Russian Journal of Bioorganic Chemistry, Vol. 23, No. 2, 1997, p. 100

Cloning of an RNA Polymerase Subunit cDNA of the Fission Yeast Schizosaccharomyces pombe by Heterospecific Complementation in Saccharomyces cerevisiae

G. V. Shpakovski* ^, **, ¹ E. N. Lebedenko*, and P. Thuriaux***

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow , GSP-7, 117871 Russia ,

** Institute of Bioorganic Chemistry, Belarussian Academy of Sciences , Minsk , Belarus ,

*** Service de Biochimie et Génétique Moléculaire, Commissariat à l’Energie Atomique (Saclay), Gif-sur-Yvette , France

Abstract: The rpb10 cDNA of the fission yeast Schizosaccharomyces pombe, encoding one of the five small subunits common to all three nuclear DNA-dependent RNA polymerases, was isolated from an expression cDNA library by two independent approaches: PCR-based screening and direct suppression by means of heterospecific complementation of a temperature-sensitive mutant defective in the corresponding gene of Saccharomyces cerevisiae. The cloned Sz. pombe cDNA encodes a protein Rpb10 of 71 amino acids with an M of 8275 Da, sharing 51 amino acids (71% identity) with the subunit ABC10 of RNA polymerases I–III from S. cerevisiae. All eukaryotic members of this protein family have the same general organization featuring two highly conserved motifs (RCFT/SCGK and RYCCRRM) around an atypical zinc finger and an additional invariant HVDLIEK motif toward the C-terminal end. The last motif is only characteristic for homologs from eukaryotes. In keeping with this remarkable structural conservation, the Sz. pombe cDNA also fully complemented aS. cerevisiae deletion mutant lacking subunit ABC10 (null allele rpb10- 1::HIS3).

Key words: eukaryotic transcription, RNA polymerases I–III, common subunits, fission yeast, rpb10 gene, heterospecific complementation, suppressors, zinc finger

Russian Journal of Bioorganic Chemistry, Vol. 23, No. 3, 1997, p. 211–214

The First Member of a Novel Family of Protein Factors of Eukaryotic Transcription Discovered by the Heterospecific Complementation

G. V. Shpakovski ¹ and E. N. Lebedenko

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, GSP-7 Moscow , 117871 Russia

Abstract–The cDNA of a previosly uncharacterized gene fet5 (factor of eukaryotic transcription, clone no. 5) of the fussion yeast Schizosaccharomyces pombe was cloned by the heterospecific complementation of a conditional mutant of Saccharomyces cerevisiae defective in the function of the RNA polymerases I–III common subunit ABC10β. The gene encodes a new factor of eukaryotic transcription, Fet5, the first member of a superfamily of proteins for which the area of functioning is determined. The Fet5-superfamily consists of three distinct families of proteins highly evolutionarily conserved and videly spread among eukaryotes. Features of the Fet5 amino acid sequence suggest that it belongs to ATP/Gtp-binding proteins.

Key words: RNA polymerases I–III common subunit ABC10β, multicopy extragenic suppressor, heterospecific suppressor, Schizosaccharomyces pombe, fet5 gene, transcription factor, purine nucleotides binding proteins, new protein superfamily

Russian Journal of Bioorganic Chemistry, Vol. 23, No. 5, 1997, p. 412

Molecular Cloning and Characterization of cDNA of the rpb10 ⁺ Gene Encoding the Smallest Subunit of Nuclear RNA Polymerases of Schizosaccharomyces pombe ¹

G. V. Shpakovski ² and E. N. Lebedenko

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow , GSP-7, 117871 Russia

Abstract: The full-length cDNA of the rpc10 ⁺ gene encoding mini-subunit Rpc10, which is common for all three nuclear RNA polymerases of the fission yeast Schizosaccharomyces pombe, was cloned and sequenced. The Rpc10 subunit of Sz. pombe and its homologs from S. cerevisiae and H. sapiens are positively charged pro-teins with a highly conserved C-terminal region and an invariant zinc-binding domain (Zn-finger) of a typical amino acid composition: YxCx₂ Cx₁₂RCx₂ CGxR. Functional tests on heterospecific complementation, using tetrad analysis or plasmid shuffling, showed that the Rpc10 subunit of Sz. pombe can successfully replace the homologous ABC10 subunit in nuclear RNA polymerases I–III of S. cerevisiae.

Key words: nuclear RNA polymerases I–III, common subunits; fission yeast, rpc10 ⁺ gene; heterospecific complementation; zinc-binding domains

Russian Journal of Bioorganic Chemistry, Vol. 23, No. 12, 1997, p. 874

Molecular Cloning of the rpb5 ⁺ , rpb7 ⁺ , and rpb11 ⁺ Genes of the Fission Yeast Schizosaccharomyces pombe : Completing the Primary Structure of All Indispensable Subunits of Its RNA Polymerase II ¹

G. V. Shpakovski ² and E. N. Lebedenko

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,ul. Miklukho-Maklaya 16/10, GSP-7 Moscow , 117871 Russia

Abstract: Genes encoding subunits Rpb5, Rpb7, and Rpb11 of RNA polymerase II of the fission yeast Schizosaccharomyces pombe have been cloned and sequenced to complete the structural characterization of all indispensable components of one of the most important enzymes of the transcription machinery of the fission yeast.