Stimulation of transcription by mutations affecting conserved regions of RNA polymerase II

Abstract

Mutations that increase the low-level transcription of the Saccharomyces cerevisiae HIS4 gene, which results from deletion of the genes encoding transcription factors BAS1, BAS2, and GCN4, were isolated previously in SIT1 (also known as RPO21, RPB1, and SUA8), the gene encoding the largest subunit of RNA polymerase II (RNAPII). Here we show that sit1 substitutions cluster in two conserved regions of the enzyme which form part of the active site. Six sit1 mutations, affect region F, a region that is involved in transcriptional elongation and in resistance to alpha-aminatin. Four sit1 substitutions lie in another region involved in transcriptional elongation, region D, which binds Mg2+ ions essential for RNA catalysis. One region D substitution is lethal unless suppressed by a substitution in region G and interacts genetically with PPR2, the gene encoding transcription elongation factor IIS. Some sit1 substitutions affect the selection of transcriptional start sites at the CYC1 promoter in a manner reminiscent of that of sua8 (sua stands for suppression of upstream ATG) mutations. Together with previous findings which indicate that regions D and G are in close proximity to the 3' end of the nascent transcript and that region F is involved in the translocation process, our results suggest that transcriptional activation by the sit1 mutations results from alteration of the RNAPII active center.

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 lethal rpb1-17 substitution (43), the sua8-1 and 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. Reconstructed sit1 alleles carrying a region F mutation or the wild-type SIT1 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 sit1 alleles carrying region D mutations or the wild-type SIT1 alleles 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 sit1-8 allele. 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 on URA3 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 sit1-8 allele; −, absence of growth.

FIG. 4

Synthetic lethality of sit1-8D and ppr2. 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 on URA3, CEN/ARS]), shown on the left side of the plate or YF2278 (MATα rpo21::ADE2 ppr2::hisG [pJS121, RPO21 on URA3, 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).