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

Mapping Intimacies ◽

10.1101/434738 ◽

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.

Download Full-text

Biphasic unbinding of a metalloregulator from DNA for transcription (de)repression in Live Bacteria

Nucleic Acids Research ◽

10.1093/nar/gkaa056 ◽

2020 ◽

Vol 48 (5) ◽

pp. 2199-2208

Author(s):

Won Jung ◽

Kushal Sengupta ◽

Brian M Wendel ◽

John D Helmann ◽

Peng Chen

Keyword(s):

Single Molecule ◽

Protein Concentration ◽

Consensus Sequence ◽

Salt Bridges ◽

Ternary Complex Formation ◽

Escherichia Coli Cells ◽

Live Bacteria ◽

Zinc Sensing ◽

Conventional Models ◽

Uptake Transporters

Abstract Microorganisms use zinc-sensing regulators to alter gene expression in response to changes in the availability of zinc, an essential micronutrient. Under zinc-replete conditions, the Fur-family metalloregulator Zur binds to DNA tightly in its metallated repressor form to Zur box operator sites, repressing the transcription of zinc uptake transporters. Derepression comes from unbinding of the regulator, which, under zinc-starvation conditions, exists in its metal-deficient non-repressor forms having no significant affinity with Zur box. While the mechanism of transcription repression by Zur is well-studied, little is known on how derepression by Zur could be facilitated. Using single-molecule/single-cell measurements, we find that in live Escherichia coli cells, Zur's unbinding rate from DNA is sensitive to Zur protein concentration in a first-of-its-kind biphasic manner, initially impeded and then facilitated with increasing Zur concentration. These results challenge conventional models of protein unbinding being unimolecular processes and independent of protein concentration. The facilitated unbinding component likely occurs via a ternary complex formation mechanism. The impeded unbinding component likely results from Zur oligomerization on chromosome involving inter-protein salt-bridges. Unexpectedly, a non-repressor form of Zur is found to bind chromosome tightly, likely at non-consensus sequence sites. These unusual behaviors could provide functional advantages in Zur's facile switching between repression and derepression.

Download Full-text

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

Biophysical Journal ◽

10.1016/j.bpj.2017.11.3096 ◽

2018 ◽

Vol 114 (3) ◽

pp. 566a

Author(s):

Niels Vandenberk

Keyword(s):

Single Molecule ◽

Reaction Pathway ◽

Protein Sorting ◽

Cellular Protein ◽

E Coli

Download Full-text

Simultaneous Binding of Multiple EF-Tu Copies to Translating Ribosomes in Live Escherichia coli

mBio ◽

10.1128/mbio.02143-17 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 14

Author(s):

Mainak Mustafi ◽

James C. Weisshaar

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Single Molecule ◽

Ternary Complex ◽

Elongation Factor ◽

Ternary Complexes ◽

Local Concentration ◽

E Coli ◽

A Site

ABSTRACT In bacteria, elongation factor Tu is a translational cofactor that forms ternary complexes with aminoacyl-tRNA (aa-tRNA) and GTP. Binding of a ternary complex to one of four flexible L7/L12 units on the ribosome tethers a charged tRNA in close proximity to the ribosomal A site. Two sequential tests for a match between the aa-tRNA anticodon and the current mRNA codon then follow. Because one elongation cycle can occur in as little as 50 ms and the vast majority of aa-tRNA copies are not cognate with the current mRNA codon, this testing must occur rapidly. We present a single-molecule localization and tracking study of fluorescently labeled EF-Tu in live Escherichia coli. Imaging at 2 ms/frame distinguishes 60% slowly diffusing EF-Tu copies (assigned as transiently bound to translating ribosome) from 40% rapidly diffusing copies (assigned as a mixture of free ternary complexes and free EF-Tu). Combining these percentages with copy number estimates, we infer that the four L7/L12 sites are essentially saturated with ternary complexes in vivo. The results corroborate an earlier inference that all four sites can simultaneously tether ternary complexes near the A site, creating a high local concentration that may greatly enhance the rate of testing of aa-tRNAs. Our data and a combinatorial argument both suggest that the initial recognition test for a codon-anticodon match occurs in less than 1 to 2 ms per aa-tRNA copy. The results refute a recent study (A. Plochowietz, I. Farrell, Z. Smilansky, B. S. Cooperman, and A. N. Kapanidis, Nucleic Acids Res 45:926–937, 2016, https://doi.org/10.1093/nar/gkw787) of tRNA diffusion in E. coli that inferred that aa-tRNAs arrive at the ribosomal A site as bare monomers, not as ternary complexes. IMPORTANCE Ribosomes catalyze translation of the mRNA codon sequence into the corresponding sequence of amino acids within the nascent polypeptide chain. Polypeptide elongation can be as fast as 50 ms per added amino acid. Each amino acid arrives at the ribosome as a ternary complex comprising an aminoacyl-tRNA (aa-tRNA), an elongation factor called EF-Tu, and GTP. There are 43 different aa-tRNAs in use, only one of which typically matches the current mRNA codon. Thus, ternary complexes must be tested very rapidly. Here we use fluorescence-based single-molecule methods that locate and track single EF-Tu copies in E. coli. Fast and slow diffusive behavior determines the fraction of EF-Tu copies that are ribosome bound. We infer simultaneous tethering of ~4 ternary complexes to the ribosome, which may facilitate rapid initial testing for codon matching on a time scale of less than 1 to 2 ms per aa-tRNA.

