scholarly journals Monomeric YoeB toxin retains RNase activity but adopts an obligate dimeric form for thermal stability

2019 ◽  
Vol 47 (19) ◽  
pp. 10400-10413 ◽  
Author(s):  
Ian J Pavelich ◽  
Tatsuya Maehigashi ◽  
Eric D Hoffer ◽  
Ajchareeya Ruangprasert ◽  
Stacey J Miles ◽  
...  
Keyword(s):  
Large Subunit ◽  
Dependent Manner ◽  
Type Ii ◽  
Substrate Selection ◽  
E Coli ◽  
Dimeric Form ◽  
The Stability ◽  
Stress Dependent ◽  
A Site ◽  
Sense Codon

Abstract Chromosomally-encoded toxin-antitoxin complexes are ubiquitous in bacteria and regulate growth through the release of the toxin component typically in a stress-dependent manner. Type II ribosome-dependent toxins adopt a RelE-family RNase fold and inhibit translation by degrading mRNAs while bound to the ribosome. Here, we present biochemical and structural studies of the Escherichia coli YoeB toxin interacting with both a UAA stop and an AAU sense codon in pre- and post-mRNA cleavage states to provide insights into possible mRNA substrate selection. Both mRNAs undergo minimal changes during the cleavage event in contrast to type II ribosome-dependent RelE toxin. Further, the 16S rRNA decoding site nucleotides that monitor the mRNA in the aminoacyl(A) site adopt different orientations depending upon which toxin is present. Although YoeB is a RelE family member, it is the sole ribosome-dependent toxin that is dimeric. We show that engineered monomeric YoeB is active against mRNAs bound to both the small and large subunit. However, the stability of monomeric YoeB is reduced ∼20°C, consistent with potential YoeB activation during heat shock in E. coli as previously demonstrated. These data provide a molecular basis for the ability of YoeB to function in response to thermal stress.

scholarly journals Regulation of RraA, a Protein Inhibitor of RNase E-Mediated RNA Decay

2006 ◽  
Vol 188 (9) ◽  
pp. 3257-3263 ◽  
Author(s):  
Meng Zhao ◽  
Li Zhou ◽  
Yasuaki Kawarasaki ◽  
George Georgiou
Keyword(s):  
Escherichia Coli ◽  
Intergenic Region ◽  
Ectopic Expression ◽  
Dependent Manner ◽  
Feedback Circuit ◽  
Rnase E ◽  
Protein Inhibitor ◽  
E Coli ◽  
The Stability ◽  
Fusion Analysis

ABSTRACT The recently discovered RraA protein acts as an inhibitor of the essential endoribonuclease RNase E, and we demonstrated that ectopic expression of RraA affects the abundance of more than 700 transcripts in Escherichia coli (K. Lee, X. Zhan, J. Gao, J. Qiu, Y. Feng, R. Meganathan, S. N. Cohen, and G. Georgiou, Cell 114:623-634, 2003). We show that rraA is expressed from its own promoter, P rraA , located in the menA-rraA intergenic region. Primer extension and lacZ fusion analysis revealed that transcription from P rraA is elevated upon entry into stationary phase in a σs-dependent manner. In addition, the stability of the rraA transcript is dependent on RNase E activity, suggesting the involvement of a feedback circuit in the regulation of the RraA level in E. coli.

scholarly journals METTL3-Mediated DLGAP1 Antisense RNA 2 Promotes Gastric Cancer Tumorigenesis Through Facilitating C-Myc-Dependent Warburg Effect

2021 ◽  
Author(s):  
Changshun Yang ◽  
Yu Zhang ◽  
Xuefei Cheng ◽  
Weihua Li
Keyword(s):  
Gastric Cancer ◽  
Antisense Rna ◽  
Warburg Effect ◽  
Aerobic Glycolysis ◽  
Dependent Manner ◽  
Rna Immunoprecipitation ◽  
The Stability ◽  
A Site ◽  
Rip Assay ◽  
Site Binding

Abstract Background The critical roles of N6-methyladenosine (m6A) modification have been demonstrated by more and more evidence. However, the cross-talking of m6A and long non-coding RNAs (lncRNAs) in gastric cancer (GC) tumorigenesis is still unclear. Here, our work focused on the functions and molecular mechanism of m6A-modified lncRNA DLGAP1 antisense RNA 2 (DLGAP1-AS2) in GC. Methods LncRNA expression profile data was derived from GEO. M6A profile was screened using Methylated RNA immunoprecipitation sequencing (MeRIP-Seq). The metabolism assays were conducted using quantitative analysis of glucose, lactate, ATP and extracellular acidification rate (ECAR). The m6A level of specific RNA was identified using MeRIP-qPCR. The molecular interaction was detected using RIP assay. Results Microarray analysis found that lncRNA DLGAP1-AS2 up-regulated in GC cells. Clinical data showed that DLGAP1-AS2 high-expression was correlated with advanced pathological stage and poor prognosis. Functionally, DLGAP1-AS2 promoted the Warburg effect (aerobic glycolysis) and knockdown of DLGAP1-AS2 suppressed the tumor growth of GC cells. Mechanistically, m6A methyltransferase METTL3 enhanced the stability of DLGAP1-AS2 via m6A site binding. Moreover, DLGAP1-AS2 interacted with YTHDF1 to enhance the stability of c-Myc mRNA through DLGAP1-AS2/m6A/YTHDF1/c-Myc mRNA. Conclusions In conclusion, our work indicates the functions of m6A-modified DLGAP1-AS2 in the GC aerobic glycolysis, disclosing a potential m6A-dependent manner for GC treatment.

