2-Amino-1,3-benzothiazole-6-carboxamide Preferentially Binds the Tandem Mismatch Motif r(UY:GA).

RNA helices are often punctuated with non-Watson-Crick features that may be targeted by chemical compounds, but progress toward identifying such compounds has been slow. We embedded a tandem UU:GA mismatch motif (5'-UG-3':5'-AU-3') within an RNA hairpin stem to identify compounds that bind the motif specifically. The three-dimensional structure of the RNA hairpin and its interaction with a small molecule identified through virtual screening are presented. The G-A mismatch forms a sheared pair upon which the U-U base pair stacks. The hydrogen bond configuration of the U-U pair involves O2 of the U adjacent to the G and O4 of the U adjacent to the A. The G-A and U-U pairs are flanked by A-U and G-C base pairs, respectively, and the stability of the mismatch is greater than when the motif is within the context of other flanking base pairs or when the 5'-3' orientation of the G-A and U-U pairs is swapped. Residual dipolar coupling constants were used to generate an ensemble of structures against which a virtual screen of 64480 small molecules was performed. The tandem mismatch was found to be specific for one compound, 2-amino-1,3-benzothiazole-6-carboxamide, which binds with moderate affinity but extends the motif to include the flanking A-U and G-C base pairs. The finding that the affinity for the UU:GA mismatch is dependent on flanking sequence emphasizes the importance of the motif context and potentially increases the number of small noncanonical features within RNA that can be specifically targeted by small molecules.

Structure and Dynamics of the Tetra-A Loop and (A-A)-U Sequence Motif within the Coliphage GA Replicase RNA Operator.

The three-dimensional structure of a RNA hairpin containing the RNA operator binding site for bacteriophage GA coat protein is presented. The phage GA operator contains the asymmetric (A-A)-U sequence motif and is capped by a four-adenine (tetra-A) loop. The uridine of the (A-A)-U motif preferentially pairs with the 5'-proximal cross-strand adenine, and the 3'-proximal adenine stacks into the helix. The tetra-A loop is well-ordered with adenine residues 2-4 forming a 3' stack. This loop conformation stands in contrast to the structure of the 5'-AUUA loop of the related phage MS2 operator in which residues 1 and 2 form a 5' stack. The context dependence of the (A-A)-U sequence motif conformation was examined using structures of 76 unique occurrences from the Protein Data Bank. The motif almost always has one adenine bulged and the other adenine adopting an A-U base pair. In the case in which the (A-A)-U motif is flanked by only one Watson-Crick base pair, the adenine adjacent to the flanking base pair tends to bulge; 80% of motifs with a 3' flanking pair have a 3' bulged adenine, and 84% of motifs with a 5' flanking pair have a 5' bulged adenine. The frequencies of 3'- and 5'-proximal adenines bulging are 33 and 67%, respectively, when the (A-A)-U motif is flanked by base pairs on both sides. Although a 3' flanking cytidine correlates (88%) with bulging of the 5'-proximal adenine, no strict dependence on flanking nucleotide identity was identified for the 5' side.

S-Warfarin Limited Sampling Models to Estimate Area Under the Concentration Versus Time Curve for Cytochrome P450 2C9 Baseline Activity and After Induction.

BACKGROUND: Phenotyping cytochrome P450 (CYP) 2C9 activity using S-warfarin has routinely required extensive blood sampling over at least 96 hours after dose to estimate the area under the concentration time curve from zero to infinity (AUC). Alternatively, S-warfarin limited sampling models (LSMs) using one or 2 concentration timepoints have been proposed to estimate AUC. This study evaluated whether S-warfarin LSMs accurately estimate CYP2C9 baseline and induction conditions in healthy adults and in advanced-stage cancer patients. METHODS: Plasma S-warfarin concentrations from healthy adults (n = 92) and in advanced-stage cancer patients (n = 22) were obtained from 6 published studies where a single 10 mg dose of oral warfarin was administered at CYP2C9 baseline and induction conditions. S-warfarin observed AUC was determined by noncompartmental analysis, whereas estimated AUC was calculated from the LSMs. Bias and precision were assessed by percent mean prediction error, percent mean absolute error, and percent root mean square error. RESULTS: Different results were observed for S-warfarin LSMs in estimating CYP2C9 baseline activity, with most studies resulting in unacceptable bias and precision. The percent mean prediction error, percent mean absolute error, and/or percent root mean square error exceeded acceptable limits for LSMs in patients with advanced-stage cancer and during CYP2C9 induction with lopinavir/ritonavir. CONCLUSIONS: The differing results during CYP2C9 baseline conditions, as well as unacceptable bias and precision in patients with advanced cancer and during CYP2C9 induction, considerably limit the widespread use of previously published S-warfarin LSMs.

An evolutionarily conserved DNA architecture determines target specificity of the TWIST family bHLH transcription factors.

Basic helix-loop-helix (bHLH) transcription factors recognize the canonical E-box (CANNTG) to regulate gene transcription; however, given the prevalence of E-boxes in a genome, it has been puzzling how individual bHLH proteins selectively recognize E-box sequences on their targets. TWIST is a bHLH transcription factor that promotes epithelial-mesenchymal transition (EMT) during development and tumor metastasis. High-resolution mapping of TWIST occupancy in human and Drosophila genomes reveals that TWIST, but not other bHLH proteins, recognizes a unique double E-box motif with two E-boxes spaced preferentially by 5 nucleotides. Using molecular modeling and binding kinetic analyses, we found that the strict spatial configuration in the double E-box motif aligns two TWIST-E47 dimers on the same face of DNA, thus providing a high-affinity site for a highly stable intramolecular tetramer. Biochemical analyses showed that the WR domain of TWIST dimerizes to mediate tetramer formation, which is functionally required for TWIST-induced EMT. These results uncover a novel mechanism for a bHLH transcription factor to recognize a unique spatial configuration of E-boxes to achieve target specificity. The WR-WR domain interaction uncovered here sets an example of target gene specificity of a bHLH protein being controlled allosterically by a domain outside of the bHLH region.