Download Full-text

Single-molecule imaging in live bacteria cells

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2012.0355 ◽

2013 ◽

Vol 368 (1611) ◽

pp. 20120355 ◽

Cited By ~ 13

Author(s):

Ken Ritchie ◽

Yoriko Lill ◽

Chetan Sood ◽

Hochan Lee ◽

Shunyuan Zhang

Keyword(s):

Single Molecule ◽

Caulobacter Crescentus ◽

Ensemble Methods ◽

Model Systems ◽

Living Systems ◽

Single Molecule Imaging ◽

E Coli ◽

The Past ◽

Live Bacteria ◽

Made In

Bacteria, such as Escherichia coli and Caulobacter crescentus , are the most studied and perhaps best-understood organisms in biology. The advances in understanding of living systems gained from these organisms are immense. Application of single-molecule techniques in bacteria have presented unique difficulties owing to their small size and highly curved form. The aim of this review is to show advances made in single-molecule imaging in bacteria over the past 10 years, and to look to the future where the combination of implementing such high-precision techniques in well-characterized and controllable model systems such as E. coli could lead to a greater understanding of fundamental biological questions inaccessible through classic ensemble methods.

Download Full-text

Convergence of DNA methylation and phosphorothioation epigenetics in bacterial genomes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1702450114 ◽

2017 ◽

Vol 114 (17) ◽

pp. 4501-4506 ◽

Cited By ~ 34

Author(s):

Chao Chen ◽

Lianrong Wang ◽

Si Chen ◽

Xiaolin Wu ◽

Meijia Gu ◽

...

Keyword(s):

Single Molecule ◽

Consensus Sequence ◽

Spectrometric Analysis ◽

Nonbridging Oxygen ◽

Temperature Sensitive ◽

Dna Modification ◽

Consensus Sequences ◽

E Coli ◽

Chemical Structures

Explosive growth in the study of microbial epigenetics has revealed a diversity of chemical structures and biological functions of DNA modifications in restriction–modification (R-M) and basic genetic processes. Here, we describe the discovery of shared consensus sequences for two seemingly unrelated DNA modification systems,6mA methylation and phosphorothioation (PT), in which sulfur replaces a nonbridging oxygen in the DNA backbone. Mass spectrometric analysis of DNA fromEscherichia coliB7A andSalmonella entericaserovar Cerro 87, strains possessing PT-based R-M genes, revealed d(GPS6mA) dinucleotides in the GPS6mAAC consensus representing ∼5% of the 1,100 to 1,300 PT-modified d(GPSA) motifs per genome, with6mA arising from a yet-to-be-identified methyltransferase. To further explore PT and6mA in another consensus sequence, GPS6mATC, we engineered a strain ofE. coliHST04 to express Dnd genes fromHahella chejuensisKCTC2396 (PT in GPSATC) and Dam methyltransferase fromE. coliDH10B (6mA in G6mATC). Based on this model, in vitro studies revealed reduced Dam activity in GPSATC-containing oligonucleotides whereas single-molecule real-time sequencing of HST04 DNA revealed6mA in all 2,058 GPSATC sites (5% of 37,698 total GATC sites). This model system also revealed temperature-sensitive restriction by DndFGH in KCTC2396 and B7A, which was exploited to discover that6mA can substitute for PT to confer resistance to restriction by the DndFGH system. These results point to complex but unappreciated interactions between DNA modification systems and raise the possibility of coevolution of interacting systems to facilitate the function of each.

Download Full-text

RoXaN, a Novel Cellular Protein Containing TPR, LD, and Zinc Finger Motifs, Forms a Ternary Complex with Eukaryotic Initiation Factor 4G and Rotavirus NSP3

Journal of Virology ◽

10.1128/jvi.78.8.3851-3862.2004 ◽

2004 ◽

Vol 78 (8) ◽

pp. 3851-3862 ◽

Cited By ~ 29

Author(s):

Damien Vitour ◽

Pierre Lindenbaum ◽

Patrice Vende ◽

Michelle M. Becker ◽

Didier Poncet

Keyword(s):

Amino Acids ◽

Zinc Finger ◽

Protein Interactions ◽

Ternary Complex ◽

Consensus Sequence ◽

Cellular Protein ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Amino Terminal ◽

