Combinatorial regulation of transcription implies flexible yet precise assembly of multiprotein regulatory complexes in response to signals. Biochemical and crystallographic analyses revealed that hormone binding leads to the formation of a hydrophobic groove within the ligand binding domain (LBD) of the thyroid hormone receptor that interacts with an LxxLL motif-containing ␣-helix from GRIP1, a coactivator. Residues immediately adjacent to the motif modulate the affinity of the interaction; the motif and the adjacent sequences are employed to different extents in binding to different receptors. Such interactions of amphipathic ␣-helices with hydrophobic grooves define protein interfaces in other regulatory complexes as well. We suggest that these common structural elements impart flexibility to combinatorial regulation, whereas side chains at the interface impart specificity.

The crystal structure of the rat alpha 1 thyroid hormone receptor ligand-binding domain bound with a thyroid hormone agonist reveals that ligand is completely buried within the domain as part of the hydrophobic core. In addition, the carboxy-terminal activation domain forms an amphipathic helix, with its hydrophobic face constituting part of the hormone binding cavity. These observations suggest a structural role for ligand, in establishing the active conformation of the receptor, that is likely to underlie hormonal regulation of gene expression for the nuclear receptors.

A method for isolation of large, translationally active RNA species is presented. The procedure involves homogenization of cells or tissues in 5 M guanidine monothiocyanate followed by direct precipitation of RNA from the guanidinium by 4 M LiCl. Modifications are described for use with tissue culture cells, yeast, tissues, or isolated nuclei. The advantages of the procedure include speed, simplicity, avoidance of an ultracentrifugation, and its applicability to large numbers of small samples. The procedure yields large mRNA precursors up to 10 kb and mRNA species which translate very well. However, small (less than 300 nucleotides) RNA species are recovered with a poor yield.

Ultra-deep sequencing identified a significantly larger proportion of HIV-infected, treatment-naive persons as harboring drug-resistant viral variants. Among participants who initiated treatment with the NNRTI strategy, the risk of VF was significantly greater for participants who had low- and high-prevalence NNRTI-resistant variants.

Current approaches to inhibit nuclear receptor (NR) activity target the hormone binding pocket but face limitations. We have proposed that inhibitors, which bind to nuclear receptor surfaces that mediate assembly of the receptor's binding partners, might overcome some of these limitations. The androgen receptor (AR) plays a central role in prostate cancer, but conventional inhibitors lose effectiveness as cancer treatments because anti-androgen resistance usually develops. We conducted functional and x-ray screens to identify compounds that bind the AR surface and block binding of coactivators for AR activation function 2 (AF-2). Four compounds that block coactivator binding in solution with IC 50 ≈ 50 μM and inhibit AF-2 activity in cells were detected: three nonsteroidal antiinflammatory drugs and the thyroid hormone 3,3′,5-triiodothyroacetic acid. Although visualization of compounds at the AR surface reveals weak binding at AF-2, the most potent inhibitors bind preferentially to a previously unknown regulatory surface cleft termed binding function (BF)-3, which is a known target for mutations in prostate cancer and androgen insensitivity syndrome. X-ray structural analysis reveals that 3,3′,5-triiodothyroacetic acid binding to BF-3 remodels the adjacent interaction site AF-2 to weaken coactivator binding. Mutation of residues that form BF-3 inhibits AR function and AR AF-2 activity. We propose that BF-3 is a previously unrecognized allosteric regulatory site needed for AR activity in vivo and a possible pharmaceutical target.