Nonradioactive, ultrasensitive site-specific protein–protein photocrosslinking: interactions of α-helix 2 of TATA-binding protein with general transcription factor TFIIA and transcriptional repressor NC2

Abstract

We have developed an approach that enables nonradioactive, ultrasensitive (attamole sensitivity) site-specific protein–protein photocrosslinking, and we have applied the approach to the analysis of interactions of α-helix 2 (H2) of human TATA-element binding protein (TBP) with general transcription factor TFIIA and transcriptional repressor NC2. We have found that TBP H2 can be crosslinked to TFIIA in the TFIIA–TBP–DNA complex and in higher order transcription–initiation complexes, and we have mapped the crosslink to the ‘connector’ region of the TFIIA α/β subunit (TFIIAα/β). We further have found that TBP H2 can be crosslinked to NC2 in the NC2–TBP–DNA complex, and we have mapped the crosslink to the C-terminal ‘tail’ of the NC2 α-subunit (NC2α). Interactions of TBP H2 with the TFIIAα/β connector and the NC2α C-terminal tail were not observed in crystal structures of TFIIA–TBP–DNA and NC2–TBP–DNA complexes, since relevant segments of TFIIA and NC2 were not present in truncated TFIIA and NC2 derivatives used for crystallization. We propose that interactions of TBP H2 with the TFIIAα/β connector and the NC2α C-terminal tail provide an explanation for genetic results suggesting importance of TBP H2 in TBP–TFIIA interactions and TBP–NC2 interactions, and provide an explanation—steric exclusion—for competition between TFIIA and NC2.

Structural basis for recognition and remodeling of the TBP:DNA:NC2 complex by Mot1

“…NC2 prevents assembly of TBP with other general transcription factors ( Meisterernst and Roeder, 1991 ; Inostroza et al, 1992 ; Cang et al, 1999 ; Kim et al, 2008 ) but is retained on DNA in the absence of functional Mot1 in vitro and in vivo ( Geisberg et al, 2002 ; Schluesche et al, 2007 ; Van Werven et al, 2008 ; de Graaf et al, 2010 ). While Mot1 forms a stable complex with DNA-bound TBP and NC2, it is fully capable of disrupting this complex in the presence of ATP.…”

The T Allele of rs8075977 in the 5′-Flanking Region of the PEDF Gene Is Associated with Reduced Risk of Coronary Artery Disease in Elderly Chinese Men

“…TFIIA, the general transcription factor, can activate accurate transcription initiation through stabilization of TATA box-bound TBP (TATA-binding protein) (Suzuki et al 2015). In addition, TFIIA can regulate responses to transcriptional activators and be recruited by transcriptional activators to specific target promoters (Kim et al 2008). Interestingly, TFIIA was reported to function in the cellular mechanism of defense against ROS, suggesting its role in the response to oxidative stress (Kraemer et al 2006), which is in agreement with the anti-oxidant property of PEDF (Rychli et al 2009).…”

Benefits of using molecular structure and abundance in phylogenomic analysis

“…The troublesome task of solving the problem of orthology and paralogy in sequence analysis (Kim et al, 2008) is inapplicable to domains at any level of structural abstraction, which by definition include all domain sequence variants (Murzin et al, 1995). According to definitions of the structural classification of proteins (SCOP), protein domains that belong to the same family can be orthologous to each other.…”