Structural features of the single-stranded DNA-binding protein MoSub1 fromMagnaporthe oryzae

Abstract

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 effect of phosphate ion on the ssDNA binding mode of MoSub1, a Sub1/PC4 homolog from rice blast fungus

“…As mentioned before, phosphorylation plays significant roles during PC4/Sub1 activity. However, the major potential phosphorylated region, the SEAC motif of PC4 and the N‐terminal of MoSub1, were absent in the crystal structures. Based on previous studies and the presence of a phosphate ion in our crystal structure, we assumed that the phosphate ion in this MoSub1–ssDNA structure may be a substitute for the phosphate group of phosphorylated amino acids.…”

“…1,8 MoSub1, a Sub1 homolog from rice blast fungus (Magnaporthe oryzae), also binds ssDNA tightly, and the crystal structures of MoSub1 and its complex with ssDNA have also been determined. 15,16 These structures show high similarity to the PC4 structure and they have a similar dimer interface and DNA-binding region, but with some differences in ssDNA conformation.…”

“…Expression and purification of the recombinant MoSub1 have been described previously. 15 The coding sequence of the MoSub1 ssDNA binding domain, MoSub1 , was sub-cloned into the pHAT2 expression plasmid (kindly supplied by Dr. Arie Geerlof, EMBO). Expression and purification were similar to those of the MoSub1 wild-type.…”

“…MoSub1 conformation in this structure was almost identical to that in a previous MoSub1-ssDNA structure (PDB entry: 4BHM), with a root-mean-square deviation of only 0.7 Å for C-alpha atoms and also close to its PC4 homolog. [13][14][15][16] Two monomers, consisting of a curved five stranded anti-parallel β-sheet followed by a 45 kinked α-helix, formed dimers using the kinked α-helix, which packed against the β-sheet of their respective partner in one ASU ( Figure 1A).…”

“…Sub1, which differs from PC4 in domain structure, particularly the presence of a C‐terminal extension, and its interaction with TFIIB but not TFIIA, shares very high homology in the DNA binding region . MoSub1, a Sub1 homolog from rice blast fungus ( Magnaporthe oryzae ), also binds ssDNA tightly, and the crystal structures of MoSub1 and its complex with ssDNA have also been determined . These structures show high similarity to the PC4 structure and they have a similar dimer interface and DNA‐binding region, but with some differences in ssDNA conformation.…”