Isolation and characterization of the fission yeast gene Sprpa12 + reveals that the conserved C-terminal zinc-finger reg
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O R I GI N A L P A P E R
Y. Imazawa á K. Imai á Y. Yao á K. Yamamoto K. Hisatake á M. Muramatsu á Y. Nogi
Isolation and characterization of the ®ssion yeast gene Sprpa12 + reveals that the conserved C-terminal zinc-®nger region is dispensable for the function of its product Received: 21 December 1999 / Accepted: 7 September 2000 / Published online: 8 December 2000 Ó Springer-Verlag 2000
Abstract RNA polymerase I of Saccharomyces cerevisiae contains a small subunit, A12.2, encoded by RPA12, that was previously shown to be involved in the assembly and/or stabilization of the largest subunit, A190, of RNA polymerase I. To examine whether an equivalent subunit is present in another eukaryotic RNA polymerase I, we have cloned a Schizosaccahromyces pombe cDNA that is able to complement the rpa12 mutation in S. cerevisiae. The gene, named Sprpa12+, encodes a polypeptide of 119 amino acids that shows 55% identity to S. cerevisiae A12. 2 over its entire length, including two zinc-®nger motifs. Disruption of the chromosomal Sprpa12+ gene shows that it is required for growth at higher temperatures but not at lower temperatures. Expression of Sprpa190+/nuc1+, which encodes the largest subunit of the S. pombe RNA polymerase I, from a multicopy plasmid can partially suppress the growth defect of the Sprpa12 disruptant at higher temperatures. These ®ndings suggest that A12.2 subunit is functionally and structurally conserved between S. cerevisiae and S. pombe. Finally, the analysis of mutants suggests that SpRPA12 requires the zinc-®nger domain in the Nterminal region but not the one in the C-terminal region for its function.
Communicated by H. Ikeda Y. Imazawa á K. Imai á Y. Yao á K. Hisatake M. Muramatsu á Y. Nogi (&) Department of Biochemistry, Saitama Medical School, 38 Morohongo, Moroyama, Iruma-Gun, Saitama, Japan E-mail: [email protected] Fax: +81-492-949751 Y. Imazawa á K. Imai CREST, Japan Science and Technology Corporation, Higasiikebukuro, Toshimaku, Tokyo, Japan K. Yamamoto Department of Oncology, Nagasaki University School of Medicine, Sakamoto, Nagasaki, Japan
Key words Schizosaccharomyces pombe á RNA polyme rase I Zinc-®nger Interspeci®c complementation
Introduction In eukaryotes, three distinct nuclear RNA polymerases, I, II and III (pol I, pol II and pol III) transcribe speci®cally ribosomal RNA (rRNA) genes, protein-encoding (mRNA) genes, and transfer RNA and 5S rRNA genes, respectively. Among eukaryotic RNA polymerases, the RNA polymerases of Saccharomyces cerevisiae have been studied most extensively; S. cerevisiae pol I consists of 14 subunits and all of the corresponding genes have been cloned and subjected to genetic and biochemical analysis (Thuriaux and Sentenac 1992 and references therein). Two large subunits, A190 and A135, are related to the b' and b subunits (Memet et al. 1986; Yano and Nomura 1991), while two smaller subunits, AC40 and AC19, shared by pol I and pol III show limited homologies to the a subunit of bacterial RNA polymerase (Mann et al. 1987; Dequard-Chablat et al. 1991). F
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