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1.
Nucleic Acids Res ; 46(16): 8651-8661, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30107527

RESUMO

The GTPase elongation factor EF-Tu delivers aminoacyl-tRNAs to the mRNA-programmed ribosome during translation. Cognate codon-anticodon interaction stimulates GTP hydrolysis within EF-Tu. It has been proposed that EF-Tu undergoes a large conformational change subsequent to GTP hydrolysis, which results in the accommodation of aminoacyl-tRNA into the ribosomal A-site. However, this proposal has never been tested directly. Here, we apply single-molecule total internal reflection fluorescence microscopy to study the conformational dynamics of EF-Tu when bound to the ribosome. Our studies show that GTP hydrolysis initiates a partial, comparatively small conformational change of EF-Tu on the ribosome, not directly along the path from the solution 'GTP' to the 'GDP' structure. The final motion is completed either concomitant with or following dissociation of EF-Tu from the ribosome. The structural transition of EF-Tu on the ribosome is slower when aa-tRNA binds to a cognate versus a near-cognate codon. The resulting longer residence time of EF-Tu on the ribosome may be important for promoting accommodation of the cognate aminoacyl-tRNA into the A-site.


Assuntos
GTP Fosfo-Hidrolases/química , Fator Tu de Elongação de Peptídeos/química , Aminoacil-RNA de Transferência/genética , Ribossomos/genética , Anticódon/genética , Códon/genética , Escherichia coli/genética , GTP Fosfo-Hidrolases/genética , Guanosina Difosfato/química , Guanosina Trifosfato/química , Hidrólise , Cinética , Fator Tu de Elongação de Peptídeos/genética , Biossíntese de Proteínas/genética , Conformação Proteica , RNA Mensageiro/química , RNA Mensageiro/genética , Aminoacil-RNA de Transferência/química , Ribossomos/química
2.
Nucleic Acids Res ; 46(16): 8641-8650, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30107565

RESUMO

According to the traditional view, GTPases act as molecular switches, which cycle between distinct 'on' and 'off' conformations bound to GTP and GDP, respectively. Translation elongation factor EF-Tu is a GTPase essential for prokaryotic protein synthesis. In its GTP-bound form, EF-Tu delivers aminoacylated tRNAs to the ribosome as a ternary complex. GTP hydrolysis is thought to cause the release of EF-Tu from aminoacyl-tRNA and the ribosome due to a dramatic conformational change following Pi release. Here, the crystal structure of Escherichia coli EF-Tu in complex with a non-hydrolysable GTP analogue (GDPNP) has been determined. Remarkably, the overall conformation of EF-Tu·GDPNP displays the classical, open GDP-bound conformation. This is in accordance with an emerging view that the identity of the bound guanine nucleotide is not 'locking' the GTPase in a fixed conformation. Using a single-molecule approach, the conformational dynamics of various ligand-bound forms of EF-Tu were probed in solution by fluorescence resonance energy transfer. The results suggest that EF-Tu, free in solution, may sample a wider set of conformations than the structurally well-defined GTP- and GDP-forms known from previous X-ray crystallographic studies. Only upon binding, as a ternary complex, to the mRNA-programmed ribosome, is the well-known, closed GTP-bound conformation, observed.


Assuntos
Escherichia coli/química , Guanosina Trifosfato/química , Fator Tu de Elongação de Peptídeos/química , Conformação Proteica , Cristalografia por Raios X , Escherichia coli/genética , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Guanosina Difosfato/química , Guanosina Trifosfato/análogos & derivados , Fator Tu de Elongação de Peptídeos/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Ribossomos/química , Ribossomos/genética
3.
Nucleic Acids Res ; 43(19): 9519-28, 2015 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-26338772

RESUMO

The G-protein EF-Tu, which undergoes a major conformational change when EF-Tu·GTP is converted to EF-Tu·GDP, forms part of an aminoacyl(aa)-tRNA·EF-Tu·GTP ternary complex (TC) that accelerates the binding of aa-tRNA to the ribosome during peptide elongation. Such binding, placing a portion of EF-Tu in contact with the GTPase Associated Center (GAC), is followed by GTP hydrolysis and Pi release, and results in formation of a pretranslocation (PRE) complex. Although tRNA movement through the ribosome during PRE complex formation has been extensively studied, comparatively little is known about the dynamics of EF-Tu interaction with either the ribosome or aa-tRNA. Here we examine these dynamics, utilizing ensemble and single molecule assays employing fluorescent labeled derivatives of EF-Tu, tRNA, and the ribosome to measure changes in either FRET efficiency or fluorescence intensity during PRE complex formation. Our results indicate that ribosome-bound EF-Tu separates from the GAC prior to its full separation from aa-tRNA, and suggest that EF-Tu·GDP dissociates from the ribosome by two different pathways. These pathways correspond to either reversible EF-Tu·GDP dissociation from the ribosome prior to the major conformational change in EF-Tu that follows GTP hydrolysis, or irreversible dissociation after or concomitant with this conformational change.


