scholarly journals The E. coli Sec Reaction Pathway for Cellular Protein Sorting under a Single Molecule Loupe

2018 ◽  
Vol 114 (3) ◽  
pp. 566a
Author(s):  
Niels Vandenberk
Keyword(s):  
Single Molecule ◽  
Reaction Pathway ◽  
Protein Sorting ◽  
Cellular Protein ◽  
E Coli

scholarly journals Biphasic unbinding of Zur from DNA for transcription (de)repression in Live Bacteria

2018 ◽  
Author(s):  
Won Jung ◽  
Peng Chen
Keyword(s):  
Single Molecule ◽  
Ternary Complex ◽  
Consensus Sequence ◽  
Salt Bridge ◽  
Cellular Protein ◽  
Protein Oligomerization ◽  
E Coli ◽  
Transcription Regulator ◽  
Ternary Complex Formation ◽  
Live Bacteria

AbstractTranscription regulator on-off binding to DNA constitutes a mechanistic paradigm in gene regulation, in which the repressors/activators bind to operator sites tightly while the corresponding non-repressors/non-activators do not. Another paradigm regards regulator unbinding from DNA to be a unimolecular process whose kinetics is independent of regulator concentration. Using single-molecule single-cell measurements, we find that the behaviors of the zinc-responsive uptake regulator Zur challenges these paradigms. Apo-Zur, a non-repressor and presumed non-DNA binder, can bind to chromosome tightly in live E. coli cells, likely at non-consensus sequence sites. Moreover, the unbinding from DNA of its apo-non-repressor and holo-repressor forms both show a biphasic, repressed-followed-by-facilitated kinetics with increasing cellular protein concentrations. The facilitated unbinding likely occurs via a ternary complex formation mechanism; the repressed unbinding is first-of-its-kind and likely results from protein oligomerization on chromosome, in which an inter-protein salt-bridge plays a key role. This biphasic unbinding could provide functional advantages in Zur's facile switching between repression and derepression.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

scholarly journals Fast and efficient Rmap assembly using the Bi-labelled de Bruijn graph

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Kingshuk Mukherjee ◽  
Massimiliano Rossi ◽  
Leena Salmela ◽  
Christina Boucher
Keyword(s):  
Single Molecule ◽  
De Bruijn Graph ◽  
Anabas Testudineus ◽  
E Coli ◽  
Genome Wide ◽  
A Genome ◽  
De Bruijn ◽  
Optical Maps ◽  
Definition Of ◽  
Numeric Representation

AbstractGenome wide optical maps are high resolution restriction maps that give a unique numeric representation to a genome. They are produced by assembling hundreds of thousands of single molecule optical maps, which are called Rmaps. Unfortunately, there are very few choices for assembling Rmap data. There exists only one publicly-available non-proprietary method for assembly and one proprietary software that is available via an executable. Furthermore, the publicly-available method, by Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006), follows the overlap-layout-consensus (OLC) paradigm, and therefore, is unable to scale for relatively large genomes. The algorithm behind the proprietary method, Bionano Genomics’ Solve, is largely unknown. In this paper, we extend the definition of bi-labels in the paired de Bruijn graph to the context of optical mapping data, and present the first de Bruijn graph based method for Rmap assembly. We implement our approach, which we refer to as rmapper, and compare its performance against the assembler of Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006) and Solve by Bionano Genomics on data from three genomes: E. coli, human, and climbing perch fish (Anabas Testudineus). Our method was able to successfully run on all three genomes. The method of Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006) only successfully ran on E. coli. Moreover, on the human genome rmapper was at least 130 times faster than Bionano Solve, used five times less memory and produced the highest genome fraction with zero mis-assemblies. Our software, rmapper is written in C++ and is publicly available under GNU General Public License at https://github.com/kingufl/Rmapper.

scholarly journals Studying Binding, Conformational Transition and Assembly of E. Coli Cytolysin a Pore Forming Toxin by Single Molecule Fluorescence

2017 ◽  
Vol 112 (3) ◽  
pp. 524a
Author(s):  
Pradeep Sathyanarayana ◽  
Satyaghosh Maurya ◽  
Ganapathy Ayappa ◽  
Sandhya S. Visweswariah ◽  
Rahul Roy
Keyword(s):  
Single Molecule ◽  
Conformational Transition ◽  
Single Molecule Fluorescence ◽  
E Coli

scholarly journals Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Xue Fei ◽  
Tristan A Bell ◽  
Simon Jenni ◽  
Benjamin M Stinson ◽  
Tania A Baker ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Functional Results ◽  
Structural Features ◽  
Specific Protein ◽  
Power Stroke ◽  
E Coli ◽  
Protein Substrates ◽  
Biophysical Studies ◽  
Sequential Models

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

scholarly journals DNA sliding and loop formation by E. coli SMC complex: MukBEF

2021 ◽  
Author(s):  
man zhou
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Dna Interaction ◽  
Loop Formation ◽  
Unknown Mechanism ◽  
E Coli ◽  
Dna Compaction ◽  
And Function ◽  
Structural Maintenance Of Chromosomes

SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.

scholarly journals Modelling reaction kinetics inside cells

2008 ◽  
Vol 45 ◽  
pp. 41-56 ◽  
Author(s):  
Ramon Grima ◽  
Santiago Schnell
Keyword(s):  
Molecular Biology ◽  
Reaction Kinetics ◽  
Single Molecule ◽  
Predictive Accuracy ◽  
Reaction Pathway ◽  
Imaging Techniques ◽  
Deterministic Models ◽  
Non Invasive ◽  
The Past ◽  
Fundamental Length

In the past decade, advances in molecular biology such as the development of non-invasive single molecule imaging techniques have given us a window into the intricate biochemical activities that occur inside cells. In this chapter we review four distinct theoretical and simulation frameworks: (i) non-spatial and deterministic, (ii) spatial and deterministic, (iii) non-spatial and stochastic and (iv) spatial and stochastic. Each framework can be suited to modelling and interpreting intracellular reaction kinetics. By estimating the fundamental length scales, one can roughly determine which models are best suited for the particular reaction pathway under study. We discuss differences in prediction between the four modelling methodologies. In particular we show that taking into account noise and space does not simply add quantitative predictive accuracy but may also lead to qualitatively different physiological predictions, unaccounted for by classical deterministic models.

Magnetic Tweezers-Based Single-Molecule Assays to Study Interaction of E. coli SSB with DNA and

2021 ◽  
pp. 93-115
Author(s):  
Debjani Bagchi ◽  
Weiting Zhang ◽  
Samar Hodeib ◽  
Bertrand Ducos ◽  
Vincent Croquette ◽  
...  
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers ◽  
E Coli
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