The decapentaplegic core promoter region plays an integral role in the spatial control of transcription.

The Drosophila melanogaster decapentaplegic (dpp) gene encodes a transforming growth factor beta-related cell signaling molecule that plays a critical role in dorsal/ventral pattern formation. The dpp expression pattern in the Drosophila embryo is dynamic, consisting of three phases. Phase I, in which dpp is expressed in a broad dorsal domain, depends on elements in the dpp second intron that interact with the Dorsal transcription factor to repress transcription ventrally. In contrast, phases II and III, in which dpp is expressed first in broad longitudinal stripes (phase II) and subsequently in narrow longitudinal stripes (phase III), depend on multiple independent elements in the dpp 5'-flanking region. Several aspects of the normal dpp expression pattern appear to depend on the unique properties of the dpp core promoter. For example, this core promoter (extending from -22 to +6) is able to direct a phase II expression pattern in the absence of additional upstream or downstream regulatory elements. In addition, a ventral-specific enhancer in the dpp 5'-flanking region that binds the Dorsal factor activates the heterologous hsp70 core promoter but not the dpp core promoter. Thus, the dpp core promoter region may contribute to spatially regulated transcription both by interacting directly with spatially restricted activators and by modifying the activity of proteins bound to enhancer elements

Mutation of Actin Tyr-53 Alters the Conformations of the DNase I-binding Loop and the Nucleotide-binding Cleft*

All but 11 of the 323 known actin sequences have Tyr at position 53, and the 11 exceptions have the conservative substitution Phe, which raises the following questions. What is the critical role(s) of Tyr-53, and, if it can be replaced by Phe, why has this happened so infrequently? We compared the properties of purified endogenous Dictyostelium actin and mutant constructs with Tyr-53 replaced by Phe, Ala, Glu, Trp, and Leu. The Y53F mutant did not differ significantly from endogenous actin in any of the properties assayed, but the Y53A and Y53E mutants differed substantially; affinity for DNase I was reduced, the rate of nucleotide exchange was increased, the critical concentration for polymerization was increased, filament elongation was inhibited, and polymerized actin was in the form of small oligomers and imperfect filaments. Growth and/or development of cells expressing these actin mutants were also inhibited. The Trp and Leu mutations had lesser but still significant effects on cell phenotype and the biochemical properties of the purified actins. We conclude that either Tyr or Phe is required to maintain the functional conformations of the DNase I-binding loop (D-loop) in both G- and F-actin, and that the conformation of the D-loop affects not only the properties that directly involve the D-loop (binding to DNase I and polymerization) but also allosterically modifies the conformation of the nucleotide-binding cleft, thus increasing the rate of nucleotide exchange. The apparent evolutionary “preference” for Tyr at position 53 may be the result of Tyr allowing dynamic modification of the D-loop conformation by phosphorylation (Baek, K., Liu, X., Ferron, F., Shu, S., Korn, E. D., and Dominguez, R. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 11748–11753) with effects similar, but not identical, to those of the Ala and Glu mutations

Actin-destabilizing factors disrupt filaments by means of a time reversal of polymerization

Actin, one of the most highly conserved and abundant eukaryotic proteins, is constantly being polymerized and depolymerized within cells as part of cellular motility, tissue formation and repair, and embryonic development. Many proteins exist that bind to monomeric or filamentous (F) forms of actin to regulate the polymerization state. It has become increasingly apparent that the ability of different proteins to bind to and regulate actin filament dynamics depends on the ability of the filament to exist in altered conformations. Yet, little is known about how these conformational changes occur at the molecular level. We have destabilized F-actin filaments by forming a disulfide that locks the “hydrophobic plug” to the body of the actin subunit or by altering the C terminus of actin with a tetramethylrhodamine label. We also examined F-actin filaments at short times after the initiation of polymerization. In all three cases, a substantial fraction of protomers can be found in a “tilted” state that also is induced by actin depolymerizing factor/cofilin proteins. These observations suggest that F-actin filaments are annealed over time into a stable filament and that actin-depolymerizing proteins can effect a time reversal of polymerization

Remodeling of actin filaments by ADF/cofilin proteins

Cofilin/ADF proteins play key roles in the dynamics of actin, one of the most abundant and highly conserved eukaryotic proteins. We used cryoelectron microscopy to generate a 9-Å resolution three-dimensional reconstruction of cofilin-decorated actin filaments, the highest resolution achieved for a complex of F-actin with an actin-binding protein. We show that the cofilin-induced change in the filament twist is due to a unique conformation of the actin molecule unrelated to any previously observed state. The changes between the actin protomer in naked F-actin and in the actin-cofilin filament are greater than the conformational changes between G- and F-actin. Our results show the structural plasticity of actin, suggest that other actin-binding proteins may also induce large but different conformational changes, and show that F-actin cannot be described by a single molecular model