Assuntos
Guanosina Difosfato/metabolismo , Elongação Traducional da Cadeia Peptídica , Fator Tu de Elongação de Peptídeos/metabolismo , Ribossomos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Guanosina Trifosfato/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Proteínas Ribossômicas/metabolismo
4.
Environ Monit Assess ; 188(8): 493, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27473107

RESUMO

Safeguarding sustainability of forest ecosystems with their habitat variability and all their functions is of highest priority. Therefore, the long-term adaptability of forest ecosystems to a changing environment must be secured, e.g., through sustainable forest management. High adaptability is based on biological variation starting at the genetic level. Thus, the ultimate goal of the Convention on Biological Diversity (CBD) to halt the ongoing erosion of biological variation is of utmost importance for forest ecosystem functioning and sustainability. Monitoring of biological diversity over time is needed to detect changes that threaten these biological resources. Genetic variation, as an integral part of biological diversity, needs special attention, and its monitoring can ensure its effective conservation. We compare forest genetic monitoring to other biodiversity monitoring concepts. Forest genetic monitoring (FGM) enables early detection of potentially harmful changes of forest adaptability before these appear at higher biodiversity levels (e.g., species or ecosystem diversity) and can improve the sustainability of applied forest management practices and direct further research. Theoretical genetic monitoring concepts developed up to now need to be evaluated before being implemented on a national and international scale. This article provides an overview of FGM concepts and definitions, discusses their advantages and disadvantages, and provides a flow chart of the steps needed for the optimization and implementation of FGM. FGM is an important module of biodiversity monitoring, and we define an effective FGM scheme as consisting of an assessment of a forest population's capacity to survive, reproduce, and persist under rapid environmental changes on a long-term scale.


Assuntos
Biodiversidade , Conservação dos Recursos Naturais/métodos , Monitoramento Ambiental/métodos , Florestas , Variação Genética , Árvores/genética , Europa (Continente) , Formulação de Políticas , Árvores/crescimento & desenvolvimento
5.
PeerJ ; 12: e17644, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39131610

RESUMO

Background: Tree ontogeny is the genetic trajectories of regenerative processes in trees, repeating in time and space, including both development and reproduction. Understanding the principles of tree ontogeny is a key priority in emulating natural ecological patterns and processes that fall within the calls for closer-to-nature forest management. By recognizing and respecting the growth and development of individual trees and forest stands, forest managers can implement strategies that align with the inherent dynamics of forest ecosystem. Therefore, this study aims to determine the ontogenetic characteristics of tree regeneration and growth in northern European hemiboreal forests. Methodology: We applied a three-step process to review i) the ontogenetic characteristics of forest trees, ii) ontogenetic strategies of trees for stand-forming species, and iii) summarise the review findings of points i and ii to propose a conceptual framework for transitioning towards closer-to-nature management of hemiboreal forest trees. To achieve this, we applied the super-organism approach to forest development as a holistic progression towards the establishment of natural stand forming ecosystems. Results: The review showed multiple aspects; first, there are unique growth and development characteristics of individual trees at the pre-generative and generative stages of ontogenesis under full and minimal light conditions. Second, there are four main modes of tree establishment, growth and development related to the light requirements of trees; they were described as ontogenetic strategies of stand-forming tree species: gap colonisers, gap successors, gap fillers and gap competitors. Third, the summary of our analysis of the ontogenetic characteristics of tree regeneration and growth in northern European hemiboreal forests shows that stand-forming species occupy multiple niche positions relative to forest dynamics modes. Conclusions: This study demonstrates the importance of understanding tree ontogeny under the pretext of closer-to-nature forest management, and its potential towards formulating sustainable forest management that emulates the natural dynamics of forest structure. We suggest that scientists and foresters can adapt closer-to-nature management strategies, such as assisted natural regeneration of trees, to improve the vitality of tree communities and overall forest health. The presented approach prioritizes ecological integrity and forest resilience, promoting assisted natural regeneration, and fostering adaptability and connectivity among plant populations in hemiboreal tree communities.


