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Protein interaction cloning in yeast of the mouse third largest RNA polymerase II subunit, mRPB31
I V Korobko1, K Yamamoto, Y Nogi
1Department of Biochemistry, Saitama Medical School, Moroyama, Iruma-gun, Japan.
Abstract:
The cDNA encoding a protein that interacts with the mouse homologue of the yeast RNA polymerase II (polII) subunit, RPB11, and the human polII subunit, hRPB14, has been isolated by protein interaction cloning. Its deduced amino acid sequence has 96% homology to the human third largest polII subunit, hRPB33 [Pati and Weissman (1990) J. Biol. Chem. 265, 8400 8405]. Therefore, we conclude that the cloned cDNA encodes the mouse third largest polII subunit, mRPB31. Isolation of cDNA by protein interaction cloning provides evidence supporting the hypothesis, first proposed for human polII assembly [Pati (1994) Gene 145, 289-292], that the mRPB31/mRPB14 heterodimer, rather than the mRPB31 homodimer, forms in the mouse polII assembly. Indeed, in the yeast two-hybrid system, mRPB31 was shown to fail to form homodimer.
Insights
Researchers identified the mouse RPB31 subunit of RNA polymerase II (polII) using protein interaction cloning. This finding supports the hypothesis that RPB31 forms a heterodimer with RPB14 during mouse polII assembly.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- RNA polymerase II (polII) is crucial for gene transcription.
- Understanding the assembly of polII subunits is key to comprehending its function.
- Previous studies suggested specific subunit interactions in human polII assembly.
Purpose of the Study:
- To isolate and identify the mouse homologue of the third largest polII subunit.
- To investigate the assembly mechanism of mouse polII, specifically the role of the mRPB31 subunit.
- To test the hypothesis that mRPB31 forms a heterodimer with mRPB14.
Main Methods:
- Protein interaction cloning was used to isolate the cDNA encoding a protein interacting with mouse RPB11 and human hRPB14.
- Sequence homology analysis was performed to identify the isolated protein.
- The yeast two-hybrid system was employed to assess homodimerization of mRPB31.
Main Results:
- The cDNA encoding mouse RPB31 (mRPB31), the third largest polII subunit, was successfully isolated.
- mRPB31 shares 96% homology with the human third largest polII subunit, hRPB33.
- mRPB31 failed to form homodimers in the yeast two-hybrid system, indicating it does not self-associate.
Conclusions:
- The cloned cDNA encodes the mouse third largest polII subunit, mRPB31.
- Protein interaction cloning provides evidence for the mRPB31/mRPB14 heterodimer model in mouse polII assembly.
- The findings support the hypothesis that heterodimer formation, not homodimer formation, is critical for mouse polII assembly.