A novel approach to investigating protein/protein interactions and their functions by TAP-tagged yeast strains and its application to examine yeast transcription machinery.
Tandem affinity purification (TAP) method combined with LC-MS/MS is the most accurate and reliable way to study the interaction of proteins or proteomics in a genome-wide scale. For the first time, we used a TAP-tag as a mutagenic tool to disrupt protein interactions at the specific site. Although lots of commonly used mutational tools exist to study functions of a gene, such as deletional mutations and site-directed mutagenesis, each method has its own demerit. To test the usefulness of a TAP-tag as a mutagenic tool, we applied a TAP-tag to RNA polymerase II, which is the key enzyme of gene expression and is controlled by hundreds of transcription factors even to transcribe a gene. Our experiment is based on the hypothesis that there will be interrupted interactions between Pol II and transcription factors owing to the TAP-tag attached at the C-terminus of each subunit of Pol II, and the abnormality caused by interrupted protein interactions can be observed by measuring a cell-cycle of each yeast strain. From ten different TAP-tagged strains, Rpb7- and Rpb12-TAP-tagged strains show severe defects in growth rate and morphology. Without a heterodimer of Rpb4/Rpb7, only the ten subunits Pol II can conduct transcription normally, and there is no previously known function of Rpb7. The observed defect of the Rpb7-TAP-tagged strain shows that Rpb7 forms a complex with other proteins or compounds and the interruption of the interaction can interfere with the normal cell cycle and morphology of the cell and nucleus. This is a novel attempt to use a TAP-tag as a proteomic tool to study protein interactions.
Jung
Junho
J
Department of Advanced Technology Fusion, Konkuk University, Seoul 143-701, Korea.
Ahn
Yeh-Jin
YJ
Kang
Lin-Woo
LW
D000939:Epitopes / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
D000939:Epitopes; D029701:Saccharomyces cerevisiae Proteins; D012319:RNA Polymerase II