The Determinant Amino Acids Of DNA Binding Domain In ERF-DNA Specific Interaction

Ethylene-responsive element (ERE) binding factors (ERFs) are the productsof a large multigene family and are involved in many critical processes of plantdevelopment through gene regulation and in mediating signal transductionpathways in response to environmental stress. ERFs were first identified intobacco, and originally designated as ERE binding proteins (EREBPs), but werelater renamed ERE binding factors (ERFs). Bioinformatics analysis of theArabidopsis genome has identified some 124 genes encoding the ERFs [in whichERFs are termed the EREBP subfamily under the AP2/EREBP family].Sincethen, an increasing number of ERFs from various plants have been reportedwhich are divergent except for a highly conserved DNA binding domain, theso-called ERF domain, of 58 or 59 amino acids. Whereas ERF domains arehighly conserved, EREBPs exhibit some divergence. At present, the transcription factor NtERF2 is the ethylene response element intobacco which has been well studied. Arabidopsis ethylene-responsive elementbinding factor 1 (AtERF1) is so far the only ERF in this super family whose ERFdomain solution structure has been determined by NMR. The fig.1.3 shows theamino acids in AtERF1 DBD that bind directly to the bases of GCC box.As we described previous, lots of EREBPs have been shown to be capable ofbinding in vitro to GCC box or any other DNA motif with similar structure, suchas DRE motif. AtERF1 was demonstrated to be able to bind to GCC box, wasalso shown to bind to another cis-acting element with the core sequence CCGAC,and named DRE. In contrast, the ERF domain fragment of NtERF2 does notinteract with the DRE motif, but restrictedly recognizes GCC box. Thus, throughsequence alignment, we selected the NtERF2 and AtERF1 as our researchobjects whose DBD are high homologous.However, there is only five amino aciddifference in the DBD between NtERF2 and AtERF1, one in the β-sheet, three inthe α-helix, and one in the relaxed region. The two transcription factors exhibitedan absolute difference in binding sequence specificity to DNA.Conservativeness and DNA binding specificity of ERF domain has been aninteresting topic in gene transcription regulation studies. In this work, the β-sheetand α-helix of NtERF2 and AtERF1 were exchanged by the recombinant DNAtechnique to study the ERF-DNA binding mechanism, and to explore the keyamino acid residues of NtERF2 that affect specific binding to GCC box. ThecDNAs of the domain-exchang mutants, namely mut1 and mut2, were clonedinto the pET15b plasmid respectively and over-expressed in Escherichia coliBL21(DE3). The expressed proteins were purified by Ni-affinitychromatography. Electrophoresis mobility shift binding assays (EMSAs) wereperformed to test whether those mutant ERF domains were able to bind GCCbox or DRE motif. Then, filter binding assay was carried out to confirm theresults from EMSA. The results showed that the Mut1 and Mut2 were able tobind both GCC box and DRE motif, suggesting that mutation of Y32 into F32(Mut1) changed the binding specificity of NtERF2 to DRE A possiblemechanism of the binding specificity determination is discussed. Currentfindings revealed that Y32 may be an important determinant of the NtERF2binding specificity. In addition, some amino acids in β-sheet and α-helix of ERFdomain are likely form local geometry in a coorperative manner similar to theAtERF1 DBD which is able to bind to GCC box and DRE motif. These resultswarrant further investigation into the differential recognition requirementsamong the different members of this ERF superfamily.