Analysis of protein -protein interactions in transcription initiation complexes: Site-specific protein-protein photocrosslinking.

摘要

TATA box binding protein (TBP) is a key factor for transcription initiation by all three eukaryotic RNA polymerases. The mechanism of transcription initiation includes direct protein-protein interactions between TBP and transcription machinery. Helix 2 of TBP's N-terminal repeat has been implicated as an important target for transcription regulation, involving interactions with TFIIA, hTAF II250, and NC2.;The overall objective of this thesis is analysis of protein-protein interactions between the H2 region of TBP and various factors using newly developed photocrosslinking reagents. Chapter II introduces ultrasensitive, nonradioactive photochemical crosslinking agents (biotin-EDA-AET and biotin-PEG-AET), and development of the crosslink detection method. The established method allows a sensitive detection of protein-protein interaction within a multiprotein complex.;Chapter III describes a crosslinking study to investigate protein-protein interactions of hTBPc-TFIIA-DNA complex. The results show that the H2 of hTBPc is in close proximity with alpha/beta subunit of TFIIA. The crosslinked sites are mapped to a nonconserved region (residues 80--95), suggesting that there is protein-protein interaction between H2 and the nonconserved region of alpha/beta subunit of TFIIA.;Chapter IV describes the identification of protein-protein interactions of the TBP-TFIIB-DNA complex. The crosslinking and subsequent mapping results show that the H2 region is in direct physical proximity to the N-terminal linker region of TFIIB. The chapter also introduces molecular interplay between TFIIA and TFIIB on the H2 region in a preinitiation complex.;Chapter V describes the identification of protein-protein interactions between H2 and NC2alpha in the TBP-NC2-DNA complex. The segment of the C-terminal part of NC2alpha is spatially close to the H2 region in the complex, implying an inhibitory role in TBP-TFIIA interaction.;Chapter VI describes an introduction to a high-resolution crosslink mapping technique using new crosslinking agent, iminobiotin-EDA-AET. The method will be useful to achieve detailed information on protein-protein interaction.