42,872 research outputs found
The Interplay between Chemistry and Mechanics in the Transduction of a Mechanical Signal into a Biochemical Function
There are many processes in biology in which mechanical forces are generated.
Force-bearing networks can transduce locally developed mechanical signals very
extensively over different parts of the cell or tissues. In this article we
conduct an overview of this kind of mechanical transduction, focusing in
particular on the multiple layers of complexity displayed by the mechanisms
that control and trigger the conversion of a mechanical signal into a
biochemical function. Single molecule methodologies, through their capability
to introduce the force in studies of biological processes in which mechanical
stresses are developed, are unveiling subtle intertwining mechanisms between
chemistry and mechanics and in particular are revealing how chemistry can
control mechanics. The possibility that chemistry interplays with mechanics
should be always considered in biochemical studies.Comment: 50 pages, 18 figure
Kinetic approaches to lactose operon induction and bimodality
The quasi-equilibrium approximation is acceptable when molecular interactions
are fast enough compared to circuit dynamics, but is no longer allowed when
cellular activities are governed by rare events. A typical example is the
lactose operon (lac), one of the most famous paradigms of transcription
regulation, for which several theories still coexist to describe its behaviors.
The lac system is generally analyzed by using equilibrium constants,
contradicting single-event hypotheses long suggested by Novick and Weiner
(1957). Enzyme induction as an all-or-none phenomenon. Proc. Natl. Acad. Sci.
USA 43, 553-566) and recently refined in the study of (Choi et al., 2008. A
stochastic single-molecule event triggers phenotype switching of a bacterial
cell. Science 322, 442-446). In the present report, a lac repressor
(LacI)-mediated DNA immunoprecipitation experiment reveals that the natural
LacI-lac DNA complex built in vivo is extremely tight and long-lived compared
to the time scale of lac expression dynamics, which could functionally
disconnect the abortive expression bursts and forbid using the standard modes
of lac bistability. As alternatives, purely kinetic mechanisms are examined for
their capacity to restrict induction through: (i) widely scattered derepression
related to the arrival time variance of a predominantly backward asymmetric
random walk and (ii) an induction threshold arising in a single window of
derepression without recourse to nonlinear multimeric binding and Hill
functions. Considering the complete disengagement of the lac repressor from the
lac promoter as the probabilistic consequence of a transient stepwise
mechanism, is sufficient to explain the sigmoidal lac responses as functions of
time and of inducer concentration. This sigmoidal shape can be misleadingly
interpreted as a phenomenon of equilibrium cooperativity classically used to
explain bistability, but which has been reported to be weak in this system
Stochastic dynamics of macromolecular-assembly networks
The formation and regulation of macromolecular complexes provides the
backbone of most cellular processes, including gene regulation and signal
transduction. The inherent complexity of assembling macromolecular structures
makes current computational methods strongly limited for understanding how the
physical interactions between cellular components give rise to systemic
properties of cells. Here we present a stochastic approach to study the
dynamics of networks formed by macromolecular complexes in terms of the
molecular interactions of their components. Exploiting key thermodynamic
concepts, this approach makes it possible to both estimate reaction rates and
incorporate the resulting assembly dynamics into the stochastic kinetics of
cellular networks. As prototype systems, we consider the lac operon and phage
lambda induction switches, which rely on the formation of DNA loops by proteins
and on the integration of these protein-DNA complexes into intracellular
networks. This cross-scale approach offers an effective starting point to move
forward from network diagrams, such as those of protein-protein and DNA-protein
interaction networks, to the actual dynamics of cellular processes.Comment: Open Access article available at
http://www.nature.com/msb/journal/v2/n1/full/msb4100061.htm
Real sequence effects on the search dynamics of transcription factors on DNA
Recent experiments show that transcription factors (TFs) indeed use the
facilitated diffusion mechanism to locate their target sequences on DNA in
living bacteria cells: TFs alternate between sliding motion along DNA and
relocation events through the cytoplasm. From simulations and theoretical
analysis we study the TF-sliding motion for a large section of the DNA-sequence
of a common E. coli strain, based on the two-state TF-model with a fast-sliding
search state and a recognition state enabling target detection. For the
probability to detect the target before dissociating from DNA the TF-search
times self-consistently depend heavily on whether or not an auxiliary operator
(an accessible sequence similar to the main operator) is present in the genome
section. Importantly, within our model the extent to which the interconversion
rates between search and recognition states depend on the underlying nucleotide
sequence is varied. A moderate dependence maximises the capability to
distinguish between the main operator and similar sequences. Moreover, these
auxiliary operators serve as starting points for DNA looping with the main
operator, yielding a spectrum of target detection times spanning several orders
of magnitude. Auxiliary operators are shown to act as funnels facilitating
target detection by TFs.Comment: 26 pages, 7 figure
Computational Investigations on Polymerase Actions in Gene Transcription and Replication Combining Physical Modeling and Atomistic Simulations
Polymerases are protein enzymes that move along nucleic acid chains and
catalyze template-based polymerization reactions during gene transcription and
replication. The polymerases also substantially improve transcription or
replication fidelity through the non-equilibrium enzymatic cycles. We briefly
