Comparative analysis of homology models of the Ah receptor ligand binding domain: Verification of structure-function predictions by site-directed mutagenesis of a nonfunctional receptor

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates the biological and toxic effects of a wide variety of structurally diverse chemicals, including the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While significant interspecies differences in AHR ligand binding specificity, selectivity, and response have been observed, the structural determinants responsible for those differences have not been determined, and homology models of the AHR ligand-binding domain (LBD) are available for only a few species. Here we describe the development and comparative analysis of homology models of the LBD of 16 AHRs from 12 mammalian and nonmammalian species and identify the specific residues contained within their ligand binding cavities. The ligand-binding cavity of the fish AHR exhibits differences from those of mammalian and avian AHRs, suggesting a slightly different TCDD binding mode. Comparison of the internal cavity in the LBD model of zebrafish (zf) AHR2, which binds TCDD with high affinity, to that of zfAHR1a, which does not bind TCDD, revealed that the latter has a dramatically shortened binding cavity due to the side chains of three residues (Tyr296, Thr386, and His388) that reduce the amount of internal space available to TCDD. Mutagenesis of two of these residues in zfAHR1a to those present in zfAHR2 (Y296H and T386A) restored the ability of zfAHR1a to bind TCDD and to exhibit TCDD-dependent binding to DNA. These results demonstrate the importance of these two amino acids and highlight the predictive potential of comparative analysis of homology models from diverse species. The availability of these AHR LBD homology models will facilitate in-depth comparative studies of AHR ligand binding and ligand-dependent AHR activation and provide a novel avenue for examining species-specific differences in AHR responsiveness. © 2013 American Chemical Society

RNA-binding specificity of E. coli NusA

The RNA sequences boxA, boxB and boxC constitute the nut regions of phage λ. They nucleate the formation of a termination-resistant RNA polymerase complex on the λ chromosome. The complex includes E. coli proteins NusA, NusB, NusG and NusE, and the λ N protein. A complex that includes the Nus proteins and other factors forms at the rrn leader. Whereas RNA-binding by NusB and NusE has been described in quantitative terms, the interaction of NusA with these RNA sequences is less defined. Isotropic as well as anisotropic fluorescence equilibrium titrations show that NusA binds only the nut spacer sequence between boxA and boxB. Thus, nutR boxA5-spacer, nutR boxA16-spacer and nutR boxA69-spacer retain NusA binding, whereas a spacer mutation eliminates complex formation. The affinity of NusA for nutL is 50% higher than for nutR. In contrast, rrn boxA, which includes an additional U residue, binds NusA in the absence of spacer. The Kd values obtained for rrn boxA and rrn boxA-spacer are 19-fold and 8-fold lower, respectively, than those for nutR boxA-spacer. These differences may explain why λ requires an additional protein, λ N, to suppress termination. Knowledge of the different affinities now describes the assembly of the anti-termination complex in quantitative terms

Phenothiourea Sensitizes Zebrafish Cranial Neural Crest and Extraocular Muscle Development to Changes in Retinoic Acid and IGF Signaling

1-phenyl 2-thiourea (PTU) is a tyrosinase inhibitor commonly used to block pigmentation and aid visualization of zebrafish development. At the standard concentration of 0.003% (200 µM), PTU inhibits melanogenesis and reportedly has minimal other effects on zebrafish embryogenesis. We found that 0.003% PTU altered retinoic acid and insulin-like growth factor (IGF) regulation of neural crest and mesodermal components of craniofacial development. Reduction of retinoic acid synthesis by the pan-aldehyde dehydrogenase inhibitor diethylbenzaldehyde, only when combined with 0.003% PTU, resulted in extraocular muscle disorganization. PTU also decreased retinoic acid-induced teratogenic effects on pharyngeal arch and jaw cartilage despite morphologically normal appearing PTU-treated controls. Furthermore, 0.003% PTU in combination with inhibition of IGF signaling through either morpholino knockdown or pharmacologic inhibition of tyrosine kinase receptor phosphorylation, disrupted jaw development and extraocular muscle organization. PTU in and of itself inhibited neural crest development at higher concentrations (0.03%) and had the greatest inhibitory effect when added prior to 22 hours post fertilization (hpf). Addition of 0.003% PTU between 4 and 20 hpf decreased thyroxine (T4) in thyroid follicles in the nasopharynx of 96 hpf embryos. Treatment with exogenous triiodothyronine (T3) and T4 improved, but did not completely rescue, PTU-induced neural crest defects. Thus, PTU should be used with caution when studying zebrafish embryogenesis as it alters the threshold of different signaling pathways important during craniofacial development. The effects of PTU on neural crest development are partially caused by thyroid hormone signaling

Structural basis for RNA recognition by NusB and NusE in the initiation of transcription antitermination

Processive transcription antitermination requires the assembly of the complete antitermination complex, which is initiated by the formation of the ternary NusB–NusE–BoxA RNA complex. We have elucidated the crystal structure of this complex, demonstrating that the BoxA RNA is composed of 8 nt that are recognized by the NusB–NusE heterodimer. Functional biologic and biophysical data support the structural observations and establish the relative significance of key protein–protein and protein–RNA interactions. Further crystallographic investigation of a NusB–NusE–dsRNA complex reveals a heretofore unobserved dsRNA binding site contiguous with the BoxA binding site. We propose that the observed dsRNA represents BoxB RNA, as both single-stranded BoxA and double-stranded BoxB components are present in the classical lambda antitermination site. Combining these data with known interactions amongst antitermination factors suggests a specific model for the assembly of the complete antitermination complex

Fundulus as the premier teleost model in environmental biology : opportunities for new insights using genomics

Author Posting. © Elsevier B.V., 2007. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Comparative Biochemistry and Physiology Part D: Genomics and Proteomics 2 (2007): 257-286, doi:10.1016/j.cbd.2007.09.001.A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.This material is based on work supported by grants from the National Science Foundation DBI-0420504 (LJB), OCE 0308777 (DLC, RNW, BBR), BES-0553523 (AW), IBN 0236494 (BBR), IOB-0519579 (DHE), IOB-0543860 (DWT), FSML-0533189 (SC); National Institute of Health NIEHS P42-ES007381(GVC, MEH), P42-ES10356 (RTD), ES011588 (MFO); and NCRR P20 RR-016463 (DWT); Natural Sciences and Engineering Research Council of Canada Discovery (DLM, TDS, WSM) and Collaborative Research and Development Programs (DLM); NOAA/National Sea Grant NA86RG0052 (LJB), NA16RG2273 (SIK, MEH,GVC, JJS); Environmental Protection Agency U91620701 (WSB), R82902201(SC) and EPA’s Office of Research and Development (DEN)