Chromatin,And Gene Regulation In Eukaryotic Cells At The Transcriptional Leve

Phytochromes are environmental sensors, historically thought of as red/far-red photoreceptors in plants. Their photoperception occurs through a covalently linked tetrapyrrole chromophore, which undergoes a light-dependent conformational change propagated through the protein to a variable output domain. The phytochrome composition is modular, typically consisting of a PAS-GAF-PHY architecture for the N-terminal photosensory core. A collection of three-dimensional structures has uncovered key features, including an unusual figure-of-eight knot, an extension reaching from the PHY domain to the chromophore-binding GAF domain, and a centrally located, long α-helix hypothesized to be crucial for intramolecular signaling. Continuing identification of phytochromes in microbial systems has expanded the assigned sensory abilities of this family out of the red and into the yellow, green, blue, and violet portions of the spectrum. Furthermore, phytochromes acting not as photoreceptors but as redox sensors have been recognized. In addition, architectures other than PAS-GAF-PHY are known, thus revealing phytochromes to be a varied group of sensory receptors evolved to utilize their modular design to perceive a signal and respond accordingly. This review focuses on the structures of bacterial phytochromes and implications for signal transmission. We also discuss the small but growing set of bacterial phytochromes for which a physiological function has been ascertained.     

Vertebrate Homologue of Drosophila GAGA Factor

Polycomb group (PcG) and trithorax group (trxG) proteins are chromatin-mediated regulators of a number of developmentally important genes including the homeotic genes. In Drosophila melanogaster, one of the trxG members, Trithorax like (Trl), encodes the essential multifunctional DNA binding protein called GAGA factor (GAF). While most of the PcG and trxG genes are conserved from flies to humans, a Trl-GAF homologue has been conspicuously missing in vertebrates. Here, we report the first identification of c-Krox/Th-POK as the vertebrate homologue of GAF on the basis of sequence similarity and comparative structural analysis. The in silico structural analysis of the zinc finger region showed preferential interaction of vertebrate GAF with GAGA sites similar to that of fly GAF. We also show by cross-immunoreactivity studies that both fly and vertebrate GAFs are highly conserved and share a high degree of structural similarity. Electrophoretic mobility shift assays show that vertebrate GAF binds to GAGA sites in vitro. Finally, in vivo studies by chromatin immunoprecipitation confirmed that vertebrate GAF binds to GAGA-rich DNA sequences present in hox clusters. Identification of vertebrate GAF and the presence of its target sites at various developmentally regulated loci, including hox complexes, highlight the evolutionarily conserved components involved in developmental mechanisms across the evolutionary lineage and answer a long-standing question of the presence of vertebrate GAF.     

Solution structure of a cyanobacterial phytochrome GAF domain in the red-light-absorbing ground state

The unique photochromic absorption behavior of phytochromes (Phys) depends on numerous reversible interactions between the bilin chromophore and the associated polypeptide. To help define these dynamic interactions, we determined by NMR spectroscopy the first solution structure of the chromophore-binding cGMP phosphodiesterase/adenylcyclase/FhlA (GAF) domain from a cyanobacterial Phy assembled with phycocyanobilin (PCB). The three-dimensional NMR structure of Synechococcus OS-B′ cyanobacterial Phy 1 in the red-light-absorbing state of Phy (Pr) revealed that PCB is bound to Cys138 of the GAF domain via the A-ring ethylidene side chain and is buried within the GAF domain in a ZZZsyn,syn,anti configuration. The D ring of the chromophore sits within a hydrophobic pocket and is tilted by approximately 80° relative to the B/C rings by contacts with Lys52 and His169. The solution structure revealed remarkable flexibility for PCB and several adjacent amino acids, indicating that the Pr chromophore has more freedom in the binding pocket than anticipated. The propionic acid side chains of rings B and C and Arg101 and Arg133 nearby are especially mobile and can assume several distinct and energetically favorable conformations. Mutagenic studies on these arginines, which are conserved within the Phy superfamily, revealed that they have opposing roles, with Arg101 and Arg133 helping stabilize and destabilize the far-red-light-absorbing state of Phy (Pfr), respectively. Given the fact that the Synechococcus OS-B′ GAF domain can, by itself, complete the Pr → Pfr photocycle, it should now be possible to determine the solution structure of the Pfr chromophore and surrounding pocket using this Pr structure as a framework.     