Zinc Finger Motifs

ABSTRACT Rotavirus mRNAs are capped but not polyadenylated, and viral proteins are translated by the cellular translation machinery. This is accomplished through the action of the viral nonstructural protein NSP3, which specifically binds the 3′ consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G I. To further our understanding of the role of NSP3 in rotavirus replication, we looked for other cellular proteins capable of interacting with this viral protein. Using the yeast two-hybrid assay, we identified a novel cellular protein-binding partner for rotavirus NSP3. This 110-kDa cellular protein, named RoXaN (rotavirus X protein associated with NSP3), contains a minimum of three regions predicted to be involved in protein-protein or nucleic acid-protein interactions. A tetratricopeptide repeat region, a protein-protein interaction domain most often found in multiprotein complexes, is present in the amino-terminal region. In the carboxy terminus, at least five zinc finger motifs are observed, further suggesting the capacity of RoXaN to bind other proteins or nucleic acids. Between these two regions exists a paxillin leucine-aspartate repeat (LD) motif which is involved in protein-protein interactions. RoXaN is capable of interacting with NSP3 in vivo and during rotavirus infection. Domains of interaction were mapped and correspond to the dimerization domain of NSP3 (amino acids 163 to 237) and the LD domain of RoXaN (amino acids 244 to 341). The interaction between NSP3 and RoXaN does not impair the interaction between NSP3 and eIF4G I, and a ternary complex made of NSP3, RoXaN, and eIF4G I can be detected in rotavirus-infected cells, implicating RoXaN in translation regulation.

Download Full-text

Binding Mechanism and Structural Insights into the Identified Protein Target of Covid-19 with In-Vitro Effective Drug Ivermectin

10.26434/chemrxiv.12463946.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Parth Sarthi Sen Gupta ◽

Satyaranjan Biswal ◽

Saroj Kumar Panda ◽

Abhik Kumar Ray ◽

Malay Kumar Rana

Keyword(s):

Ternary Complex ◽

Bound State ◽

Molecular Target ◽

Cooperative Interaction ◽

Effective Drug ◽

Rna Dependent Rna Polymerase ◽

Protein Target ◽

Ternary Complex Formation ◽

Structural Insights

While an FDA approved drug Ivermectin was reported to dramatically reduce the cell line of SARS-CoV-2 by ~5000 folds within 48 hours, the precise mechanism of action and the COVID-19 molecular target involved in interaction with this in-vitro effective drug are unknown yet. Among 12 different COVID-19 targets studied here, the RNA dependent RNA polymerase (RdRp) with RNA and Helicase NCB site show the strongest affinity to Ivermectin amounting -10.4 kcal/mol and -9.6 kcal/mol, respectively. Molecular dynamics of corresponding protein-drug complexes reveals that the drug bound state of RdRp with RNA has better structural stability than the Helicase NCB site, with MM/PBSA free energy of -135.2 kJ/mol, almost twice that of Helicase (-76.6 kJ/mol). The selectivity of Ivermectin to RdRp is triggered by a cooperative interaction of RNA-RdRp by ternary complex formation. Identification of the target and its interaction profile with Ivermectin can lead to more powerful drug designs for COVID-19 and experimental exploration.

Download Full-text

Coaction of Electrostatic and Hydrophobic Interactions: Dynamic Constraints on Disordered TrkA Juxtamembrane Domain

10.26434/chemrxiv.9847190 ◽

2019 ◽

Author(s):

Zichen Wang ◽

Huaxun Fan ◽

Xiao Hu ◽

John Khamo ◽

Jiajie Diao ◽

...

Keyword(s):

Single Molecule ◽

Protein Function ◽

Hydrophobic Interactions ◽

Transmembrane Helix ◽

Point Mutations ◽

Salt Bridge ◽

Receptor Activation ◽

Membrane Dynamics ◽

Juxtamembrane Domain ◽

Joint Work

The receptor tyrosine kinase family transmits signals into cell via a single transmembrane helix and a flexible juxtamembrane domain (JMD). Membrane dynamics makes it challenging to study the structural mechanism of receptor activation experimentally. In this study, we employ all-atom molecular dynamics with Highly Mobile Membrane-Mimetic to capture membrane interactions with the JMD of tropomyosin receptor kinase A (TrkA). We find that PIP2 lipids engage in lasting binding to multiple basic residues and compete with salt bridge within the peptide. We discover three residues insertion into the membrane, and perturb it through computationally designed point mutations. Single-molecule experiments indicate the contribution from hydrophobic insertion is comparable to electrostatic binding, and in-cell experiments show that enhanced TrkA-JMD insertion promotes receptor ubiquitination. Our joint work points to a scenario where basic and hydrophobic residues on disordered domains interact with lipid headgroups and tails, respectively, to restrain flexibility and potentially modulate protein function.

Download Full-text