Zero-Length Crosslinking Study on Interactions of TBP, GAL4-AH, and TFIIB in the Preinitiation Complex

전사개시전 복합체에서 TBP, GAL4-AH, TFIIB의 상호작용에 대한 Zero-Length Crosslinking 실험

A zero-Iength croessinking procedure for studying protein-protein interactlons in preinitiation complex has heen developed. Preinltiadon complexes were assembled with immobilized DNA templates coupled to metal beads. Pudfied complexes were dIrectly crosslinked by 1-ethyl-3.(3-dimethylaminopropyl)carbodlimide (EDC). The reaction was stopped by addition of $\beta$-mercaptoethanol, and the complexes were isolated from EDC immedIately. An appllcatlion of this method with a preinltlation complex assembled with TBP, TFIIB, and GAL4-AH demonstrated that ThP dIrectly interacts with GAL4-AH and TFIIB in the prelnitlatlon complex. However, croeslinked produd between GAL4-AH and ThilB was not observed. These resutts lndlcate that GAL4-AH does not stably Interact with TFIIB In the GAL4-AH-TFIIB-TBP-DNA prelnitlatlon complex.

전사개시전 복합체(preinitiation complex)에서 단백질간의 상호작용을 연구하시 위해 zero-Iength croessinking방법을 이용하였다. DNA template가 결합한 금속 지지체를 이용하여 in vitro에서 전사개시전 복합체를 형성시키고, 이렇게 만든 복합체를 1-ethyl-3-(3-dimethylaminopropyl) carbodlimide(EDC)로 croessinking시켰다. $\beta$-mercaptoethanol를 첨가하여 croessinking반응을 멈추게 한 다음, EDC로부터 전사개시전 복합체를 분리하였다. TBF,TFIIB,GAL4-AH등으로 구성된 전사개시전 복합체에 이러한 방법을 적용함으로써 TBF가 GAL4-AH,TFIIB와 각각 직접적으로 결합하고 있음을 규명하였다. 반면에 GAL4-AH와 TFIIB가 croessinking된 산물은 확인할 수 없었다. 이러한 결과들은 GAL4-AH,TFIIB,TBP,DNA로 구성된 전사개시전 복합체에서 GAL4-AH는 TFIIB와 안정적인 결합을 하고 있지 않음을 암시한다.