• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biochemie
• mikrobiologie, lékařská mikrobiologie

Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation.

• MeSH
• elongace translace peptidového řetězce * MeSH
• eukaryotický iniciační faktor 4E metabolismus   MeSH
• eukaryotický iniciační faktor 4G metabolismus   MeSH
• iniciace translace peptidového řetězce * MeSH
• messenger RNA genetika   metabolismus   MeSH
• multiproteinové komplexy metabolismus   MeSH
• poly A MeSH
• polyribozomy metabolismus   MeSH
• proteiny vázající selen metabolismus   MeSH
• proteosyntéza MeSH
• ribozomy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

• Publikační typ
• abstrakty MeSH

In hemolymph of insect species, compounds with remarkable properties for pharmaceutical industry are present. At the first line, there were found compounds of low molecular mass, less than 1 kDa. One of such compounds, β-alanyl-tyrosine (252 Da), was isolated from larval hemolymph of some species of holometabolous insects (e.g. Neobellieria bullata). Its paralytic activity and antimicrobial properties were described until now. In this study, we present the effect of elongation of β-alanyl-tyrosine by repeating of this motive on the biological and physical properties of prepared analogues. For assessment of antimicrobial properties of these new compounds strains of Gram-positive, Gram-negative bacteria and fungi were used, we also followed the haemolytic activity and toxic effect on human cell culture HepG2. On the base of ECD spectroscopy measurement, subsequent molecular modelling and known secondary structure of original β-alanyl-tyrosine dipeptide, the secondary structures of repeating sequences of β-AY were specified. The repeating structures of β-alanyl-tyrosine show increase in antimicrobial activity; for Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, minimal inhibitory concentration was decreased from 30 to 15 mM for 2xβ-AY, 0.4 mM for 4xβ-AY and 0.25 mM for 6xβ-AY.

• MeSH
• aminokyselinové motivy MeSH
• antiinfekční látky chemie   farmakologie   MeSH
• biologické toxiny chemie   farmakologie   MeSH
• buňky Hep G2 MeSH
• dipeptidy chemie   farmakologie   MeSH
• houby účinky léků   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• proliferace buněk účinky léků   MeSH
• Staphylococcus aureus účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• alanin MeSH
• bentonit MeSH
• glycin MeSH
• peptidy MeSH

Translation reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream ORFs to prevent recycling of the post-termination 40S subunit in order to resume scanning for reinitiation downstream. Its efficiency decreases with the increasing uORF length, or by the presence of secondary structures, suggesting that the time taken to translate a uORF is more critical than its length. This led to a hypothesis that some initiation factors needed for reinitiation are preserved on the 80S ribosome during early elongation. Here, using the GCN4 mRNA containing four short uORFs, we developed a novel in vivo RNA-protein Ni2+-pull down assay to demonstrate for the first time that one of these initiation factors is eIF3. eIF3 but not eIF2 preferentially associates with RNA segments encompassing two GCN4 reinitiation-permissive uORFs, uORF1 and uORF2, containing cis-acting 5΄ reinitiation-promoting elements (RPEs). We show that the preferred association of eIF3 with these uORFs is dependent on intact RPEs and the eIF3a/TIF32 subunit and sharply declines with the extended length of uORFs. Our data thus imply that eIF3 travels with early elongating ribosomes and that the RPEs interact with eIF3 in order to stabilize the mRNA-eIF3-40S post-termination complex to stimulate efficient reinitiation downstream.

• MeSH
• 5' nepřekládaná oblast MeSH
• elongace translace peptidového řetězce MeSH
• eukaryotický iniciační faktor 3 metabolismus   MeSH
• genetické techniky MeSH
• iniciace translace peptidového řetězce * MeSH
• malé podjednotky ribozomu eukaryotické metabolismus   MeSH
• otevřené čtecí rámce * MeSH
• regulace genové exprese * MeSH
• ribozomy metabolismus   MeSH
• terminace translace peptidového řetězce MeSH
• terminační kodon MeSH
• Publikační typ
• časopisecké články MeSH

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.

• MeSH
• biokatalýza MeSH
• elektronová kryomikroskopie MeSH
• elongace translace peptidového řetězce genetika   MeSH
• elongační faktor G chemie   genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• konformace proteinů MeSH
• messenger RNA chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• posun čtecího rámce na ribozómech genetika   MeSH
• proteiny z Escherichia coli chemie   genetika   metabolismus   MeSH
• ribozomy genetika   metabolismus   ultrastruktura   MeSH
• RNA ribozomální 16S chemie   genetika   metabolismus   MeSH
• RNA transferová chemie   genetika   metabolismus   MeSH
• tRNA-methyltransferasy genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

• Klíčová slova
• induced-fit ribozym,
• MeSH
• elongace translace peptidového řetězce * MeSH
• kodon fyziologie   MeSH
• messenger RNA MeSH
• podjednotky ribozomu MeSH
• proteosyntéza * MeSH
• ribozomy * fyziologie   ultrastruktura   MeSH
• RNA katalytická MeSH
• RNA transferová MeSH
• RNA MeSH

Programmed stop codon readthrough is a post-transcription regulatory mechanism specifically increasing proteome diversity by creating a pool of C-terminally extended proteins. During this process, the stop codon is decoded as a sense codon by a near-cognate tRNA, which programs the ribosome to continue elongation. The efficiency of competition for the stop codon between release factors (eRFs) and near-cognate tRNAs is largely dependent on its nucleotide context; however, the molecular mechanism underlying this process is unknown. Here, we show that it is the translation initiation (not termination) factor, namely eIF3, which critically promotes programmed readthrough on all three stop codons. In order to do so, eIF3 must associate with pre-termination complexes where it interferes with the eRF1 decoding of the third/wobble position of the stop codon set in the unfavorable termination context, thus allowing incorporation of near-cognate tRNAs with a mismatch at the same position. We clearly demonstrate that efficient readthrough is enabled by near-cognate tRNAs with a mismatch only at the third/wobble position. Importantly, the eIF3 role in programmed readthrough is conserved between yeast and humans.

• MeSH
• aminoacyl-tRNA metabolismus   MeSH
• elongace translace peptidového řetězce * MeSH
• eukaryotický iniciační faktor 3 metabolismus   MeSH
• HeLa buňky MeSH
• iniciace translace peptidového řetězce MeSH
• kvasinky genetika   MeSH
• lidé MeSH
• paromomycin farmakologie   MeSH
• regulace genové exprese MeSH
• ribozomy účinky léků   metabolismus   MeSH
• terminační kodon * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.

• MeSH
• elektronová kryomikroskopie MeSH
• elongační faktory chemie   genetika   metabolismus   ultrastruktura   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• iniciační faktory chemie   metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• konformace proteinů MeSH
• messenger RNA chemie   genetika   metabolismus   MeSH
• mutace MeSH
• peptidy chemie   metabolismus   MeSH
• proteiny vázající RNA chemie   metabolismus   MeSH
• proteiny z Escherichia coli chemie   genetika   metabolismus   ultrastruktura   MeSH
• proteosyntéza MeSH
• ribozomy chemie   metabolismus   ultrastruktura   MeSH
• RNA transferová chemie   genetika   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• simulace molekulového dockingu MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH