1,720,968 research outputs found
A detailed molecular analysis of the events leading to translation initiation complex formation in Prokaryotes
No full textLa sintesi proteica è un processo biologico universalmente conservato che concretizza l’informazione genetica in proteine. La traduzione ha luogo sui ribosomi, particelle ribonucleoproteiche costituite da due subunità di diversa grandezza: la subunità minore sulla quale avviene la selezione del mRNA e dei tRNA corrispondenti ad ogni codone (processo di decoding), e la subunità maggiore che contiene il sito catalitico che media la formazione del legame peptidico. In E. coli la selezione del mRNA e del tRNA iniziatore è mediata da tre fattori d’inizio (IF1, IF2 ed IF3) che, formando con la subunità minore (30S), il mRNA e i fMet-tRNA il cosiddetto complesso d’inizio 30S (30S IC), coordinano l’interazione tra codone e anticodone e favoriscono l’associazione della subunità maggiore (50S) a formare il complesso d’inizio 70S pronto per iniziare il ciclo di traduzione.
Lo scopo di questo progetto di Dottorato è stato quello di approfondire alcuni aspetti della formazione del complesso d’inizio 70S.
Nella prima sezione sono presentati esperimenti di formazione di complessi d’inizio 70S in diverse condizioni, utilizzando tecniche di cinetica rapida quali il light scattering e il FRET e componenti dell’apparato traduzionale purificati. I risultati hanno permesso di collocare in un preciso ordine temporale gli eventi che preparano il ribosoma alla fase di estensione. In questo modo è stata ricavata la sequenza con cui i fattori d’inizio vengono rilasciati.
Nella seconda sezione è riportata l’analisi degli effetti di due mutazioni a livello del RNA ribosomale (rRNA) 16S (C1484U e GGGG773-6CGAU) che interrompono due delle dodici giunzioni (B3 e B7b) che tengono insieme le subunità durante la fase di estensione. I risultati ottenuti dimostrano che entrambe le mutazioni destabilizzano la struttura del ribosoma riducendo la capacità di associazione delle subunità in vivo e in vitro, e modificando l’affinità per il fattore d’inizio IF3 con conseguenze sulla fedeltà della traduzione
Translational GTPases.
Full text linkTranslational GTPases (trGTPases) play key roles in facilitating protein synthesis on the ribosome. Despite the high degree of evolutionary conservation in the sequences of their GTP-binding domains, the rates of GTP hydrolysis and nucleotide exchange vary broadly between different trGTPases. EF-Tu, one of the best-characterized model G proteins, evolved an exceptionally rapid and tightly regulated GTPase activity, which ensures rapid and accurate incorporation of amino acids into the nascent chain. Other trGTPases instead use the energy of GTP hydrolysis to promote movement or to ensure the forward commitment of translation reactions. Recent data suggest the GTPase mechanism of EF-Tu and provide an insight in the catalysis of GTP hydrolysis by its unusual activator, the ribosome. Here we summarize these advances in understanding the functional cycle and the regulation of trGTPases, stimulated by the elucidation of their structures on the ribosome and the progress in dissecting the reaction mechanism of GTPase
Activities of the peptidyl transferase center of ribosomes lacking protein L27.
Full text linkThe ribosome is the molecular machine responsible for protein synthesis in all living organisms. Its catalytic core, the peptidyl transferase center (PTC), is built of rRNA, although several proteins reach close to the inner rRNA shell. In the Escherichia coli ribosome, the flexible N-terminal tail of the ribosomal protein L27 contacts the A- and P-site tRNA. Based on computer simulations of the PTC and on previous biochemical evidence, the N-terminal α-amino group of L27 was suggested to take part in the peptidyl-transfer reaction. However, the contribution of this group to catalysis has not been tested experimentally. Here we investigate the role of L27 in peptide-bond formation using fast kinetics approaches. We show that the rate of peptide-bond formation at physiological pH, both with aminoacyl-tRNA or with the substrate analog puromycin, is independent of the presence of L27; furthermore, translation of natural mRNAs is only marginally affected in the absence of L27. The pH dependence of the puromycin reaction is unaltered in the absence of L27, indicating that the N-terminal α-amine is not the ionizing group taking part in catalysis. Likewise, L27 is not required for the peptidyl-tRNA hydrolysis during termination. Thus, apart from the known effect on subunit association, which most likely explains the phenotype of the deletion strains, L27 does not appear to be a key player in the core mechanism of peptide-bond formation on the ribosome
Ribosome dynamics during decoding.
