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J. Microbiol. Biotechnol. 2008; 18(4): 631-638

Published online April 28, 2008

Copyright © The Korean Society for Microbiology and Biotechnology.

A Novel Approach to Investigating Protein/Protein Interactions and Their Functions by TAP-tagged Yeast Strains and its Application to Examine Yeast Transcription Machinery

Jung, Junho , Yeh-Jin Ahn 1 and Lin-Woo Kang 1*

Department of Advanced Technology Fusion, Konkuk University, Seoul 143-701, Korea, 1Division of Life Science, College of Natural Sciences, Sangmyung University, Seoul 110-743, Korea

Abstract

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.

Keywords: Tandem affinity purification (TAP), RNA polymerase II, protein/protein interactions (PPI), transcription, structural proteomics