RESUMEN
Actin stress fibers (SFs) in live cells consist of series of dynamic individual sarcomeric units. Within a group of consecutive SF sarcomeres, individual sarcomeres can spontaneously shorten or lengthen without changing the overall length of this group, but the underlying mechanism is unclear. We used a computational model to test our hypothesis that this dynamic behavior is inherent to the heterogeneous mechanical properties of the sarcomeres and the cytoplasmic viscosity. Each sarcomere was modeled as a discrete element consisting of an elastic spring, a viscous dashpot and an active contractile unit all connected in parallel, and experiences forces as a result of actin filament elastic stiffness, myosin II contractility, internal viscoelasticity, or cytoplasmic drag. When all four types of forces are considered, the simulated dynamic behavior closely resembles the experimental observations, which include a low-frequency fluctuation in individual sarcomere length and compensatory lengthening and shortening of adjacent sarcomeres. Our results suggest that heterogeneous stiffness and viscoelasticity of actin fibers, heterogeneous myosin II contractility, and the cytoplasmic drag are sufficient to cause spontaneous fluctuations in SF sarcomere length. Our results shed new light to the dynamic behavior of SF and help design experiments to further our understanding of SF dynamics.
RESUMEN
The Zap1 transcriptional activator from Saccharomyces cerevisiae induces expression of a series of genes containing an 11 base pair conserved promoter element (ZRE) under conditions of zinc deficiency. This work shows that Zap1 uses four of its seven zinc finger domains to contact the ZRE and that two of these dominate the interaction by contacting the essential ACC-GGT ends. Two Zn finger domains (ZF1 and ZF2) do not contact DNA, and a third ZF3 may be more important for interfinger protein-protein interactions. Zn finger domains important for ZRE contact were identified from triple mutations in Zap1, changing three residues in the alpha helix in each finger known to be important for DNA contacts in Zn finger proteins. Replacement of -1, 3, and 6 helix residues in ZF4 and ZF7 reduced the affinity of Zap1 for the wild-type ZRE. In contrast, triple mutations within the intervening ZF5 and ZF6 domains had minimal effect. The data argue that fingers 4 and 7 contact the ACC-GGT ends while fingers 5 and 6 contact the 5 bp central ZRE sequence. This conclusion is corroborated by decreased Zap1 affinity for a ZRE DNA duplex containing mutations of the AC-GT ends of the ZRE, whereas transversion mutations within the central 5 bp of the ZRE had minimal effect on Zap1 binding affinity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/metabolismo , Dedos de Zinc , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Conejos , Elementos de Respuesta/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Resonancia por Plasmón de Superficie , Transactivadores/química , Transactivadores/genética , Factores de Transcripción , Dedos de Zinc/genéticaRESUMEN
The Zap1 transcriptional activator of Saccharomyces cerevisiae plays a major role in zinc homeostasis by inducing the expression of several genes under zinc-limited growth conditions. This activation of gene expression is mediated by binding of the protein to one or more zinc-responsive elements present in the promoters of its target genes. To better understand how Zap1 functions, we mapped its DNA binding domain using a combined in vivo and in vitro approach. Our results show that the Zap1 DNA binding domain maps to the carboxyl-terminal 194 amino acids of the protein; this region contains five of its seven potential zinc finger domains. Fusing this region to the Gal4 activation domain complemented a zap1Delta mutation for low zinc growth and also conferred high level expression on a zinc-responsive element-lacZ reporter. In vitro, the purified 194-residue fragment bound to DNA with a high affinity (dissociation constant in the low nanomolar range) similar to that of longer fragments of Zap1. Furthermore, by deletion and site-directed mutagenesis, we demonstrated that each of the five carboxyl-terminal zinc fingers are required for high affinity DNA binding.