Plants use light as a major source of information for optimizing growth and development. The photoreceptor phytochrome A (phyA) mediates various far-red light induced responses. Here, we show that Arabidopsis FHY3 and FAR1, which encode two proteins related to Mutator-like transposases, act together to modulate phyA signaling by directly activating the transcription of FHY1 and FHL, whose products are essential for light-induced phyA nuclear accumulation and subsequent light responses. FHY3 and FAR1 possess separable DNA-binding and transcriptional activation domains that are highly conserved in Mutator-like transposases. Further, expression of FHY3 and FAR1 is negatively regulated by phyA signaling. We propose that FHY3 and FAR1 define a novel class of transcription factors co-opted from an ancient Mutator-like transposase(s) to modulate phyA signaling homeostasis in higher plants.Plants constantly monitor their light environment in order to grow and develop optimally, using a battery of photoreceptors. Phytochromes are a family of photoreceptors that monitors the incident red (R, 600-700 nm) and far-red (FR, 700-750 nm) light wavelengths by switching reversibly between the R-absorbing, biologically inactive Pr form and the FR-absorbing, biologically active Pfr form (1,2). Upon photoactivation, phyA, the primary photoreceptor for FR light, is translocated from the cytoplasm into the nucleus to induce FR-responsive gene expression required for various photoresponses, such as seed germination, seedling deetiolation, FR-preconditioned blocking of greening, and flowering (3). Genetic studies have identified two pairs of homologous genes essential for phyA signaling: FAR1 (far-red-impaired response 1) and FHY3 (far-red elongated hypocotyl 3); FHY1 (far-red elongated hypocotyl 1) and FHL (FHY1-like) (4-7). FHY1 and FHL have been implicated in mediating the lightdependent nuclear accumulation of phyA (8,9). However, the biochemical function of FHY3 and FAR1 remains to be elucidated. FHY3 and FAR1 share extensive sequence homology with MURA, the transposase encoded by the maize Mutator element, and the predicted transposase of the maize mobile element Jittery (10,11). Both of these transposons are members of the superfamily of Mutator-like elements (MULEs) (12). Database mining and phylogenetic analysis revealed that FHY3/ FAR1-like sequences are present in various angiosperms and fall into several phylogenetic clusters intermingled with MULE transposases (13,table S1 and fig. S1). These proteins share an N-terminal C2H2-type zinc-chelating motif of the WRKY-GCM1 family, a central putative core transposase domain, and a C-terminal SWIM motif (14,15), with highly conserved predicted secondary/tertiary structures ( fig. S2 and S3). To investigate the molecular function

Tetradecameric Clp protease core complexes in nonphotosynthetic plastids of roots, flower petals, and in chloroplasts of leaves of Arabidopsis thaliana were purified based on native mass and isoelectric point and identified by mass spectrometry. The stoichiometry between the subunits was determined. The protease complex consisted of one to three copies of five different serine-type protease Clp proteins (ClpP1,3-6) and four non-proteolytic ClpR proteins (ClpR1-4). Three-dimensional homology modeling showed that the ClpP/R proteins fit well together in a tetradecameric complex and also indicated unique contributions for each protein.Lateral exit gates for proteolysis products are proposed. In addition, ClpS1,2, unique to land plants, tightly interacted with this core complex, with one copy of each per complex. The three-dimensional modeling show that they do fit well on the axial sites of the ClpPR cores. In contrast to plastids, plant mitochondria contained a single ϳ320-kDa homo-tetradecameric ClpP2 complex, without association of ClpR or ClpS proteins. It is surprising that the Clp core composition appears identical in all three plastid types, despite the remarkable differences in plastid proteome composition. This suggests that regulation of plastid proteolysis by the Clp machinery is not through differential regulation of ClpP/R/S gene expression, but rather through substrate recognition mechanisms and regulated interaction of chaperone-like molecules (ClpS1,2 and others) to the ClpP/R core.Plastids are essential organelles of prokaryotic origin that are present in every plant cell and differentiate from proplastids into non-photosynthetic plastids in roots and flowers and photosynthetic plastids in leafs and stems. Plastids are responsible for synthesis of key molecules required for the architecture and functions of plant cells.To maintain a correct stoichiometry between different proteins and pathways, to remove and recycle damaged or misfolded proteins, and to control gene expression by proteolysis of transcription or translation factors, different proteolytic systems are present in the plastid. Members of at least five protease families are present in plastids, but their structures, functions, substrates, and biological importance are poorly understood (2).A very prominent group of proteases in plants is the Clp protease family. Our latest analysis of the Arabidopsis thaliana nuclear genome indicates the presence of at least 26 Clp-related genes, with 15 genes encoding for plastid-localized proteins (3) (Fig.