scholarly journals AtaT Improves the Stability of Pore-Forming Protein EspB by Acetylating Lysine 206 to Enhance Strain Virulence

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhili He ◽  
Tao Li ◽  
Jianxin Wang ◽  
Deyan Luo ◽  
Nianzhi Ning ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Animal Models ◽  
Type Iii Secretion ◽  
Underlying Mechanism ◽  
Secretion System ◽  
Host Cells ◽  
Type Ii ◽  
Pathogenic Species ◽  
E Coli ◽  
The Stability

A novel type II toxin of toxin–antitoxin systems (TAs), Gcn5-related N-acetyltransferase (GNAT) family, was reported recently. GNAT toxins are mainly present in pathogenic species, but studies of their involvement in pathogenicity are rare. This study discovered that the GANT toxin AtaT in enterohemorrhagic Escherichia coli (EHEC) can significantly enhance strain pathogenicity. First, we detected the virulence of ΔataT and ΔataR in cell and animal models. In the absence of ataT, strains showed a lower adhesion number, and host cells presented weaker attaching and effacing lesions, inflammatory response, and pathological injury. Next, we screened the acetylation substrate of AtaT to understand the underlying mechanism. Results showed that E. coli pore-forming protein EspB, which acts as a translocon in type III secretion system (T3SS) in strains, can be acetylated specifically by AtaT. The acetylation of K206 in EspB increases protein stability and maintains the efficiency of effectors translocating into host cells to cause close adhesion and tissue damage.

scholarly journals Mechanism of aminoacyl-tRNA acetylation by an aminoacyl-tRNA acetyltransferase AtaT from enterohemorrhagic E. coli

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuka Yashiro ◽  
Yuriko Sakaguchi ◽  
Tsutomu Suzuki ◽  
Kozo Tomita
Keyword(s):  
Translation Initiation ◽  
Stress Adaptation ◽  
Amino Group ◽  
Type Ii ◽  
Substrate Selection ◽  
Base Pairs ◽  
E Coli ◽  
Acceptor Stem ◽  
Global Translation

Abstract Toxin-antitoxin systems in bacteria contribute to stress adaptation, dormancy, and persistence. AtaT, a type-II toxin in enterohemorrhagic E. coli, reportedly acetylates the α-amino group of the aminoacyl-moiety of initiator Met-tRNAfMet, thus inhibiting translation initiation. Here, we show that AtaT has a broader specificity for aminoacyl-tRNAs than initially claimed. AtaT efficiently acetylates Gly-tRNAGly, Trp-tRNATrp, Tyr-tRNATyr and Phe-tRNAPhe isoacceptors, in addition to Met-tRNAfMet, and inhibits global translation. AtaT interacts with the acceptor stem of tRNAfMet, and the consecutive G-C pairs in the bottom-half of the acceptor stem are required for acetylation. Consistently, tRNAGly, tRNATrp, tRNATyr and tRNAPhe also possess consecutive G-C base-pairs in the bottom halves of their acceptor stems. Furthermore, misaminoacylated valyl-tRNAfMet and isoleucyl-tRNAfMet are not acetylated by AtaT. Therefore, the substrate selection by AtaT is governed by the specific acceptor stem sequence and the properties of the aminoacyl-moiety of aminoacyl-tRNAs.

scholarly journals IMMOBILIZATION AND APPLICATION OF PARTIAL PURIFIED LOVASTATIN PRODUCED FROM LOCAL ISOLATE ASPERGILLUS TERREUS A50 USING SOLID STATE FERMENTATION

2021 ◽  
Vol 52 (2) ◽  
Author(s):  
A. J. R. Al-Sa'ady ◽  
G. M. Aziz
Keyword(s):  
Cell Line ◽  
Storage Time ◽  
Specific Activity ◽  
Dependent Manner ◽  
Hmg Coa Reductase ◽  
Minimum Fungicidal Concentration ◽  
E Coli ◽  
The Stability ◽  
Coa Reductase ◽  
And Storage

