block substitutions were isolated individually as described under “Materials and Methods” and then tested for lethality using the 5-FOA screen. Screening of all the B block point mutations identified C4T as an additional lethal mutant. Another mutant, G5C, appeared to have low viability on 5-FOA-containing plates (Fig. 1C). One interpretation of this low viability is that the mutation causes a reduction in U6 RNA accumulation that is severe enough to be lethal at single copy, but a fraction of the cells are able to compensate by increasing the copy number of the plasmid. Increased copy number results from plasmid missegregation, which occurs in about 2–3% of cell divisions for centromere plasmids of similar size (20Sikorski R.S. Heiter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar, 33Heiter P.C. Mann C. Snyder M. Davis R.W. Cell. 1985; 40: 381-392Abstract Full Text PDF PubMed Scopus (299) Google Scholar). To stably maintain a single copy of the snr6 allele, the G5C mutation was introduced into the chromosomal copy of SNR6 by the pop-in/pop-out procedure (28Scherer S. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4951-4955Crossref PubMed Scopus (485) Google Scholar). The recipient strain contained the same URA3/SNR6 plasmid used for the lethality screen. The G5C integrant strain did not grow on 5-FOA-containing medium (Fig. 1C), indicating that the G5C allele is lethal at single copy. Thus the plasmid shuffle assay identified five lethal point mutations at three adjacent positions in the B block: T3C, C4A, C4G, C4T, and G5C. The C4G change is equivalent to the G56 mutation of tRNA genes that severely reduces TFIIIC binding (34Baker R.E. Gabrielsen O. Hall B.D. J. Biol. Chem. 1986; 261: 5275-5282Abstract Full Text PDF PubMed Google Scholar) and has previously been shown to abolish transcription from an SNR6 minigene in vivo (35Chalker D.L. Sandmeyer S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4927-4931Crossref PubMed Scopus (33) Google Scholar). The lethality of the other two substitutions at position 4 indicates that a C residue at this position is absolutely required for in vivo function. The lethal G5C mutation is equivalent to the C57 mutant of tRNA genes, which also severely reduces transcription and TFIIIC binding (17Allison D.S. Goh S.H. Hall B.D. Cell. 1983; 34: 655-664Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 18Nichols M. Bell J. Klekamp M.S. Weil P.A. Söll D. J. Biol. Chem. 1989; 264: 17084-17090Abstract Full Text PDF PubMed Google Scholar, 34Baker R.E. Gabrielsen O. Hall B.D. J. Biol. Chem. 1986; 261: 5275-5282Abstract Full Text PDF PubMed Google Scholar). The lethality of the T3C mutant is perhaps the most surprising since the only mutation examined at that position in a yeast tRNA gene B block, the T3A change, only modestly reduced transcription (18Nichols M. Bell J. Klekamp M.S. Weil P.A. Söll D. J. Biol. Chem. 1989; 264: 17084-17090Abstract Full Text PDF PubMed Google Scholar). Interestingly, the lethal mutations are not symmetrical about the dyad axis of the palindromic SNR6 B block (between residues 4 and 5). This indicates that TFIIIC does not bind the SNR6 B block symmetrically. To obtain a more quantitative measure of the effect of B block mutations in vivo, we examined the steady state levels of U6 RNA in the B block mutant strains. To allow detection of transcripts from lethal snr6 alleles, and to preclude selection for increased copy number of plasmids bearing detrimental SNR6 B block mutations, we introduced all the U6 alleles into a strain that carries a pseudo-wild type U6 gene, which produces a shortened but functional U6 RNA (23Madhani H.D. Bordonné R. Guthrie C. Genes & Dev. 1990; 4: 2264-2277Crossref PubMed Scopus (131) Google Scholar). In a primer extension experiment, the pseudo-wild type U6 RNA can be distinguished from the wild type U6 RNA made from the genes with mutant B blocks. Fig. 2A shows the results for mutants with substitutions at positions 3–6. The gels were quantitated as described under “Materials and Methods,” normalizing for RNA recovery to U4 RNA in the first set of assays and to U5 RNA in the duplicate assays. The data for all the mutants is shown in Fig. 2B as a fraction of the wild type level plus or minus the standard deviation for two determinations. Most of the point mutations result in no more than a 2-fold reduction in steady state U6 RNA levels. In contrast, the mutations that were lethal on a plasmid, T3C and all three C4 substitutions, resulted in a 15–50-fold reduction in the steady state U6 RNA level. The G5C mutation, which is lethal only at single copy, resulted in 10% of the wild type level of U6 RNA. The G5T strain gave the next lowest level of U6 RNA, 33% of wild type. We introduced this mutation into the chromosomal copy of SNR6 to determine if it would be lethal at single copy. Unlike G5C, the G5T mutation is not lethal when on the chromosome (Fig. 1C). Growth curves indicated that the strain containing the integrated G5T allele grows at a wild type rate in liquid culture at 30 °C (data not shown). This strain produces 27% of the wild type amount of U6 RNA, approximately equivalent to the centromere-plasmid borne G5T allele in the presence of the pseudo-wild type SNR6. Thus, a cell can grow normally when it contains less than one-third the wild type amount of U6 RNA. We also conclude from these data that the lethality of the T2G/C9T and T3G/C9T double mutants indicates a synergistic interaction between the indivi" @default.
• W2068397766 created "2016-06-24" @default.
• W2068397766 creator A5066704185 @default.
• W2068397766 creator A5073367012 @default.
• W2068397766 date "1995-05-01" @default.
• W2068397766 modified "2023-09-26" @default.
• W2068397766 title "Lethal Mutations in a Yeast U6 RNA Gene B Block Promoter Element Identify Essential Contacts with Transcription Factor-IIIC" @default.
• W2068397766 cites W1500459843 @default.
• W2068397766 cites W1503994537 @default.
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