Dominique Payet

HMG-D complexed to a bulge DNA: An NMR model

An NMR model is presented for the structure of HMG‐D, one of the Drosophila counterparts of mammalian HMG1/2 proteins, bound to a particular distorted DNA structure, a dA2 DNA bulge. The complex is in fast to intermediate exchange on the NMR chemical shift time scale and suffers substantial linebroadening for the majority of interfacial resonances. This essentially precludes determination of a high‐resolution structure for the interface based on NMR data alone. However, by introducing a small number of additional constraints based on chemical shift and linewidth footprinting combined with analogies to known structures, an ensemble of model structures was generated using a computational strategy equivalent to that for a conventional NMR structure determination. We find that the base pair adjacent to the dA2 bulge is not formed and that the protein recognizes this feature in forming the complex; intermolecular NOE enhancements are observed from the sidechain of Thr 33 to all four nucleotides of the DNA sequence step adjacent to the bulge. Our results form the first experimental demonstration that when binding to deformed DNA, non‐sequence‐specific HMG proteins recognize the junction between duplex and nonduplex DNA. Similarities and differences of the present structural model relative to other HMG–DNA complex structures are discussed.

The chromosomal protein HMG-D binds to the TAR and RBE RNA of HIV-1

The high mobility group protein HMG‐D is known to bind preferentially to DNA of irregular structures with little or no sequence specificity. Upon binding to DNA, this HMG‐box protein widens the minor groove of the double helix and induces a significant bending of the helix. We show here that HMG‐D can strongly bind to double‐stranded RNA. Electrophoretic mobility shift assays show that HMG‐D100 interacts with the transactivation response region (TAR) RNA from HIV‐1. Strong interaction with a high affinity Rev protein binding element (RBE) RNA was also characterized. Gel shift experiments performed with several TAR RNA constructs lacking the lateral pyrimidine bulge or with modified apical loop regions indicate that the protein does not recognize the single‐strand domains of the RNA but apparently interacts directly with the double‐stranded stem regions. No protein–RNA complexes could be detected when using single‐stranded oligoribonucleotides. HMG‐D protein could bind to the wide minor groove of the A‐form TAR RNA. The comparison of the amino acid sequence of HMG‐D with that of known RNA binding proteins suggests that the interaction of the protein with a double‐stranded RNA implicates the basic region of HMG‐D as well as its HMG‐box domain. From the in vitro data reported here, we propose a novel functional role for proteins of the HMG‐1 family. The results suggest that architectural HMG proteins can be recruited by double‐stranded RNA for the development of HIV‐1 in the host cell.