Article Figures & Data
Figures
- Fig. 1.
Locations of sit1 amino acid substitutions. The amino acid sequences of regions F, D, and G of S. cerevisiae RNAPII (S. cerevisiae) are compared to those of other eukaryotes: Schizosaccharomyces pombe RNAPII (S. pombe), Arabidopsis thaliana RNAPII (A. thaliana), Caenorhabditis elegans RNAPII (C. elegans), D. melanogaster RNAPII (D. melano.), Mus musculus RNAPII (M. musculus),Sulfolobus acidocaldarius (S. acidocal.) subunit A or C as indicated, E. coli RNAP, and S. cerevisiae RNAPIII (S. c. RNAPIII). Residues identical to those of S. cerevisiae RNAPII are indicated by dots; gaps are indicated by hyphens. For each sit1 mutation, the substituted amino acid is indicated above the wild-type amino acid and the designation of the allele is given in parentheses. A second substitution in the sit1-290 polypeptide that is located upstream of region F is not shown (Table 1). Amino acid substitutions that confer resistance to the transcriptional inhibitor α-amanitin (Ama) in C. elegans (19), D. melanogaster (19), and M. musculus (10, 11) are also indicated. Single amino acid substitutions that affect elongation and termination by E. coli RNAP (49) are written below the sequence of the largest subunit of E. coli RNAP (E. coli mut.). Substitutions that confer streptolydigin resistance in E. coli (E. coli St1) (44) and Bacillus subtilis(B. sub. St1) (51) are indicated. The double substitutions in the S. cerevisiae RNAPIII C160-270 mutant (RNAPIII C160-270) (47) and C160-112 mutant (RNAPIII C160-112) (23) are indicated. The invariant Mg2+ binding motif NADFDGD in region D is underlined. Also indicated are the locations of the conditional lethalrpb1-17 substitution (43), the sua8-1and sua8-2 substitutions (13), and the mutations (rpb1-501 and -502) that confer an Spt phenotype (25).
- Fig. 2.
Region F and D substitutions are necessary to confer the Sit phenotype. (A) Region F substitutions. Reconstructedsit1 alleles carrying a region F mutation or the wild-typeSIT1 alleles were was assayed for the ability to confer the Sit phenotype by introduction into the diploid strain YF2201 (MATa/MATα sit1::LEU2/SIT1 gcn4-2/gcn4-2 bas1-2/bas1-2 bas2-2/bas2-2 ura3-52/ura3-52 leu2/leu2 trp1::hisG/trp1::hisG). The resulting transformants were then sporulated, 10 or more tetrads were dissected, and spores were allowed to germinate on YPD medium (45). Viable Trp+ (sit1 allele on plasmid) Leu+ (sit1::LEU2) haploid progeny were then tested by streaking onto solid SD medium (45) containing (+ His) or lacking (− His) histidine. Cells were allowed to grow for the indicated number of days at 30°C.sit1-9 required a longer incubation to show visible single colonies. (B) Region D substitutions. Reconstructed sit1alleles carrying region D mutations or the wild-type SIT1alleles were assayed for the ability to confer the Sit phenotype as described above. Cells were allowed to grow on solid SD medium (45) containing (+ His) or lacking (− His) histidine for the indicated number of days at 30°C. sit1-8G did not grow on medium lacking histidine, even when incubation was extended to 24 days. A region F substitution (sit1-4) is shown for comparison.
- Fig. 3.
Localization of mutations in the sit1-8allele. Schematic representation of the SIT1 locus (EcoRI-HindIII fragment) showing the positions (in the numbering system of Allison et al. [3]) of various endonuclease sites that were used in the construction of the chimeric genes. The encoded SIT1 protein is diagrammed below the restriction map. The gray boxes represent regions (A to H) of the polypeptide that are most conserved evolutionarily (27), and the diagonally striped box represents the carboxy-terminal domain. The structures of the chimeric genes are indicated by open and filled boxes representing wild-type (RPO21) and mutant (sit1-8) sequences, respectively. Each hybrid gene is designated by a letter (a to i). The ability of each chimeric gene to confer a semidominant Sit phenotype was assayed by introducing it into yeast strain YF2047 (MATα gcn4-2 bas1-2, bas2-2 ura3-52 leu2 trp1::hisG sit1::LEU2 [pJS121; SIT1 onURA3 CEN/ARS plasmid] [4]) and testing the resulting transformants for the ability to grow on solid SD medium (45) lacking histidine: ++, growth rate similar to that of a cell carrying the entire sit1-8 allele; +, growth rate slower than that of a cell carrying the entire sit1-8allele; −, absence of growth.
- Fig. 4.
Synthetic lethality of sit1-8D andppr2. TRP1 CEN/ARS plasmids bearing SIT1(RPO21) alleles (indicated by white letters on a black background) were introduced into YF2277 (MATα rpo21::ADE2 [pJS121, RPO21 onURA3, CEN/ARS]), shown on the left side of the plate or YF2278 (MATα rpo21::ADE2 ppr2::hisG [pJS121, RPO21 onURA3, CEN/ARS]), shown on the right side of the plate, and grown on SD medium (45) lacking tryptophan and containing 5′-fluoroorotic acid for 3 days at 30°C in order to select for loss of the URA3 maintenance plasmid (pJS121).
Tables
- Table 1.
Nucleotide and amino acid sequence changes insit1 alleles
Region and allele Nucleotide changea Amino acid substitution Region F sit1-7 G 2501 to A Gly 730 to Asp sit1-5 G 2587 to C Ala 759 to Pro sit1-290 G 2603 to T Cys 764 to Phe sit1-290 G 2314 to A Asp 668 to Asn sit1-9 G 2771 to A Gly 820 to Asp sit1-4 C 2807 to T Ala 832 to Val sit1-246 C 2807 to T Ala 832 to Val Region D sit1-8D A 1747 to T Asn 479 to Tyr sit1-252 A 1771 to T Met 487 to Leu sit1-261 A 1771 to T Met 487 to Leu sit1-278 A 1646 to C Asn 445 to Thr Region G sit1-8G C 3539 to T Ala 1076 to Val ↵a The positions of the nucleotide changes are given in the numbering system of Allison et al. (3).
- Table 2.
Effects of sit1 mutations on expression ofCYC1 and cyc1-5000
Allele Affected region(s) Mean β-galactosidase activity (U)b cyc1-5000/CYC1 ratio (%) CYC1 cyc1-5000 SUA8 NAa 53 (3) 1.24 (0.01) 2.3 sua8-1 D 29 (5) 6.3 (0.2) 22 SIT1 NA 15 (2) 0.32 (0.05) 2.1 sit1-8 D, G 8 (1) 0.38 (0.04) 4.8 sit1-278 D 4.04 (0.02) 0.76 (0.04) 19 sit1-4 F 3.8 (0.7) 0.32 (0.01) 8.4 sit1-5 F 5.1 (0.4) 0.35 (0.07) 6.9