Full text linkElongation factors Tu (EF-Tu) and SelB are translational GTPases that deliver aminoacyl-tRNAs (aa-tRNAs) to the ribosome. In each canonical round of translation elongation, aa-tRNAs, assisted by EF-Tu, decode mRNA codons and insert the respective amino acid into the growing peptide chain. Stop codons usually lead to translation termination; however, in special cases UGA codons are recoded to selenocysteine (Sec) with the help of SelB. Recruitment of EF-Tu and SelB together with their respective aa-tRNAs to the ribosome is a multistep process. In this review, we summarize recent progress in understanding the role of ribosome dynamics in aa-tRNA selection. We describe the path to correct codon recognition by canonical elongator aa-tRNA and Sec-tRNASec and discuss the local and global rearrangements of the ribosome in response to correct and incorrect aa-tRNAs. We present the mechanisms of GTPase activation and GTP hydrolysis of EF-Tu and SelB and summarize what is known about the accommodation of aa-tRNA on the ribosome after its release from the elongation factor. We show how ribosome dynamics ensures high selectivity for the cognate aa-tRNA and suggest that conformational fluctuations, induced fit and kinetic discrimination play major roles in maintaining the speed and fidelity of translation.This article is part of the themed issue 'Perspectives on the ribosome'
Visualization of translation termination intermediates trapped by the Apidaecin 137 peptide during RF3-mediated recycling of RF1
No full textDeutsche Forschungsgemeinschaft (German Research Foundation
The mTOR/4E-BP1/eIF4E signalling pathway as source of cancer drug targets
No full text: The mechanistic/mammalian target of rapamycin (mTOR) is the crucial hub of signalling pathways that regulate essential steps in the cell life cycle. Once incorporated in the mTORC1 complex, mTOR phosphorylates the eukaryotic initiation factor 4E (eIF4E)- binding protein 1 (4E-BP1), which then releases eIF4E. When not bound to 4E-BPs, eIF4E recognizes the mRNA 5'-cap structure and, together with eIF4A and eIF4G, it forms the eIF4F complex that recruits the ribosome on the mRNA. Under normal conditions, the cellular concentration of eIF4E is very low, making eIF4E the limiting factor in the initiation of protein synthesis. The vast majority of cancer types are characterized by the simultaneous deregulation of the mTOR/4E-BP1 signaling pathway and upregulation of eIF4E, which lead to an increased expression of cancer-promoting genes and deregulated cellular growth. Over the last decades, a growing number of selective inhibitors of the mTOR/4E-BP1/eIF4E pathway have been discovered or designed. Several inhibitors with encouraging preclinical results have been tested in clinical trials. This review summarizes the most recent research on drug development against mTOR, 4E-BP1 and eIF4E, describing the design rationale and the available structural and functional data on the most promising compounds
Tissue-specific regulation of translational readthrough tunes functions of the traffic jam transcription factor
No full textTranslational readthrough (TR) occurs when the ribosome decodes a stop codon as a sense codon, resulting in two protein isoforms synthesized from the same mRNA. TR has been identified in several eukaryotic organisms; however, its biological significance and mechanism remain unclear. Here, we quantify TR of several candidate genes in Drosophila melanogaster and characterize the regulation of TR in the large Maf transcription factor Traffic jam (Tj). Using CRISPR/Cas9-generated mutant flies, we show that the TR-generated Tj isoform is expressed in a subset of neural cells of the central nervous system and is excluded from the somatic cells of gonads. Control of TR in Tj is critical for preservation of neuronal integrity and maintenance of reproductive health. The tissue-specific distribution of a release factor splice variant, eRF1H, plays a critical role in modulating differential TR of leaky stop codon contexts. Fine-tuning of gene regulatory functions of transcription factors by TR provides a potential mechanism for cell-specific regulation of gene expression
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
- …
