Isolation and characterization of temperature-sensitive RNA polymerase II mutants of Saccharomyces cerevisiae.
Open Access
- 1 June 1987
- journal article
- research article
- Published by Taylor & Francis in Molecular and Cellular Biology
- Vol. 7 (6), 2155-2164
- https://doi.org/10.1128/mcb.7.6.2155
Abstract
Three independent, recessive, temperature-sensitive (Ts-) conditional lethal mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II (RNAP II) have been isolated after replacement of a portion of the wild-type gene (RPO21) by a mutagenized fragment of the cloned gene. Measurements of cell growth, viability, and total RNA and protein synthesis showed that rpo21-1, rpo21-2, and rpo21-3 mutations caused a slow shutoff of RNAP II activity in cells shifted to the nonpermissive temperature (39 degrees C). Each mutant displayed a distinct phenotype, and one of the mutant enzymes (rpo21-1) was completely deficient in RNAP II activity in vitro. RNAP I and RNAP III in vitro activities were not affected. These results were consistent with the notion that the genetic lesions affect RNAP II assembly or holoenzyme stability. DNA sequencing revealed that in each case the mutations involved nonconservative amino acid substitutions, resulting in charge changes. The lesions harbored by all three rpo21 Ts- alleles lie in DNA sequence domains that are highly conserved among genes that encode the largest subunits of RNAP from a variety of eucaryotes; one mutation lies in a possible Zn2+ binding domain.This publication has 58 references indexed in Scilit:
- Characterization and mutational analysis of a cluster of three genes expressed preferentially during sporulation of Saccharomyces cerevisiae.Molecular and Cellular Biology, 1986
- An RNA polymerase I enhancer in Saccharomyces cerevisiae.Molecular and Cellular Biology, 1986
- Homology between RNA polymerases of poxviruses, prokaryotes, and eukaryotes: nucleotide sequence and transcriptional analysis of vaccinia virus genes encoding 147-kDa and 22-kDa subunits.Proceedings of the National Academy of Sciences, 1986
- Potential Metal-Binding Domains in Nucleic Acid Binding ProteinsScience, 1986
- Isolation of the SUP45 omnipotent suppressor gene of Saccharomyces cerevisiae and characterization of its gene product.Molecular and Cellular Biology, 1985
- Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMPNature, 1985
- Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectorsGene, 1985
- A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistanceMolecular Genetics and Genomics, 1984
- Construction and genetic characterization of temperature-sensitive mutant alleles of the yeast actin gene.Proceedings of the National Academy of Sciences, 1984
- Identification, molecular cloning, and mutagenesis of Saccharomyces cerevisiae RNA polymerase genes.Proceedings of the National Academy of Sciences, 1984