During translation, a conserved GTPase elongation factor-EF-G in bacteria or eEF2 in eukaryotes-translocates tRNA and mRNA through the ribosome. EF-G has been proposed to act as a flexible motor that propels tRNA and mRNA movement, as a rigid pawl that biases unidirectional translocation resulting from ribosome rearrangements, or by various combinations of motor- and pawl-like mechanisms. Using time-resolved cryo-EM, we visualized GTP-catalyzed translocation without inhibitors, capturing elusive structures of ribosome•EF-G intermediates at near-atomic resolution. Prior to translocation, EF-G binds near peptidyl-tRNA, while the rotated 30S subunit stabilizes the EF-G GTPase center. Reverse 30S rotation releases Pi and translocates peptidyl-tRNA and EF-G by ~20 Å. An additional 4-Å translocation initiates EF-G dissociation from a transient ribosome state with highly swiveled 30S head. The structures visualize how nearly rigid EF-G rectifies inherent and spontaneous ribosomal dynamics into tRNA-mRNA translocation, whereas GTP hydrolysis and Pi release drive EF-G dissociation.

• MeSH
• aminoacyl-tRNA metabolismus   MeSH
• elektronová kryomikroskopie * MeSH
• elongační faktor G chemie   metabolismus   MeSH
• Escherichia coli chemie   metabolismus   MeSH
• fosfáty metabolismus   MeSH
• guanosintrifosfát chemie   metabolismus   MeSH
• malé podjednotky ribozomu bakteriální chemie   metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• proteosyntéza MeSH
• ribozomy chemie   metabolismus   MeSH
• RNA transferová metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS-antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.

• MeSH
• adeninnukleotidy metabolismus   MeSH
• Bacteria růst a vývoj   metabolismus   MeSH
• bakteriální proteiny metabolismus   MeSH
• databáze genetické MeSH
• fyziologický stres fyziologie   MeSH
• guanosinpentafosfát metabolismus   MeSH
• guanosintetrafosfát metabolismus   MeSH
• guanosintrifosfát metabolismus   MeSH
• ligasy metabolismus   MeSH
• pyrofosfatasy metabolismus   MeSH
• regulace genové exprese u bakterií genetika   MeSH
• signální transdukce MeSH
• systémy toxin-antitoxin fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

G-Quadruplexes are noncanonical nucleic acid structures made up of stacked guanosine tetrads connected by short loops. They are frequently used building blocks in synthetic biology and thought to play widespread biological roles. Multimerization can change the functional properties of G-quadruplexes, and understanding the factors that modulate this process remains an important goal. Here, we report the discovery of a novel mechanism by which the formation of multimeric G-quadruplexes can be controlled using GTP. We show that GTP likely inhibits multimer formation by becoming incorporated into a tetrad in the monomeric form of the structure and define the sequence requirements of G-quadruplexes that form GTP-dependent structures. These experiments provide new insights into the small molecule control of G-quadruplex multimerization. They also suggest possible roles for GTP-dependent multimeric G-quadruplexes in both synthetic and natural biological systems.

• MeSH
• biochemické jevy MeSH
• DNA genetika   metabolismus   MeSH
• G-kvadruplexy * MeSH
• guanosintrifosfát metabolismus   MeSH
• lidé MeSH
• mutace MeSH
• Pan troglodytes MeSH
• Pongo MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mutations that alter signaling of RAS/MAPK-family proteins give rise to a group of Mendelian diseases known as RASopathies. However, among RASopathies, the matrix of genotype-phenotype relationships is still incomplete, in part because there are many RAS-related proteins and in part because the phenotypic consequences may be variable and/or pleiotropic. Here, we describe a cohort of ten cases, drawn from six clinical sites and over 16,000 sequenced probands, with de novo protein-altering variation in RALA, a RAS-like small GTPase. All probands present with speech and motor delays, and most have intellectual disability, low weight, short stature, and facial dysmorphism. The observed rate of de novo RALA variants in affected probands is significantly higher (p = 4.93 x 10(-11)) than expected from the estimated random mutation rate. Further, all de novo variants described here affect residues within the GTP/GDP-binding region of RALA; in fact, six alleles arose at only two codons, Val25 and Lys128. The affected residues are highly conserved across both RAL- and RAS-family genes, are devoid of variation in large human population datasets, and several are homologous to positions at which disease-associated variants have been observed in other GTPase genes. We directly assayed GTP hydrolysis and RALA effector-protein binding of the observed variants, and found that all but one tested variant significantly reduced both activities compared to wild-type. The one exception, S157A, reduced GTP hydrolysis but significantly increased RALA-effector binding, an observation similar to that seen for oncogenic RAS variants. These results show the power of data sharing for the interpretation and analysis of rare variation, expand the spectrum of molecular causes of developmental disability to include RALA, and provide additional insight into the pathogenesis of human disease caused by mutations in small GTPases.

