The well studied general transcription cofactor Sub1/PC4 has multiple functions in transcription. It plays both a negative and a positive role in transcription initiation and is involved in elongation and downstream transcription processes and as a transcription reinitiation factor. MoSub1, a Sub1/PC4 orthologue from rice blast fungus, binds the single-stranded DNA dT(12) tightly with an affinity of 186 nM. The crystal structure of MoSub1 has been solved to 1.79 Å resolution. The structure of the protein shows high similiarity to the structure of PC4 and it has a similar dimer interface and DNA-binding region to PC4, indicating that MoSub1 could bind DNA using the same motif as other proteins of the Sub1/PC4 family. There are two novel features in the MoSub1 structure: a region N-terminal to the DNA-binding domain and a C-terminal extension. The region N-terminal to the DNA-binding domain of MoSub1 turns back towards the DNA-binding site and may interact directly with DNA or the DNA-binding site. The C-terminal extension region, which is absent in PC4, may not be capable of interacting with DNA and is one possible reason for the differences between Sub1 and PC4.

The MBP1 family proteins are the DNA binding subunits of MBF cell-cycle transcription factor complexes and contain an N terminal winged helix-turn-helix (wHTH) DNA binding domain (DBD). Although the DNA binding mechanism of MBP1 from Saccharomyces cerevisiae has been extensively studied, the structural framework and the DNA binding mode of other MBP1 family proteins remains to be disclosed. Here, we determined the crystal structure of the DBD of PCG2, the Magnaporthe oryzae orthologue of MBP1, bound to MCB–DNA. The structure revealed that the wing, the 20-loop, helix A and helix B in PCG2–DBD are important elements for DNA binding. Unlike previously characterized wHTH proteins, PCG2–DBD utilizes the wing and helix-B to bind the minor groove and the major groove of the MCB–DNA whilst the 20-loop and helix A interact non-specifically with DNA. Notably, two glutamines Q89 and Q82 within the wing were found to recognize the MCB core CGCG sequence through making hydrogen bond interactions. Further in vitro assays confirmed essential roles of Q89 and Q82 in the DNA binding. These data together indicate that the MBP1 homologue PCG2 employs an unusual mode of binding to target DNA and demonstrate the versatility of wHTH domains.

PC4, a well-known general transcription cofactor, has multiple functions in transcription and DNA repair. Residue W89, is engaged in stacking interactions with DNA in PC4, but substituted by tyrosine in some PC4 orthologous proteins. In order to understand the consequences and reveal the molecular details of this substitution we have determined the crystal structures of the PC4 orthologue MoSub1 and a PC4 W89Y mutant in complex with DNA. In the structure of MoSub1-DNA complex, Y74 interacts directly with a single nucleotide of oligo DNA. By comparison, the equivalent residue, W89 in wild type PC4 interacts with two nucleotides and the base of the second nucleotide has distinct orientation relative to that of the first one. A hydrophobic patch around W89 that favours interaction with two nucleotides is not formed in the PC4 W89Y mutant. Therefore, the change of the surface hydrophobicity around residue 89 results in a difference between the modes of DNA interaction. These results indicate that the conserved Y74 in MoSub1 or W89 in PC4, are not only key residues in making specific interactions with DNA but also required to determine the DNA binding mode of PC4 proteins.