Mutational analysis of the RNase-like domain in subunit 2 of fission yeast RNA polymerase II.
Local sequence similarity exists between the subunit 2 of eukaryotic RNA polymerases II and the barnase-type bacterial RNases. The RNase-like domain from the Rpb2 of Schizosaccharomyces pombe was expressed in Escherichia coli as a GST fusion protein and examined for its RNase activity. When the GST fusion protein was incubated in vitro with 32P-labeled RNA, the RNA degradation activity was less than 0.1%, if any, of the level of synthetic barnase. In order to check the in vivo function of this region, we constructed two mutant rpb2 alleles, rpb2E357A and rpb2H386L, each carrying a single amino acid substitution at the site corresponding to one of the three essential amino acid residues forming the catalytic site in barnase (mutation of barnase at the corresponding sites results in complete loss of RNase activity) and five other mutant rpb2 alleles, each carrying a single mutation at various positions within the RNase-like domain but outside the putative catalytic site for RNase activity. When these mutant rpb2 alleles were expressed in an rpb2-disrupted S. pombe strain, all the mutants grew as well as the wild-type parent and did not show any clear defective phenotypes. These results suggest either that the RNase-like domain in Rpb2 does not function as an RNase in vivo or that the RNase activity of this domain, if present at all, is not essential for cell growth.
Kawagishi-Kobayashi
M
M
Department of Molecular Genetics, National Institute of Genetics, Shizuoka, Japan.
Yamamoto
M
M
Ishihama
A
A
D001483:Base Sequence; D001665:Binding Sites; D004271:DNA, Fungal / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
D004271:DNA, Fungal; D011993:Recombinant Fusion Proteins; D012319:RNA Polymerase II; D012260:Ribonucleases; D001616:beta-Galactosidase