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PMID: 3299061 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't

Isolation and characterization of temperature-sensitive RNA polymerase II mutants of Saccharomyces cerevisiae.

Molecular and cellular biology ·Vol. 7 ·No. 6 ·1987-06-00 ·Pages 2155-64

Himmelfarb HJ, Simpson EM, Friesen JD

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.

MeSH Terms
Amino Acid Sequence Animals Base Sequence Genes Genes, Fungal Genes, Lethal Kinetics Mutation Plasmids RNA Polymerase II/genetics,metabolism Saccharomyces cerevisiae/enzymology,genetics,growth & development Sequence Homology, Nucleic Acid Species Specificity Temperature
Chemicals
RNA Polymerase II
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Himmelfarb H J
Simpson E M
Friesen J D
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54 references, click to expand
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.7.6.2155-2164.1987
Published
1987-06-00
Pages
2155-64
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC365338
Subset
IM
External Links
PubMed source DOI: 10.1128/mcb.7.6.2155-2164.1987 PMC Full Text View journal details
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