Identification of the Single-stranded DNA Binding Surface of the Transcriptional Coactivator PC4 by NMR

Abstract

The C-terminal domain of the eukaryotic transcriptional cofactor PC4 (PC4 CTD ) is known to bind with nanomolar affinity to single-stranded (ss)DNA. Here, NMR is used to study DNA binding by this domain in more detail. Amide resonance shifts that were observed in a 1 H 15 N-HSQC-monitored titration of 15 N-labeled protein with the oligonucleotide dT 18 indicate that binding of the nucleic acid occurs by means of two anti-parallel channels that were previously identified in the PC4 CTD crystal structure. The ␤-sheets and loops that make up these channels exhibit above average flexibility in the absence of ssDNA, which is reflected in higher values of T 1 , reduced heteronuclear nuclear Overhauser effects and faster deuterium exchange rates for the amides in this region. Upon ssDNA binding, this excess flexibility is significantly reduced. The binding of ssDNA by symmetry-related channels reported here provides a structural rationale for the preference of PC4 CTD for juxtaposed single-stranded regions (e.g. in heteroduplexes) observed in earlier work.The eukaryotic general transcriptional cofactor PC4 is known to enhance activated in vitro and in vivo transcription from various RNA polymerase II promoters, in concert with members of all major classes of transcriptional activators (1-3). The activator-dependent stimulation of transcription by PC4 has been shown to originate mostly from increased recruitment of basal transcription factors during the early stages of preinitiation complex (PIC) 1 formation (4). It is likely that this effect is caused by the interactions that have been reported between PC4 and the activation domains of promoter-binding activator proteins on the one hand, and basal factors (including TFIIA) on the other (1, 3). Thus, PC4 appears to act as a bridging factor that stabilizes the preinitiation complex.Recently it has become clear that PC4 has several additional functions. In the first place, the protein was found to be able to repress transcription under specific conditions (5-7). Repression at moderate PC4 concentrations is observed in minimal in vitro transcription systems, that normally do not require the basal factor TFIIH (5, 6), and with respect to aspecific transcription initiation by RNA polymerase II from DNA ends and unwound regions (6). Repression is not observed in full-factor in vitro transcription, because TFIIH is able to overcome repression by PC4 (5, 6). Thus, PC4 appears to act as what could be called a fidelity-enhancing factor for RNA polymerase II transcription initiation by repressing adventitious transcription from incomplete PICs on the one hand, and on the other hand enhancing transcription from fully assembled PICs, i.e. those that include TFIIH, generally believed to be the last factor to enter the PIC during assembly (8). Surprisingly, PC4 has recently also been found to associate in vivo with the RNA polymerase III transcription factor TFIIIC and to stimulate transcription by RNA polymerase III by increasing the rate of reinitiation (9). Interestingly, copurific...

Strange bedfellows: polyadenylation factors at the promoter

“…The ability of PC4 to interact with GTFs is supported further by observations that the protein facilitates early steps in preinitiation complex assembly, but represses transcription from such incomplete complexes, suggesting that PC4 may have multiple roles leading to formation of activated transcription complexes (Malik et al 1998;Werten et al 1999). PC4 also binds both single-and double-stranded DNA, with an especially high affinity for melted regions in duplex DNA (Werten et al 1998(Werten et al , 1999, although how this contributes to activity remains unknown.…”

“…As mentioned above, PC4 was discovered as a coactivator; that is, as a protein that allows sequence-specific transcription activators to function in reconstituted transcription reactions, presumably by facilitating interactions between the activator and GTFs (Ge and Roeder 1994;Kretzschmar et al 1994). The ability of PC4 to interact with GTFs is supported further by observations that the protein facilitates early steps in preinitiation complex assembly, but represses transcription from such incomplete complexes, suggesting that PC4 may have multiple roles leading to formation of activated transcription complexes (Malik et al 1998;Werten et al 1999). PC4 also binds both single-and double-stranded DNA, with an especially high affinity for melted regions in duplex DNA (Werten et al 1998(Werten et al , 1999, although how this contributes to activity remains unknown.…”

Structural Properties of the Promiscuous VP16 Activation Domain

“…The VP16 and PC4 proteins were prepared and quantified essentially as described before (Jonker et al, submitted for publication, 34,35). Uniformly labeled ( 15 N, 15 N/ 13 C) VP16ad (residues 412-490) was obtained by using 0. .…”

The C-terminal Domains of Vertebrate CstF-64 and Its Yeast Orthologue Rna15 Form a New Structure Critical for mRNA 3′-End Processing

“…The analysis of the 15 N-HSQC NMR spectrum of full-length PC4 was consistent with a structured domain at the C terminus (57,58), whereas the resonances corresponding to the N-terminal fragment indicated an unfolded polypeptide. We then mixed 15 N-labeled PC4 with unlabeled CstF-64, and vice versa, in several possible combinations (supplemental Table II), but the data revealed no significant spectral changes (data not shown).…”