This work was designed to study the free and immobilized partial purified lovastatin in various applications. The results of HMG-CoA reductase inhibition showed enzyme inhibition at 10 mM of standard and partial purified lovastatin with specific activity 0.056 and 0.062 U/mg protein respectively, compared with specific activity 0.277 U/mg protein without inhibitor. The results of the thermal stability and storage time on lovastatin for inhibition of HMG-CoA reductase demonstrated that the standard and partial purified lovastatin were stabled in temperatures between 20-40 ᵒC, then the stability begun to decrease at 45 ᵒC, while lovastatin was stable in storage time between 1- 8 hours, then the stability begun to decrease after ten hours at 40 ºC. The results of MIC for lovastatin were demonstrated that most tested concentration were showed antibacterial activity of free and immobilized partial purified lovastatin against Candida albicans, Escherichia coli, and Staphylococcus aureus with MIC values ranging from 15 to 75 µg/ml. Whereas the results of minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) showed that C. albicans, E. coli, and S. aureus had no growth with concentration ranging from 55 to 75, 55 to 75, and 30 to 75 µg/ml, respectively. As well as the results of the cytotoxic impact using MTT experiment indicated that partial purified lovastatin caused a reduction in cells viability (p ≤ 0.05) at a dose-dependent manner on MCF-7 cell lines, with a calculating IC50 of 138.1 µg/ml, compare with normal cell line (WRL 68 Cell Line) at IC50 of 198.7 µg/ml.

Depletion and Reconstitution of Active E. Coli Ribosomes

1975 ◽  
Vol 33 ◽  
pp. 640-641
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Protein Biosynthesis ◽  
Large Subunit ◽  
High Resolution Electron Microscopy ◽  
Small Subunit ◽  
Structure And Function ◽  
Biologically Active ◽  
Biological Macromolecules ◽  
E Coli ◽  
Resolution Electron ◽  
And Function

Correlations between structure and function of biological macromolecules have been studied intensively for many years, mostly by indirect methods. High resolution electron microscopy is a unique tool which can provide such information directly by comparing the conformation of biopolymers in their biologically active and inactive state. We have correlated the structure and function of ribosomes, ribonucleoprotein particles which are the site of protein biosynthesis. 70S E. coli ribosomes, used in this experiment, are composed of two subunits - large (50S) and small (30S). The large subunit consists of 34 proteins and two different ribonucleic acid molecules. The small subunit contains 21 proteins and one RNA molecule. All proteins (with the exception of L7 and L12) are present in one copy per ribosome.This study deals with the changes in the fine structure of E. coli ribosomes depleted of proteins L7 and L12. These proteins are unique in many aspects.

Rationalizing the pH-Activity Response for Escherichia Coli’s Glycinamide Ribonucleotidetransformylase Through Computational Methods

2019 ◽  
Author(s):  
Adrian Roitberg ◽  
Pancham Lal Gupta
Keyword(s):  
Transfer Reaction ◽  
Catalytic Mechanism ◽  
Ph Dependence ◽  
Ph Effects ◽  
Dependent Manner ◽  
E Coli ◽  
Gar Tfase ◽  
Activity Curve ◽  
Ph Dependent ◽  
Formyl Transfer

<div>Human Glycinamide ribonucleotide transformylase (GAR Tfase), a regulatory enzyme in the de novo purine biosynthesis pathway, has been established as an anti-cancer target. GAR Tfase catalyzes the formyl transfer reaction from the folate cofactor to the GAR ligand. In the present work, we study E. coli GAR Tfase, which has high sequence similarity with the human GAR Tfase with most functional residues conserved. E. coli GAR Tfase exhibits structural changes and the binding of ligands that varies with pH which leads to change the rate of the formyl transfer reaction in a pH-dependent manner. Thus, the inclusion of pH becomes essential for the study of its catalytic mechanism. Experimentally, the pH-dependence of the kinetic parameter kcat is measured to evaluate the pH-range of enzymatic activity. However, insufficient information about residues governing the pH-effects on the catalytic activity leads to ambiguous assignments of the general acid and base catalysts and consequently its catalytic mechanism. In the present work, we use pH-replica exchange molecular dynamics (pH-REMD) simulations to study the effects of pH on E. coli GAR Tfase enzyme. We identify the titratable residues governing the pH-dependent conformational changes in the system. Furthermore, we filter out the protonation states which are essential in maintaining the structural integrity, keeping the ligands bound and assisting the catalysis. We reproduce the experimental pH-activity curve by computing the population of key protonation states. Moreover, we provide a detailed description of residues governing the acidic and basic limbs of the pH-activity curve.</div>

scholarly journals SVIP is a molecular determinant of lysosomal dynamic stability, neurodegeneration and lifespan