Structure determination of noncanonical RNA motifs guided by ¹H NMR chemical shifts.

Structured noncoding RNAs underlie fundamental cellular processes, but determining their three-dimensional structures remains challenging. We demonstrate that integrating ¹H NMR chemical shift data with Rosetta de novo modeling can be used to consistently determine high-resolution RNA structures. On a benchmark set of 23 noncanonical RNA motifs, including 11 'blind' targets, chemical-shift Rosetta for RNA (CS-Rosetta-RNA) recovered experimental structures with high accuracy (0.6-2.0 Å all-heavy-atom r.m.s. deviation) in 18 cases.

Solution NMR determination of hydrogen bonding and base pairing between the glyQS T box riboswitch Specifier domain and the anticodon loop of tRNA(Gly).

In Gram-positive bacteria the tRNA-dependent T box riboswitch regulates the expression of many amino acid biosynthetic and aminoacyl-tRNA synthetase genes through a transcription attenuation mechanism. The Specifier domain of the T box riboswitch contains the Specifier sequence that is complementary to the tRNA anticodon and is flanked by a highly conserved purine nucleotide that could result in a fourth base pair involving the invariant U33 of tRNA. We show that the interaction between the T box Specifier domain and tRNA consists of three Watson-Crick base pairs and that U33 confers stability to the complex through intramolecular hydrogen bonding. Enhanced packing within the Specifier domain loop E motif may stabilize the complex and contribute to cognate tRNA selection.

Twist1 mediates repression of chondrogenesis by ß-catenin to promote cranial bone progenitor specification.

The bones of the mammalian skull vault form through intramembranous ossification. Skull bones ossify directly, in a process regulated by ß-catenin, instead of passing through a cartilage intermediate. We tested whether ß-catenin is necessary for fate selection of intramembranous bone progenitors in the skull. Here, we show in mice that removal of ß-catenin from skull bone progenitors results in the near complete transformation of the skull bones to cartilage, whereas constitutive ß-catenin activation inhibits skull bone fate selection. ß-catenin directly activated Twist1 expression in skull progenitors, conditional Twist1 deletion partially phenocopied the absence of ß-catenin, and Twist1 deletion partially restored bone formation in the presence of constitutive ß-catenin activation. Finally, Twist1 bound robustly to the 3'UTR of Sox9, the central initiator of chondrogenesis, suggesting that Twist1 might directly repress cartilage formation through Sox9. These findings provide insight into how ß-catenin signaling via Twist1 actively suppresses the formation of cartilage and promotes intramembranous ossification in the skull.

Solution nuclear magnetic resonance analyses of the anticodon arms of proteinogenic and nonproteinogenic tRNA(Gly).

Although the fate of most tRNA molecules in the cell is aminoacylation and delivery to the ribosome, some tRNAs are destined to fulfill other functional roles. In addition to their central role in translation, tRNA molecules participate in processes such as regulation of gene expression, bacterial cell wall biosynthesis, viral replication, antibiotic biosynthesis, and suppression of alternative splicing. In bacteria, glycyl-tRNA molecules with anticodon sequences GCC and UCC exhibit multiple extratranslational functions, including transcriptional regulation and cell wall biosynthesis. We have determined the high-resolution structures of three glycyl-tRNA anticodon arms with anticodon sequences GCC and UCC. Two of the tRNA molecules are proteinogenic (tRNA(Gly,GCC) and tRNA(Gly,UCC)), and the third is nonproteinogenic (np-tRNA(Gly,UCC)) and participates in cell wall biosynthesis. The UV-monitored thermal melting curves show that the anticodon arm of tRNA(Gly,UCC) with a loop-closing C-A(+) base pair melts at a temperature 10 °C lower than those of tRNA(Gly,GCC) and np-tRNA(Gly,UCC). U-A and C-G pairs close the loops of the latter two molecules and enhance stem stability. Mg(2+) stabilizes the tRNA(Gly,UCC) anticodon arm and reduces the T(m) differential. The structures of the three tRNA(Gly) anticodon arms exhibit small differences among one another, but none of them form the classical U-turn motif. The anticodon loop of tRNA(Gly,GCC) becomes more dynamic and disordered in the presence of multivalent cations, whereas metal ion coordination in the anticodon loops of tRNA(Gly,UCC) and np-tRNA(Gly,UCC) establishes conformational homogeneity. The conformational similarity of the molecules is greater than their functional differences might suggest. Because aminoacylation of full-length tRNA molecules is accomplished by one tRNA synthetase, the similar structural context of the loop may facilitate efficient recognition of each of the anticodon sequences.

Twist1-induced invadopodia formation promotes tumor metastasis.

The Twist1 transcription factor is known to promote tumor metastasis and induce Epithelial-Mesenchymal Transition (EMT). Here, we report that Twist1 is capable of promoting the formation of invadopodia, specialized membrane protrusions for extracellular matrix degradation. Twist1 induces PDGFRα expression, which in turn activates Src, to promote invadopodia formation. We show that Twist1 and PDGFRα are central mediators of invadopodia formation in response to various EMT-inducing signals. Induction of PDGFRα and invadopodia is essential for Twist1 to promote tumor metastasis. Consistent with PDGFRα being a direct transcriptional target of Twist1, coexpression of Twist1 and PDGFRα predicts poor survival in breast tumor patients. Therefore, invadopodia-mediated matrix degradation is a key function of Twist1 in promoting tumor metastasis.