Sequence divergence of the RNA polymerase shared subunit ABC14.5 (Rpb8) selectively affects RNA polymerase III assembly in Saccharomyces cerevisiae

Nucleic Acids Res. 1999 Feb 15;27(4):1047-55. doi: 10.1093/nar/27.4.1047.

Abstract

ABC14.5 (Rpb8) is a eukaryotic subunit common to all three nuclear RNA polymerases. In Saccharomyces cerevisiae, ABC14.5 (Rpb8) is essential for cell viability, however its function remains unknown. We have cloned and characterised the Schizosaccharomyces pombe rpb8(+) cDNA. We found that S.pombe rpb8, unlike the similarly diverged human orthologue, cannot substitute for S.cerevisiae ABC14. 5 in vivo. To obtain information on the function of this RNA polymerase shared subunit we have used S.pombe rpb8 as a naturally altered molecule in heterologous expression assays in S.cerevisiae. Amino acid residue differences within the 67 N-terminal residues contribute to the functional distinction of the two yeast orthologues in S.cerevisiae. Overexpression of the S.cerevisiae largest subunit of RNA polymerase III C160 (Rpc1) allows S.pombe rpb8 to functionally replace ABC14.5 in S.cerevisiae, suggesting a specific genetic interaction between the S.cerevisiae ABC14.5 (Rpb8) and C160 subunits. We provide further molecular and biochemical evidence showing that the heterologously expressed S.pombe rpb8 molecule selectively affects RNApolymerase III but not RNA polymerase I complex assembly. We also report the identification of a S.cerevisiae ABC14.5-G120D mutant which affects RNA polymerase III.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • DNA, Fungal
  • Gene Expression
  • Humans
  • Molecular Sequence Data
  • Mutagenesis
  • RNA Polymerase I / genetics
  • RNA Polymerase I / metabolism
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism*
  • RNA Polymerase III / genetics
  • RNA Polymerase III / metabolism*
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Schizosaccharomyces / enzymology*
  • Schizosaccharomyces / genetics
  • Sequence Homology, Amino Acid

Substances

  • DNA, Fungal
  • RNA Polymerase II
  • RNA Polymerase I
  • RNA Polymerase III

Associated data

  • GENBANK/Y07643
  • GENBANK/Y07644