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Short hairpin RNAs (shRNAs) have been used to achieve stable target knockdown in a variety of biological systems. Here, we report the development of a tightly regulated tetracycline-responsive human U6 promoter for shRNA expression. By engineering two copies of the tet operators flanking the TATA box of the human U6 promoter, we created a U6 promoter derivative (2O2) that exhibited much lower basal transcriptional activity compared with recently reported inducible pol III dependent promoters. As a consequence of its tighter regulation, the 2O2 system greatly improved the success rate in making inducible knockdown cell lines.

p53 exerts its tumor suppressor activity through both transcription-dependent and transcription-independent processes. Although the transcription-dependent activity of p53 has been extensively studied, the mechanism for transcription-independent p53-mediated tumor suppression is less well known. Recently, it was reported that p53 can directly induce mitochondrial permeabilization and promote apoptosis. This occurs through complexation of the DNA-binding region of p53 with the anti-apoptotic proteins Bcl-x(L) and Bcl-2 (Mihara, M. et al. (2003) Mol. Cell 11, 577-590). Using nuclear magnetic resonance (NMR) spectroscopy we show that the interaction surface on p53 involves the same region that is used by the protein to contact DNA. The p53-binding site on Bcl-x(L) consists of the carboxy-terminus of the first alpha-helix, the loop between alpha3 and alpha4, and the loop between alpha5 and alpha6 of Bcl-x(L). Furthermore, the interaction of p53 with Bcl-x(L) is blocked by the binding of a 25-residue peptide derived from the BH3 region of the pro-apoptotic protein referred to as Bad.

Two multi-dimensional heteronuclear NMR experiments are described for assigning the resonances in uniformly 15N- and 13C-labeled proteins. In one experiment (HCNH-TOCSY), the amide nitrogen and proton are correlated to the side-chain protons and carbons of the same and preceding residue. In a second triple resonance experiment (HC(CO)NH-TOCSY), the amide nitrogen and proton of one residue is correlated exclusively with the side-chain proton and carbon resonances of the preceding residue by transferring magnetization through the intervening carbonyl. The utility of these two experiments for making sequential resonance assignments in proteins is illustrated for [U-15N,13C]FKBP (107 residues) complexed to the immunosuppressant, ascomycin.

The 3D structure of two unlabeled FK506 analogs, (R)- and (S)-[18-OH]ascomycin, when bound to [U-13C,15N]FKBP were determined by isotope-filtered 2D NMR experiments. The structures for the R and S isomers that bind tightly to FKBP but lack immunosuppressive activity are compared to each other and to the conformation of the potent immunosuppressant, ascomycin, when bound to FKBP. The results are interpreted in terms of calcineurin binding to the FKBP/ascomycin complex.

3JH alpha,H beta and 3JN,H beta coupling constants were measured for isotopically labeled FKBP when bound to the immunosuppressant, ascomycin, using a 1H-coupled 3D HCCH-TOCSY and 15N-coupled 3D HSQC-TOCSY experiment, respectively. From an analysis of these two sets of coupling constants, stereospecific beta-proton assignments and chi 1 rotamers for FKBP have been obtained. All of the chi 1 rotamers were consistent with the chi 1 angles measured in the X-ray crystal structure of the FKBP/FK506 complex, suggesting that the structures of the two complexes are similar.

NMR data (1H and 13C chemical shifts, NOEs) on [U-13C]cyclosporin A bound to cyclophilin B were compared to previously published data on the [U-13C]CsA/CyPA complex [Fesik et al., (1991) Biochemistry 30, 6574-6583]. Despite only 64% sequence identity between CyPA and CyPB, the conformation and active site environment of CsA when bound to CyPA and CyPB are nearly identical as judged by the similarity of the NMR data.