The association of three subunits with yeast RNA polymerase is stabilized by A14.
RNA polymerase I of Saccharomyces cerevisiae is composed of 14 subunits. All of the corresponding genes have been cloned with the exception of the RPA14 gene encoding A14, a specific polypeptide of this enzyme. We report the cloning and the characterization of RPA14. The A14 polypeptide was separated from the other RNA polymerase I subunits by reverse-phase high pressure liquid chromatography and digested with proteinase K. Based on the amino acid sequence of one of the resulting peptides, a degenerate oligonucleotide was synthesized and used to isolate the RPA14 gene from a yeast subgenomic DNA library. RPA14 is a single copy gene that maps to chromosome IV and is flanked by CYP1 and HOM2. Disruption of RPA14 is not lethal, but growth of the rpa14::URA3 mutant strain is impaired at 37 and 38 degrees C. RNA polymerase I was purified from the rpa14::URA3 strain. After two purification steps, the enzyme did not contain the subunits A14, ABC23, and A43. This form of the enzyme was not active in a nonspecific in vitro transcription assay. These results demonstrate that A14 is a genuine subunit of RNA polymerase I and suggest that A14 plays a role in the stability of a subgroup of subunits.
Smid
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Service de Biochimie et de Génétique Moléculaire, CEA Saclay, Gif-sur-Yvette, France.
Riva
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Bouet
F
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Sentenac
A
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Carles
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C
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004795:Enzyme Stability; D005804:Genes, Lethal; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012318:RNA Polymerase I / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins
D004271:DNA, Fungal; D011506:Proteins; C093847:RPA14 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012318:RNA Polymerase I