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内容記述 |
Three types of the nuclear RNA polymerase in eukaryotes are all multi-subunit enzymes, each consisting of more than 10 subunits. The RNA polymerase II is involved in the synthesis of mRNA and plays a key role in transcription of protein-coding genes. The RNA polymerase II from the fission yeast Schizosaccharomyces pombe is composed of 12 putative subunits. Sequence analysis indicated that, as in the case of other eukaryotic RNA polymerases, the largest subunit Rpb1 and the second largest subunit Rpb2 of S. pombe RNA polymerase II have notable homology with the β' and β subunits, respectively, of the prokaryotic RNA polymerases. The third largest subunit Rpb3 also has homology with the amino (N)-terminal domain of prokaryotic RNA polymerase α subunit (αNTD), which plays a key role in subunit assembly of this complex enzyme by providing the contact surfaces for both β and β' subunits. The Schizosaccharomyces pombe Rpb3 protein forms a core subassembly together with Rpb2 (the β homologue) and Rpb11 (the second αhomologue) subunits as in the case of the prokaryotic α2β complex. Several lines of evidence indicate that in addition to these core subunits, Rpb1, Rpb5, Rpb7 and Rpb8 also interact with Rpb3.<br />Sequence comparison between prokaryotes and eukaryotes also indicated Rpb3 has four conserved regions A to D. Among which two (A and D) are conserved among Rpb3 homologues from both prokaryotes (α subunits) and eukaryotes and two (B and C) are conserved only within eukaryotes. The regions A and D correspond to the N-terminal proximal and the C-terminal proximal regions of the αNTD, each playing critical roles in the contact with α and β subunits (motif-1) or the contact withα and β' subunits (motif- 2), respectively. On the other hands, two regions in the middle part of Rpb3 protein (Region B and C) do not exist in the prokaryotic RNA polymerases, Region B is specifically conserved in eukaryotic RNA polymerase II, and Region C is conserved among all three types of eukaryotic RNA polymerases. Therefore these two regions have been considered to be involved in eukaryote specific function(s). <br />In order to get insight into in vivo roles of Rpb3 in the assembly and function of RNA polymerase II, we have performed mutant studies for the S. pombe rpb3 gene of RNA polymerase II. First, we carried out a systematic search for temperature-sensitive (Ts-) or cold-sensitive(Cs-)S. pombe mutants with mutations throughout the rpb3 gene. After PCR mutagenesis of the entire rpb3 sequence, we isolated 9 Ts- and 3 Cs- mutants. After the sequence analysis of mutant rpb3 gene, each mutant was found to carry a single (or double in a few cases) mutation in one of the four regions (A to D) conserved among the eukaryotic subunit 3 homologues. The 3 Cs- mutations were all located in the region A, in agreement with its most important role in the assembly of prokaryotic RNA polymerase, while the Ts- mutations were scattered in all four regions. Since the metabolic stability of most Ts- mutant Rpb3 proteins was markedly reduced at a non-permissive temperature, we predict that these mutant Rpb3 proteins are defective in the assembly or the mutant RNA polymerases containing the mutant Rpb3 are thermolabile. The assembly state of mutant RNA polymerase II was tested by treating purified mutant RNA ploymerases with low concentrations of urea. One representative Ts- mutant complex was indeed dissociated more easily than the wild-type RNA polymerase. Moreover, the Ts- phenotype of all the mutants were suppressed to various extents by overexpression of Rpb11, the pairing subunit in the initial stage of RNA polymerase II assembly. We conclude that the majority of rpb3 mutations affect the subunit assembly of Rpb3, even though the extent of influence on the subunit assembly is different depending on the location of mutations.<br />Since the conserved regions B and C are unique for eukaryotic Rpb3 homologues, we thought that eukaryotic-specific transcription factors interact with one or both of these regions. We started the analysis of factor-dependent in vitro transcription activity for the mutant RNA polymerases. For this purpose, we constructed activator-dependent in vitro transcription system of S. pombe using S.cerevisiae GAL4-VP16. Results of the preliminary experiments indicate loss of the factor dependent transcription activity after heat treatment of the cell extracts from rpb3 mutants carrying mutations in the B or C region. |
要旨・審査要旨 / Abstract, Screening Result (310.3 kB)