• MeSH
• faciální stigmatizace MeSH
• fenotyp MeSH
• genotyp MeSH
• guanosindifosfát metabolismus   MeSH
• guanosintrifosfát metabolismus   MeSH
• interakční proteinové domény a motivy genetika   MeSH
• konformace proteinů MeSH
• lidé MeSH
• mentální retardace genetika   MeSH
• missense mutace MeSH
• mitochondriální proteiny chemie   genetika   MeSH
• molekulární modely MeSH
• mutace * MeSH
• ral proteiny vázající GTP chemie   genetika   MeSH
• ras proteiny chemie   genetika   MeSH
• vývojové poruchy u dětí genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA.

• MeSH
• eukaryotický iniciační faktor 3 genetika   metabolismus   MeSH
• guanosintrifosfát metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• mutační analýza DNA MeSH
• mutantní proteiny genetika   metabolismus   MeSH
• podjednotky proteinů genetika   metabolismus   MeSH
• proteosyntéza MeSH
• ribozomy metabolismus   MeSH
• RNA transferová Met metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH

UNLABELLED: The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEf synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEf also efficiently utilized GMP to form GMP 3'-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEf was activated only by ppGpp. Furthermore, enzymatic activity of RelQEf is insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp. IMPORTANCE: Accumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ of Enterococcus faecalis (RelQEf), we found that, in addition to (p)ppGpp, RelQEf is an efficient producer of pGpp (GMP 3'-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQEf and suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• deoxyguanosin analogy a deriváty   biosyntéza   chemie   MeSH
• dipeptidy biosyntéza   chemie   MeSH
• Enterococcus faecalis účinky léků   enzymologie   genetika   metabolismus   MeSH
• fyziologický stres MeSH
• guanosindifosfát metabolismus   MeSH
• guanosinpentafosfát metabolismus   MeSH
• guanosintetrafosfát biosyntéza   MeSH
• guanosintrifosfát metabolismus   MeSH
• hořčík MeSH
• ligasy genetika   metabolismus   MeSH
• molekulární struktura MeSH
• regulace genové exprese enzymů MeSH
• regulace genové exprese u bakterií MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Electromagnetic fields are usually absent in the picture of processes taking place in living cells which is dominated by biochemistry, molecular genetics and microscopic morphology. Yet experimental and theoretical studies suggest that this omission is not justified. At the end of 1960's H. Fröhlich elaborated a semi-phenomenological model of polar oscillating units that are metabolically driven, exchange energy with the cell's internal heat reservoir, and store part of the energy in excited vibrational modes in such way, that mode with the lowest frequency becomes highly excited, while the higher-order modes remain near thermal equilibrium. This affords energy-hungry chemical reactions to take place while the rest of the cell is not exposed to heat stress. At present, part of the cytoskeleton – microtubules – are deemed to fulfil the role of oscillating units. The paper provides an introduction to the Fröhlich ideas for readers with background in medicine and biology in that it avoids mathematical formulas and relies on figures to convey information about the basic properties of the model. The essential features of the Fröhlich model – most notably the energy condensation – are demonstrated on ensemble encompassing three coupled vibration modes that can be exactly described using original diagrammatic method.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• biomedicínský výzkum metody   trendy   MeSH
• elektromagnetické jevy MeSH
• energetický metabolismus fyziologie   MeSH
• financování organizované MeSH
• fotosyntéza fyziologie   MeSH
• fyziologie buňky fyziologie   genetika   MeSH
• guanosintrifosfát metabolismus   MeSH
• lidé MeSH
• mikrotubuly fyziologie   metabolismus   MeSH
• mitochondrie fyziologie   metabolismus   MeSH
• skelet buněčné stěny fyziologie   chemie   metabolismus   MeSH
• statistika jako téma MeSH
• teoretické modely MeSH
• Check Tag
• lidé MeSH

