Molecular dissection of translation termination mechanism identifies two new critical regions in eRF1

Translation termination in eukaryotes is completed by two interacting factors eRF1 and eRF3. In Saccharomyces cerevisiae, these proteins are encoded by the genes SUP45 and SUP35, respectively. The eRF1 protein interacts directly with the stop codon at the ribosomal A-site, whereas eRF3—a GTPase protein—probably acts as a proofreading factor, coupling stop codon recognition to polypeptide chain release. We performed random PCR mutagenesis of SUP45 and screened the library for mutations resulting in increased eRF1 activity. These mutations led to the identification of two new pockets in domain 1 (P1 and P2) involved in the regulation of eRF1 activity. Furthermore, we identified novel mutations located in domains 2 and 3, which confer stop codon specificity to eRF1. Our findings are consistent with the model of a closed-active conformation of eRF1 and shed light on two new functional regions of the protein

GTP-dependent structural rearrangement of the eRF1:eRF3 complex and eRF3 sequence motifs essential for PABP binding

Translation termination in eukaryotes is governed by the concerted action of eRF1 and eRF3 factors. eRF1 recognizes the stop codon in the A site of the ribosome and promotes nascent peptide chain release, and the GTPase eRF3 facilitates this peptide release via its interaction with eRF1. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay through its association with cytoplasmic poly(A)-binding protein (PABP) via PAM2-1 and PAM2-2 motifs in the N-terminal domain of eRF3. We have studied complex formation between full-length eRF3 and its ligands (GDP, GTP, eRF1 and PABP) using isothermal titration calorimetry, demonstrating formation of the eRF1:eRF3:PABP:GTP complex. Analysis of the temperature dependence of eRF3 interactions with G nucleotides reveals major structural rearrangements accompanying formation of the eRF1:eRF3:GTP complex. This is in contrast to eRF1:eRF3:GDP complex formation, where no such rearrangements were detected. Thus, our results agree with the established active role of GTP in promoting translation termination. Through point mutagenesis of PAM2-1 and PAM2-2 motifs in eRF3, we demonstrate that PAM2-2, but not PAM2-1 is indispensible for eRF3:PABP complex formation

The candidate antimalarial drug MMV665909 causes oxygen-dependent mRNA mistranslation and synergises with quinoline-derived antimalarials

To cope with growing resistance to current antimalarials, new drugs with novel modes of action are urgently needed. Molecules targeting protein synthesis appear to be promising candidates. We identified a compound (MMV665909) from the MMV Malaria Box of candidate antimalarials that could produce synergistic growth inhibition with the aminoglycoside antibiotic paromomycin, suggesting a possible action of the compound in mRNA mistranslation. This mechanism of action was substantiated with the yeast cell model using available reporters of mistranslation and other genetic tools. Mistranslation induced by MMV665909 was oxygen-dependent, suggesting a role for reactive oxygen species (ROS). Overexpression of Rli1 (a ROS-sensitive, conserved FeS protein essential in mRNA translation) rescued inhibition by MMV665909, consistent with the drug’s action on translation fidelity being mediated through Rli1. The MMV drug also synergised with major quinoline-derived antimalarials which can perturb amino acid availability or promote ROS stress: chloroquine, amodiaquine and primaquine. The data collectively suggest translation-fidelity as a novel target of antimalarial action and support MMV665909 as a promising drug candidate

Adenine and guanine recognition of stop codon is mediated by different N domain conformations of translation termination factor eRF1

Positioning of release factor eRF1 toward adenines and the ribose-phosphate backbone of the UAAA stop signal in the ribosomal decoding site was studied using messenger RNA (mRNA) analogs containing stop signal UAA/UAAA and a photoactivatable cross-linker at definite locations. The human eRF1 peptides cross-linked to these analogs were identified. Cross-linkers on the adenines at the 2nd, 3rd or 4th position modified eRF1 near the conserved YxCxxxF loop (positions 125–131 in the N domain), but cross-linker at the 4th position mainly modified the tripeptide 26-AAR-28. This tripeptide cross-linked also with derivatized 3′-phosphate of UAA, while the same cross-linker at the 3′-phosphate of UAAA modified both the 26–28 and 67–73 fragments. A comparison of the results with those obtained earlier with mRNA analogs bearing a similar cross-linker at the guanines indicates that positioning of eRF1 toward adenines and guanines of stop signals in the 80S termination complex is different. Molecular modeling of eRF1 in the 80S termination complex showed that eRF1 fragments neighboring guanines and adenines of stop signals are compatible with different N domain conformations of eRF1. These conformations vary by positioning of stop signal purines toward the universally conserved dipeptide 31-GT-32, which neighbors guanines but is oriented more distantly from adenines

