Functional analysis of RNA polymerase II Rpb3 mutants of the fission yeast Schizosaccharomyces pombe
J Mitobe1, H Mitsuzawa, A Ishihama
1Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan.
Abstract:
The RNA polymerase II (Pol II) of Schizosaccharomyces pombe is composed of 12 subunits. Subunit Rpb3 has sequence homology with the N-terminal domain of the prokaryotic alpha subunit, which plays a key role in RNA polymerase assembly. Together with the Rpb2 (the beta homologue) and Rpb11 (the second alpha homologue) subunits, Rpb3 constitutes a core subassembly (Rpb2-Rpb3-Rpb11) which corresponds to the the alpha2beta assembly intermediate of prokaryotic RNA polymerase. For the functional mapping of Rpb3, we made a collection of 12 heat-sensitive (Ts) or cold-sensitive (Cs) S. pombe mutants, each carrying a single mutation in one of the four conserved regions of Rpb3. The altered functions of six representative Pol II mutants containing the mutant Rpb3 were analyzed in vitro using an improved version of the GAL4-VP16 activator-dependent transcription system catalyzed by S. pombe cell extracts. The transcription activity by the extracts from Rpb3 mutants decreased to varying extents after heat treatment; but the extracts from Rpb3 mutants which had mutations in the eukaryote-specific conserved regions B and C regained their activity by the addition of GAL4-VP16, to a larger extent than those from the region A and D mutants. We propose that both terminal regions (A and D) play important roles in RNA polymerase assembly, while the central portion (regions B and C) is involved in activated transcription.
More Related Videos
09:04Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis
Published on: July 26, 2018
08:07Analysis of the Expression and Complexes Assembly of the Mitochondrial Respiratory Chain Proteins in the Fission Yeast Schizosaccharomyces pombe
Published on: May 2, 2025
Related Concept Videos
Bacterial RNA Polymerase
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
Eukaryotic RNA Polymerases
All three eukaryotic RNAPs require specific transcription factors, of which the...
RNA Polymerase II Accessory Proteins
Transcription Initiation
The promoters and enhancers and their accessory proteins allow tight regulation of...
Bacterial RNA Polymerase
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
Eukaryotic RNA Polymerases
All three eukaryotic RNAPs require specific transcription factors, of which the...