Assuntos
Agricultura Florestal , Florestas , Árvores , Árvores/crescimento & desenvolvimento , Agricultura Florestal/métodos , Conservação dos Recursos Naturais/métodos , Europa (Continente) , Ecossistema
6.
Sci Rep ; 14(1): 2713, 2024 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-38302512

RESUMO

We investigated the presence of spatial genetic groups within forest tree populations and determined if the genetic divergence among these groups is greater than that between populations using Scots pine (Pinus sylvestris) as a model species. We genotyped 890 adult trees of Scots pine in six natural populations in Lithuania at 11 nuclear microsatellite loci. We used a Bayesian clustering approach to identify the within-population genetic groups within each of the six populations. We calculated the differentiation indexes among the genetic groups within each population and among the six populations by ignoring the genetic groups. The Bayesian clustering revealed 2 to 6 distinct genetic groups of varying size as the most likely genetic structures within populations. The genetic differentiation indexes among the genetic groups within populations were nearly tenfold greater (FST = 0.012-0.070) than those between the populations (FST = 0.003). We conclude on the existence of markedly stronger structuring of genetic variation within populations than between populations of Scots pine in large forest tracts of northern Europe. Such genetic structures serve as a contributing factor to large within population genetic diversity in northern conifers. We assume that within population mating in Scots pine is not completely random but rather is stratified into genetic clusters. Our study provides pioneering novel key insights into structuring of genetic variation within populations. Our findings have implications for examining within-population genetic diversity and genetic structure, conservation, and management of genetic resources.


Assuntos
Pinus sylvestris , Pinus sylvestris/genética , Teorema de Bayes , Deriva Genética , Florestas , Genética Populacional , Variação Genética
7.
Biochemistry ; 51(13): 2642-51, 2012 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-22409271

RESUMO

During translation, the nucleic acid language employed by genes is translated into the amino acid language used by proteins. The translator is the ribosome, while the dictionary employed is known as the genetic code. The genetic information is presented to the ribosome in the form of a mRNA, and tRNAs connect the two languages. Translation takes place in three steps: initiation, elongation, and termination. After a protein has been synthesized, the components of the translation apparatus are recycled. During each phase of translation, the ribosome collaborates with specific translation factors, which secure a proper balance between speed and fidelity. Notably, initiation, termination, and ribosomal recycling occur only once per protein produced during normal translation, while the elongation step is repeated a large number of times, corresponding to the number of amino acids constituting the protein of interest. In bacteria, elongation factor Tu plays a central role during the selection of the correct amino acids throughout the elongation phase of translation. Elongation factor Tu is the main subject of this review.


Assuntos
Fator Tu de Elongação de Peptídeos/metabolismo , Ribossomos/metabolismo , Modelos Moleculares , Fator Tu de Elongação de Peptídeos/química , Biossíntese de Proteínas
8.
Ecol Evol ; 11(11): 6260-6275, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34141216

RESUMO

Tilia cordata Mill. is a valuable tree species enriching the ecological values of the coniferous-dominated boreal forests in Europe. Following the historical decline, spreading of Tilia sp. is challenged by the elevated inbreeding and habitat fragmentation. We studied the geographical distribution of genetic diversity of Tilia cordata populations in Lithuania. We used 14 genomic microsatellite markers to genotype 543 individuals from 23 wild-growing populations. We found that Tilia cordata retained high levels of genetic diversity (population F IS = 0-0.15, H o = 0.53-0.69, H e = 0.56-0.75). AMOVA, Bayesian clustering, and Monmonier's barrier detection indicate weak but significant differentiation among the populations (F ST = 0.037***) into geographically interpretable clusters of (a) western Lithuania with high genetic heterogeneity but low genetic diversity, bottleneck effects, (b) relatively higher genetic diversity of Tilia cordata on rich and most soils of midland lowland, and (c) the most differentiated populations on poor soils of the coolest northeastern highland possessing the highest rare allele frequency but elevated inbreeding and bottleneck effects. Weak genetic differentiation among the Tilia cordata populations in Lithuania implies common ancestry, absence of strong adaptive gradients, and effective genetic exchange possible mediated via the riparian networks. A hypothesis on riparian networks as gene flow mediators in Tilia cordata was raised based on results of this study.

9.
ACS Chem Biol ; 9(10): 2421-31, 2014 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-25126896

RESUMO

The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process. We use these assays to examine: (i) the relative rates of EF-Tu movement away from the GAC and from aa-tRNA during decoding, (ii) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput screening of ribosomal antibiotics.


Assuntos
Fator Tu de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas/fisiologia , Aminoacil-RNA de Transferência/química , Proteínas Ribossômicas/metabolismo , Ribossomos/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Modelos Moleculares , Mutação/genética , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/genética , Conformação Proteica , Aminoacil-RNA de Transferência/metabolismo
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