Solution NMR views of dynamical ordering of biomacromolecules

Background

To understand the mechanisms related to the ‘dynamical ordering’ of macromolecules and biological systems, it is crucial to monitor, in detail, molecular interactions and their dynamics across multiple timescales. Solution nuclear magnetic resonance (NMR) spectroscopy is an ideal tool that can investigate biophysical events at the atomic level, in near-physiological buffer solutions, or even inside cells.

Chromophore Structure of Cyanobacterial Phytochrome Cph1 in the Pr State: Reconciling Structural and Spectroscopic Data by QM/MM Calculations

A quantum mechanics (QM)/molecular mechanics (MM) hybrid method was applied to the Pr state of the cyanobacterial phytochrome Cph1 to calculate the Raman spectra of the bound PCB cofactor. Two QM/MM models were derived from the atomic coordinates of the crystal structure. The models differed in the protonation site of His²⁶⁰ in the chromophore-binding pocket such that either the δ-nitrogen (M-HSD) or the ɛ-nitrogen (M-HSE) carried a hydrogen. The optimized structures of the two models display small differences specifically in the orientation of His²⁶⁰ with respect to the PCB cofactor and the hydrogen bond network at the cofactor-binding site. For both models, the calculated Raman spectra of the cofactor reveal a good overall agreement with the experimental resonance Raman (RR) spectra obtained from Cph1 in the crystalline state and in solution, including Cph1 adducts with isotopically labeled PCB. However, a distinctly better reproduction of important details in the experimental spectra is provided by the M-HSD model, which therefore may represent an improved structure of the cofactor site. Thus, QM/MM calculations of chromoproteins may allow for refining crystal structure models in the chromophore-binding pocket guided by the comparison with experimental RR spectra. Analysis of the calculated and experimental spectra also allowed us to identify and assign the modes that sensitively respond to chromophore-protein interactions. The most pronounced effect was noted for the stretching mode of the methine bridge A-B adjacent to the covalent attachment site of PCB. Due a distinct narrowing of the A-B methine bridge bond angle, this mode undergoes a large frequency upshift as compared with the spectrum obtained by QM calculations for the chromophore in vacuo. This protein-induced distortion of the PCB geometry is the main origin of a previous erroneous interpretation of the RR spectra based on QM calculations of the isolated cofactor.Abbreviations: Agp1, phytochrome from Agrobacterium tumefaciens; α-CPC, α-subunit of C-phycocyanin; BV, biliverdin IXα; B3LYP, three-parameter exchange functional according to Becke, Lee, Yang, and Parr; DFT, density functional theory; DrBphP, phytochrome from Deinococcus radiodurans; GAF, domain found in cGMP-specific phosphodiesterases; MM, molecular mechanics; MD, molecular dynamics; N-H ip, N-H in-plane bending; PCB, phycocyanobilin; PED, potential energy distribution; phyA, plant phytochrome; Pr, Pfr, red- and far-red absorbing parent states of phytochrome; PΦB, phytochromobilin; QM, quantum mechanics; RMSD, root mean-square deviation; RR, resonance Raman     

蓝细菌光敏色素的分子结构、生物合成和可逆光致变色效应

蓝细菌光敏色素(CBCRs)是蓝细菌中感受光的重要光受体,能够响应从紫外光到红外光范围内的光信号,进而影响蓝细菌的光化学行为。蓝细菌光敏色素通过N-末端GAF(cGMP phosphodiesterase,adenylyl cyclase and FhlA domain)结构域中保守性半胱氨酸共价结合藻胆色素,形成具有感光生理功能的色素蛋白质。本文重点在分子水平上综述了蓝细菌光敏色素的分子结构、生物合成和可逆光致变色效应机理,并基于最新的研究进展,就蓝细菌光敏色素今后的研究方向进行了展望。