Incomplete homogenization of 18 S ribosomal DNA coding regions in Arabidopsis thaliana

<p>Abstract</p> <p>Background</p> <p>As a result of concerted evolution, coding regions of ribosomal DNA sequences are highly conserved within species and variation is generally thought to be limited to a few nucleotides. However, rDNA sequence variation has not been systematically examined in plant genomes, including that of the model plant <it>Arabidopsis thaliana </it>whose genome was the first to be sequenced.</p> <p>Findings</p> <p>Both genomic and transcribed 18 S sequences were sampled and revealed that most deviation from the consensus sequence was limited to single nucleotide substitutions except for a variant with a 270 bp deletion from position 456 to 725 in <it>Arabidopsis </it>numbering. The deletion maps to the functionally important and highly conserved 530 loop or helix18 in the structure of <it>E. coli </it>16 S. The expression of the deletion variant is tightly controlled during developmental growth stages. Transcripts were not detectable in young seedlings but could be amplified from RNA extracts of mature leaves, stems, flowers and roots of <it>Arabidopsis thaliana </it>ecotype Columbia. We also show polymorphism for the deletion variant among four <it>Arabidopsis </it>ecotypes examined.</p> <p>Conclusion</p> <p>Despite a strong purifying selection that might be expected against functionally impaired rDNAs, the newly identified variant is maintained in the <it>Arabidopsis </it>genome. The expression of the variant and the polymorphism displayed by <it>Arabidopsis </it>ecotypes suggest a transition state in concerted evolution.</p

Selectivity of stop codon recognition in translation termination is modulated by multiple conformations of GTS loop in eRF1

Translation termination in eukaryotes is catalyzed by two release factors eRF1 and eRF3 in a cooperative manner. The precise mechanism of stop codon discrimination by eRF1 remains obscure, hindering drug development targeting aberrations at translation termination. By solving the solution structures of the wild-type N-domain of human eRF1 exhibited omnipotent specificity, i.e. recognition of all three stop codons, and its unipotent mutant with UGA-only specificity, we found the conserved GTS loop adopting alternate conformations. We propose that structural variability in the GTS loop may underline the switching between omnipotency and unipotency of eRF1, implying the direct access of the GTS loop to the stop codon. To explore such feasibility, we positioned N-domain in a pre-termination ribosomal complex using the binding interface between N-domain and model RNA oligonucleotides mimicking Helix 44 of 18S rRNA. NMR analysis revealed that those duplex RNA containing 2-nt internal loops interact specifically with helix α1 of N-domain, and displace C-domain from a non-covalent complex of N-domain and C-domain, suggesting domain rearrangement in eRF1 that accompanies N-domain accommodation into the ribosomal A site

Understanding the Origins of Bacterial Resistance to Aminoglycosides through Molecular Dynamics Mutational Study of the Ribosomal A-Site

Paromomycin is an aminoglycosidic antibiotic that targets the RNA of the bacterial small ribosomal subunit. It binds in the A-site, which is one of the three tRNA binding sites, and affects translational fidelity by stabilizing two adenines (A1492 and A1493) in the flipped-out state. Experiments have shown that various mutations in the A-site result in bacterial resistance to aminoglycosides. In this study, we performed multiple molecular dynamics simulations of the mutated A-site RNA fragment in explicit solvent to analyze changes in the physicochemical features of the A-site that were introduced by substitutions of specific bases. The simulations were conducted for free RNA and in complex with paromomycin. We found that the specific mutations affect the shape and dynamics of the binding cleft as well as significantly alter its electrostatic properties. The most pronounced changes were observed in the U1406C∶U1495A mutant, where important hydrogen bonds between the RNA and paromomycin were disrupted. The present study aims to clarify the underlying physicochemical mechanisms of bacterial resistance to aminoglycosides due to target mutations