review computational efforts that have been made toward understanding
mechano-chemical coupling and fidelity control mechanisms of the polymerase
elongation. The polymerases are regarded as molecular information motors during
the elongation process. It requires a full spectrum of computational approaches
from multiple time and length scales to understand the full polymerase
functional cycle. We keep away from quantum mechanics based approaches to the
polymerase catalysis due to abundant former surveys, while address only
statistical physics modeling approach and all-atom molecular dynamics
simulation approach. We organize this review around our own modeling and
simulation practices on a single-subunit T7 RNA polymerase, and summarize
commensurate studies on structurally similar DNA polymerases. For multi-subunit
RNA polymerases that have been intensively studied in recent years, we leave
detailed discussions on the simulation achievements to other computational
chemical surveys, while only introduce very recently published representative
studies, including our own preliminary work on structure-based modeling on
yeast RNA polymerase II. In the end, we quickly go through kinetic modeling on
elongation pauses and backtracking activities. We emphasize the fluctuation and
control mechanisms of the polymerase actions, highlight the non-equilibrium
physical nature of the system, and try to bring some perspectives toward
understanding replication and transcription regulation from single molecular
details to a genome-wide scale
Concentration and Length Dependence of DNA Looping in Transcriptional Regulation
In many cases, transcriptional regulation involves the binding of transcription factors at sites on the DNA that are not immediately adjacent to the promoter of interest. This action at a distance is often mediated by the formation of DNA loops: Binding at two or more sites on the DNA results in the formation of a loop, which can bring the transcription factor into the immediate neighborhood of the relevant promoter. These processes are important in settings ranging from the historic bacterial examples (bacterial metabolism and the lytic-lysogeny decision in bacteriophage), to the modern concept of gene regulation to regulatory processes central to pattern formation during development of multicellular organisms. Though there have been a variety of insights into the combinatorial aspects of transcriptional control, the mechanism of DNA looping as an agent of combinatorial control in both prokaryotes and eukaryotes remains unclear. We use single-molecule techniques to dissect DNA looping in the lac operon. In particular, we measure the propensity for DNA looping by the Lac repressor as a function of the concentration of repressor protein and as a function of the distance between repressor binding sites. As with earlier single-molecule studies, we find (at least) two distinct looped states and demonstrate that the presence of these two states depends both upon the concentration of repressor protein and the distance between the two repressor binding sites. We find that loops form even at interoperator spacings considerably shorter than the DNA persistence length, without the intervention of any other proteins to prebend the DNA. The concentration measurements also permit us to use a simple statistical mechanical model of DNA loop formation to determine the free energy of DNA looping, or equivalently, the J-factor for looping
Influence of mutations of Val226 on the catalytic rate of haloalkane dehalogenase
Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols. The 3D structure, reaction mechanism and kinetic mechanism have been studied. The steady state kcat with 1,2-dichloroethane and 1,2-dibromoethane is limited mainly by the rate of release of the halide ion from the buried active-site cavity. During catalysis, the halogen that is cleaved off (Clα) from 1,2-dichloroethane interacts with Trp125 and the Clβ interacts with Phe172. Both these residues have van der Waals contacts with Val226. To establish the effect of these interactions on catalysis, and in an attempt to change enzyme activity without directly mutating residues involved in catalysis, we mutated Val226 to Gly, Ala and Leu. The Val226Ala and Val226Leu mutants had a 2.5-fold higher catalytic rate for 1,2-dibromoethane than the wild-type enzyme. A pre-steady state kinetic analysis of the Val226Ala mutant enzyme showed that the increase in kcat could be attributed to an increase in the rate of a conformational change that precedes halide release, causing a faster overall rate of halide dissociation. The kcat for 1,2-dichloroethane conversion was not elevated, although the rate of chloride release was also faster than in the wild-type enzyme. This was caused by a 3-fold decrease in the rate of formation of the alkyl-enzyme intermediate for 1,2-dichloroethane. Val226 seems to contribute to leaving group (Clα or Brα) stabilization via Trp125, and can influence halide release and substrate binding via an interaction with Phe172. These studies indicate that wild-type haloalkane dehalogenase is optimized for 1,2-dichloroethane, although 1,2-dibromoethane is a better substrate.
An integrative approach for modeling and simulation of Heterocyst pattern formation in Cyanobacteria strands
A comprehensive approach to cellular differentiation in cyanobacteria is
developed. To this aim, the process of heterocyst cell formation is studied
under a systems biology point of view. By relying on statistical physics
techniques, we translate the essential ingredients and mechanisms of the
genetic circuit into a set of differential equations that describes the
continuous time evolution of combined nitrogen, PatS, HetR and NtcA
concentrations. The detailed analysis of these equations gives insight into the
single cell dynamics. On the other hand, the inclusion of diffusion and noisy
conditions allows simulating the formation of heterocysts patterns in
cyanobacteria strains. The time evolution of relevant component concentrations
are calculated allowing for a comparison with experiments. Finally, we discuss
the validity and the possible improvements of the model.Comment: 20 pages (including the supporting information), 8 figure
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