The enzyme acetylcholinesterase generates a strong electrostatic field that can attract the cationic substrate acetylcholine to the active site. However, the long and narrow active site gorge seems inconsistent with the enzyme's high catalytic rate. A molecular dynamics simulation of acetylcholinesterase in water reveals the transient opening of a short channel, large enough to pass a water molecule, through a thin wall of the active site near tryptophan-84. This simulation suggests that substrate, products, or solvent could move through this "back door," in addition to the entrance revealed by the crystallographic structure. Electrostatic calculations show a strong field at the back door, oriented to attract the substrate and the reaction product choline and to repel the other reaction product, acetate. Analysis of the open back door conformation suggests a mutation that could seal the back door and thus test the hypothesis that thermal motion of this enzyme may open multiple routes of access to its active site.

We have carried out conformational energy calculations on alanine-based copolymers with the sequence Ac-AAAAAXAAAA-NH2 in water, where X stands for lysine or glutamine, to identify the underlying source of stability of alanine-based polypeptides containing charged or highly soluble polar residues in the absence of charge-charge interactions. The results indicate that ionizable or neutral polar residues introduced into the sequence to make them soluble sequester the water away from the CO and NH groups of the backbone, thereby enabling them to form internal hydrogen bonds. This solvation effect dictates the conformational preference and, hence, modifies the conformational propensity of alanine residues. Even though we carried out simulations for specific amino acid sequences, our results provide an understanding of some of the basic principles that govern the process of folding of these short sequences independently of the kind of residues introduced to make them soluble. In addition, we have investigated through simulations the effect of the bulk dielectric constant on the conformational preferences of these peptides. Extensive conformational Monte Carlo searches on terminally blocked 10-mer and 16-mer homopolymers of alanine in the absence of salt were carried out assuming values for the dielectric constant of the solvent of 80, 40, and 2. Our simulations show a clear tendency of these oligopeptides to augment the ␣-helix content as the bulk dielectric constant of the solvent is lowered. This behavior is due mainly to a loss of exposure of the CO and NH groups to the aqueous solvent. Experimental evidence indicates that the helical propensity of the amino acids in water shows a dramatic increase on addition of certain alcohols, such us trifluoroethanol. Our results provide a possible explanation of the mechanism by which alcohol͞water mixtures affect the free energy of helical alanine oligopeptides relative to nonhelical ones. T he ␣-helical conformation of short homopolypeptides containing only alanines is unstable in water at room temperature (1). This behavior is consistent with the value of the Zimm-Bragg parameters (2) s and determined from experiments on random copolymers (3). If charged (4) or neutral polar (5) residues are introduced into a chain of 13 alanine residues to solubilize it in water, the helix becomes stable, more so when charged residues rather than neutral polar residues are incorporated. This behavior has been attributed to desolvation of the backbone NH and CO groups of the alanines by the preferentially hydrated charged or polar groups (6) rather than to a high intrinsic tendency of alanines to form an ␣-helix. By being deprived of a high degree of hydration, the backbone NH and CO groups can form the ␣-helical hydrogen bonds.Previous experiments (4, 5) and calculations (6, 7) treated polyalanine chains containing several charged or neutral polar guest groups, e.g., three lysines or glutamines in the copolymers AAAAKAAAAKAAAAKA and AAQAAAAQAAAAQAAY, respectively, in which the helix-stab...