Physical processes in living cells were not taken into consideration among the essentials of biological activity, regardless of the fact that they establish a state far from thermodynamic equilibrium. In biological system chemical energy is transformed into the work of physical forces for various biological functions. The energy transformation pathway is very likely connected with generation of the endogenous electrodynamic field as suggested by experimentally proved electrodynamic activity of biological systems connected with mitochondrial and microtubule functions. Besides production of ATP and GTP (adenosine and guanosine triphosphate) mitochondria form a proton space charge layer,

• MeSH
• adenosintrifosfát metabolismus   MeSH
• apoptóza fyziologie   genetika   imunologie   MeSH
• biomedicínský výzkum metody   trendy   MeSH
• elektromagnetická pole škodlivé účinky   MeSH
• financování organizované MeSH
• fyziologie buňky fyziologie   genetika   imunologie   MeSH
• glykolýza fyziologie   genetika   imunologie   MeSH
• guanosintrifosfát metabolismus   MeSH
• kyselina dichloroctová aplikace a dávkování   škodlivé účinky   terapeutické užití   MeSH
• lidé MeSH
• mikrotubuly fyziologie   metabolismus   patologie   MeSH
• mitochondrie fyziologie   metabolismus   patologie   MeSH
• nádorová transformace buněk genetika   imunologie   účinky léků   MeSH
• nádory etiologie   metabolismus   terapie   MeSH
• Check Tag
• lidé MeSH

There are five subtypes of muscarinic receptors that serve various important physiological functions in the central nervous system and the periphery. Mental functions like attention, learning, and memory are attributed to the muscarinic M1 subtype. These functions decline during natural aging and an early deficit is typical for Alzheimer s disease. In addition, stimulation of the M1 receptor increases non-amyloidogenic processing of the amyloid precursor protein and thus prevents accumulation of noxious beta-amyloid fragments. The selectivity of classical muscarinic agonists among receptor subtypes is very low due to the highly conserved nature of the orthosteric binding site among receptor subtypes. Herein we summarize some recent studies with the functionally-selective M1 agonist xanomeline that indicate complex pharmacological profile of this drug that includes interactions with and activation of receptor from both orthosteric and ectopic binding sites, and the time-dependent changes of ligand binding and receptor activation. These findings point to potential profitability of exploitation of ectopic ligands in the search for truly selective muscarinic receptor agonists.

• MeSH
• acetylcholin metabolismus   MeSH
• agonisté muskarinových receptorů farmakologie   MeSH
• guanosintrifosfát metabolismus   MeSH
• karbachol farmakologie   MeSH
• lidé MeSH
• mozek metabolismus   MeSH
• pyridiny farmakologie   MeSH
• racionální návrh léčiv * MeSH
• receptor muskarinový M1 agonisté   metabolismus   MeSH
• thiadiazoly farmakologie   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Thermogenic uncoupling has been proven only for UCP1 in brown adipose tissue. All other isoforms of UCPs are potentially acting in suppression of mitochondrial reactive oxygen species (ROS) production. In this contribution we show that BAT mitochondria can be uncoupled by lauric acid in the range of approximately 100 nM when endogenous fatty acids are combusted by carnitine cycle and beta-oxidation is properly separated from the uncoupling effect. Respiration increased up to 3 times when related to the lowest fatty acid content (BSA present plus carnitine cycle). We also illustrated that any effect leading to more coupled states leads to enhanced H2O2 generation and any effect resulting in uncoupling gives reduced H2O2 generation in BAT mitochondria. Finally, we report doubling of plant UCP transcript in cells as well as amount of protein detected by 3H-GTP-binding sites in mitochondria of shoots and roots of maize seedlings subjected to the salt stress.

• MeSH
• buněčné dýchání MeSH
• financování organizované MeSH
• guanosintrifosfát metabolismus   MeSH
• hnědá tuková tkáň metabolismus   MeSH
• iontové kanály MeSH
• karnitin metabolismus   MeSH
• kořeny rostlin metabolismus   MeSH
• kukuřice setá metabolismus   MeSH
• kyseliny laurové farmakologie   metabolismus   MeSH
• mastné kyseliny metabolismus   MeSH
• membránové proteiny fyziologie   MeSH
• mitochondriální proteiny MeSH
• mitochondrie metabolismus   MeSH
• oxidační stres MeSH
• peroxid vodíku metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• rostlinné proteiny fyziologie   MeSH
• rozpřahující látky farmakologie   metabolismus   MeSH
• savci MeSH
• transportní proteiny fyziologie   MeSH
• vazebná místa MeSH
• výhonky rostlin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• přehledy MeSH
• techniky in vitro MeSH