ABSTRACT
We have previously shown that a critical region of the gata2 promoter contains an inverted CCAAT box and adopts a partial A-form DNA structure in vitro. At gastrula stages of development transcription requires binding of CBTF (CCAAT box transcription factor), a multi-subunit transcription factor, to this region. Xilf3 is one component of CBTF and the double stranded RNA binding domains (dsRBDs) of Xilf3 must be active for both binding to, and transcription from, this promoter. Here we determine the contribution of DNA sequence and structure at the gata2 promoter to transcriptional activity. In all the constructs we tested a CCAAT box was a requirement for full activity. However, base substitutions that increase B-form structure propensity in the sequences flanking the CCAAT box are equally able to decrease activity even if a CCAAT box is present. In contrast, mutations that maintain A-form propensity in these regions also maintain, or increase, transcription factor binding and transcriptional activity. We propose a two-component model for the interaction of CBTF with the gata2 promoter, requiring both a CCAAT sequence and flanking A-form DNA structures. These results support a novel role for dsRBDs in transcriptional regulation and suggest a function for A-form DNA in vivo.
Subject(s)
DNA, A-Form/metabolism , Embryo, Nonmammalian/metabolism , GATA2 Transcription Factor/genetics , Promoter Regions, Genetic/genetics , Xenopus Proteins/genetics , Xenopus laevis/genetics , Animals , Base Sequence , Binding Sites/genetics , CCAAT-Binding Factor/genetics , CCAAT-Binding Factor/metabolism , Circular Dichroism , DNA, A-Form/chemistry , DNA, A-Form/genetics , Electrophoretic Mobility Shift Assay , Embryo, Nonmammalian/embryology , GATA2 Transcription Factor/metabolism , Gene Expression Regulation, Developmental , Luciferases/genetics , Luciferases/metabolism , Mutation , Nuclear Factor 90 Proteins/genetics , Nuclear Factor 90 Proteins/metabolism , Nucleic Acid Conformation , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Xenopus Proteins/metabolism , Xenopus laevis/embryologyABSTRACT
Triplet repeats that cause human genetic diseases have been shown to exhibit unusual compact structures in DNA, and in this paper we show that similar structures exist in shorter "normal length" CNG RNA. CUG and control RNAs were made chemically and by in vitro transcription. We find that "normal" short CUG RNAs migrate anomalously fast on non-denaturing gels, compared with control oligos of similar base composition. By contrast, longer tracts approaching clinically relevant lengths appear to form higher order structures. The CD spectrum of shorter tracts is similar to triplex and pseudoknot nucleic acid structures and different from classical hairpin spectra. A model is outlined that enables the base stacking features of poly(r(G-C))(2).poly(r(U)) or poly(d(G-C))(2).poly(d(T)) triplexes to be achieved, even by a single 15-mer.
