Random walks in the stringent response
Date
2016-11-02
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Poomisvastus on võtmetähtsusega adaptiivsete mehhanismide regulatsioonil, mis aitavad bakteritel ebasoodsaid keskkonnatingimusi üle elada. Soolekepikeses (Escherichia coli) on selles protsessis oluliseks ensüümiks RelA, mis vastusena aminohappenäljale sünteesib signaalmolekuli (p)ppGpp. See signaalmolekul mõjutab transkriptsiooni, translatsiooni ja rakkude jagunemist.
Meie töötasime välja ühe molekuli jälgimise mikroskoopia metoodika, mis võimaldab mõõta molekulide difusiooni rakus. Kasutasime seda metoodikat erineva kiirusega liikuvate molekulide kirjeldamiseks. Rakus vabalt difundeeruva valgu näiteks oli fluorestseeruv valk mEos2. Hoopis teistsuguste omadustega valguks osutus mitokondri membraanivalk Tom40, mille liikumine on ühte asukohta piiratud. RelA puhul täheldasime nii vabu, kiirelt difundeeruvaid molekule kui ka ribosoomile seondunud ja seetõttu aeglaselt liikuvaid molekule.
Kombineerides ühe molekuli jälgimise tulemusi biokeemiliste andmetega, pakume välja RelA valgu töötsükli mudeli. Kuhjuv (p)ppGpp põhjustab samuti RelA aktivatsiooni. Sellisel viisil tekib positiivse tagasisidestusega regulatsioonisüsteem ja signaalmolekuli kontsentratsioon tõuseb kiiresti.
The stringent response plays key role in the activation and regulation of the adaptive mechanisms that bacteria employ in order to accommodate to the adverse conditions. In E.coli the process is governed by the stringent factor RelA, transferring the amino-acid starvation signals by synthesize (p)ppGpp altering cell replication, transcription and translation. We have developed in vivo single-molecule tracking microscopy assay that allows us to track fast and slowly diffusive cytosolic (stringent factor RelA and free GFP variant mEos2) or membrane bound (mitochondrial membrane channel Tom40) proteins. The fluorescently labeled Tom40-Dendra2 complex in the mitochondrial membrane showed highly mobile but confined diffusion properties By combining biochemical and single-molecule microscopy approaches we have suggested different (p)ppGpp synthesizing mechanism from the standard hopping model where many (p)ppGpp molecules are produced upon dissociation of enzymatically active RelA from the ribosome and (p)ppGpp production is directly responsible for enhancement of the RelA enzymatic activity by positive feedback loop acting at the enzymatic level.
The stringent response plays key role in the activation and regulation of the adaptive mechanisms that bacteria employ in order to accommodate to the adverse conditions. In E.coli the process is governed by the stringent factor RelA, transferring the amino-acid starvation signals by synthesize (p)ppGpp altering cell replication, transcription and translation. We have developed in vivo single-molecule tracking microscopy assay that allows us to track fast and slowly diffusive cytosolic (stringent factor RelA and free GFP variant mEos2) or membrane bound (mitochondrial membrane channel Tom40) proteins. The fluorescently labeled Tom40-Dendra2 complex in the mitochondrial membrane showed highly mobile but confined diffusion properties By combining biochemical and single-molecule microscopy approaches we have suggested different (p)ppGpp synthesizing mechanism from the standard hopping model where many (p)ppGpp molecules are produced upon dissociation of enzymatically active RelA from the ribosome and (p)ppGpp production is directly responsible for enhancement of the RelA enzymatic activity by positive feedback loop acting at the enzymatic level.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Keywords
valgud, translatsioon (biol.), ensüümid, molekulid, proteins, translation (biol.), enzymes, molecules