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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 °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.
Volume 270,
Number 22,
Issue of June 2, pp. 13534-13540, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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