[Mapping the interaction site of Rpb2 and Rpb3 subunit of fission yeast RNA polymerase II].
To map the interacting site of subunit Rpb2 to subunit Rpb3 of RNA polymerase II in fission yeast Schizosaccharomyces pombe, the yeast two-hybrid system was employed in this paper to screen the interacting clones between Rpb2 and Rpb3.4 fragments of Rpb2 cDNA were cloned into the Ga14 BD vector pAS2. The 4 clones were named as pAS2 Rpb2-1, 2-2, 2-3 and 2-4, respectively. The complete cDNA of Rpb3 was cloned into the Gal 4 AD vector pGADGH. The clone was named as pGADGH Rpb3. The two-hybrid plasmids pGADGH Rpb3 and pAS2Rpb2-1, 2-2, 2-3 or 2-4 respectively were cotransformed into host cell yeast Y190. The interaction positive cotransformants were identified by beta-gal activity assay. The beta-gal positive cotransformants were selected from pGADGH Rpb3 and pAS2Rpb2-4 two-hybrid system. DNA sequencing and alignment results showed that the interacting site of Rpb2 to Rpb3 located within the fragment from base 2701 to 2966 of Rpb2 cDNA, or within the C-termini polypeptide from amino acid 902 to 989 of Rpb2 protein.
Qu
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Cancer Institute, Zhejiang University, Hangzhou 310009, China.
Zheng
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Gu
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H
Shi
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B
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid; D014169:Transformation, Bacterial
D018076:DNA, Complementary; D004271:DNA, Fungal; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D012319:RNA Polymerase II; C112110:RPB3 protein, S cerevisiae; C491782:rpb3 protein, S pombe