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alyssa E. Johnson ◽  
Brian O. Orr ◽  
Richard D. Fetter ◽  
Armen J. Moughamian ◽  
Logan A. Primeaux ◽  
...  
Keyword(s):  
Dynamic Stability ◽  
Muscle Wasting ◽  
Dependent Manner ◽  
Missense Mutations ◽  
Over Expression ◽  
System A ◽  
Molecular Determinant ◽  
Intracellular Organelles ◽  
Stress Dependent ◽  
Lateral Sclerosis

AbstractMissense mutations in Valosin-Containing Protein (VCP) are linked to diverse degenerative diseases including IBMPFD, amyotrophic lateral sclerosis (ALS), muscular dystrophy and Parkinson’s disease. Here, we characterize a VCP-binding co-factor (SVIP) that specifically recruits VCP to lysosomes. SVIP is essential for lysosomal dynamic stability and autophagosomal–lysosomal fusion. SVIP mutations cause muscle wasting and neuromuscular degeneration while muscle-specific SVIP over-expression increases lysosomal abundance and is sufficient to extend lifespan in a context, stress-dependent manner. We also establish multiple links between SVIP and VCP-dependent disease in our Drosophila model system. A biochemical screen identifies a disease-causing VCP mutation that prevents SVIP binding. Conversely, over-expression of an SVIP mutation that prevents VCP binding is deleterious. Finally, we identify a human SVIP mutation and confirm the pathogenicity of this mutation in our Drosophila model. We propose a model for VCP disease based on the differential, co-factor-dependent recruitment of VCP to intracellular organelles.

Arabidopsis AtCNGC10 rescues potassium channel mutants of E. coli, yeast and Arabidopsis and is regulated by calcium/calmodulin and cyclic GMP in E. coli

2005 ◽  
Vol 32 (7) ◽  
pp. 643 ◽  
Author(s):  
Xinli Li ◽  
Tamás Borsics ◽  
H. Michael Harrington ◽  
David A. Christopher
Keyword(s):  
Channel Blocker ◽  
K Channel ◽  
Structure Characteristic ◽  
Dependent Manner ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
E Coli ◽  
Diverse Species ◽  
The Family ◽  
Low K

We have isolated and characterised AtCNGC10, one of the 20 members of the family of cyclic nucleotide (CN)-gated and calmodulin (CaM)-regulated channels (CNGCs) from Arabidopsis thaliana (L.) Heynh. AtCNGC10 bound CaM in a C-terminal subregion that contains a basic amphiphillic structure characteristic of CaM-binding proteins and that also overlaps with the predicted CN-binding domain. AtCNGC10 is insensitive to the broad-range K+ channel blocker, tetraethylammonium, and lacks a typical K+-signature motif. However, AtCNGC10 complemented K+ channel uptake mutants of Escherichia coli (LB650), yeast (Saccharomyces cerevisiae CY162) and Arabidopsis (akt1-1). Sense 35S-AtCNGC10 transformed into the Arabidopsis akt1-1 mutant, grew 1.7-fold better on K+-limited medium relative to the vector control. Coexpression of CaM and AtCNGC10 in E. coli showed that Ca2+ / CaM inhibited cell growth by 40%, while cGMP reversed the inhibition by Ca2+ / CaM, in a AtCNGC10-dependent manner. AtCNGC10 did not confer tolerance to Cs+ in E. coli, however, it confers tolerance to toxic levels of Na+ and Cs+ in the yeast K+ uptake mutant grown on low K+ medium. Antisense AtCNGC10 plants had 50% less potassium than wild type Columbia. Taken together, the studies from three evolutionarily diverse species demonstrated a role for the CaM-binding channel, AtCNGC10, in mediating the uptake of K+ in plants.

Predator-dependent transmissible disease spreading in prey under Holling type-II functional response

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Dipankar Ghosh ◽  
Prasun K. Santra ◽  
Abdelalim A. Elsadany ◽  
Ghanshaym S. Mahapatra
Keyword(s):  
Functional Response ◽  
Prey Species ◽  
Linearization Method ◽  
Logistic Growth ◽  
Type Ii ◽  
Disease Spreading ◽  
Predator Interactions ◽  
The Stability ◽  
Infected Prey ◽  
Type Ii Functional Response

Abstract This paper focusses on developing two species, where only prey species suffers by a contagious disease. We consider the logistic growth rate of the prey population. The interaction between susceptible prey and infected prey with predator is presumed to be ruled by Holling type II and I functional response, respectively. A healthy prey is infected when it comes in direct contact with infected prey, and we also assume that predator-dependent disease spreads within the system. This research reveals that the transmission of this predator-dependent disease can have critical repercussions for the shaping of prey–predator interactions. The solution of the model is examined in relation to survival, uniqueness and boundedness. The positivity, feasibility and the stability conditions of the fixed points of the system are analysed by applying the linearization method and the Jacobian matrix method.

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