The 23S ribosomal RNA from Pyrococcus furiosus is circularly permuted

Synthesis and assembly of ribosomal components are fundamental cellular processes and generally well-conserved within the main groups of organisms. Yet, provocative variations to the general schemes exist. We have discovered an unusual processing pathway of pre-rRNA in extreme thermophilic archaea exemplified by Pyrococcus furiosus. The large subunit (LSU) rRNA is produced as a circularly permuted form through circularization followed by excision of Helix 98. As a consequence, the terminal domain VII that comprise the binding site for the signal recognition particle is appended to the 5´ end of the LSU rRNA that instead terminates in Domain VI carrying the Sarcin-Ricin Loop, the primary interaction site with the translational GTPases. To our knowledge, this is the first example of a true post-transcriptional circular permutation of a main functional molecule and the first example of rRNA fragmentation in archaea.publishedVersio

The Myxobacterial Antibiotic Myxovalargin: Biosynthesis, Structural Revision, Total Synthesis, and Molecular Characterization of Ribosomal Inhibition

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent

Peptide inhibitors of bacterial protein synthesis with broad spectrum and SbmA-independent bactericidal activity against clinical pathogens.

Proline-rich antimicrobial peptides (PrAMPs) are promising lead compounds for developing new antimicrobials, however their narrow spectrum of action is limiting. PrAMPs kill bacteria binding to their ribosomes and inhibiting protein synthesis. In this study, 133 derivatives of the PrAMP Bac7(1-16) were synthesized to identify the crucial residues for ribosome inactivation and antimicrobial activity. Then, five new Bac7(1-16) derivatives were conceived and characterized by antibacterial and membrane permeabilization assays, by X-ray crystallography and molecular dynamics simulations. Some derivatives displayed broad spectrum activity, encompassing Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa and Staphylococcus aureus. Two peptides out of five, acquired a weak membrane-perturbing activity, while maintaining the ability to inhibit protein synthesis. These derivatives became independent of the SbmA transporter, commonly used by native PrAMPs, suggesting that they obtained a novel route to enter bacterial cells. PrAMP-derived compounds could become new-generation antimicrobials to combat the antibiotic-resistant pathogens

Molecular modelling of the GIR1 branching ribozyme gives new insight into evolution of structurally related ribozymes

Twin-ribozyme introns contain a branching ribozyme (GIR1) followed by a homing endonuclease (HE) encoding sequence embedded in a peripheral domain of a group I splicing ribozyme (GIR2). GIR1 catalyses the formation of a lariat with 3 nt in the loop, which caps the HE mRNA. GIR1 is structurally related to group I ribozymes raising the question about how two closely related ribozymes can carry out very different reactions. Modelling of GIR1 based on new biochemical and mutational data shows an extended substrate domain containing a GoU pair distinct from the nucleophilic residue that dock onto a catalytic core showing a different topology from that of group I ribozymes. The differences include a core J8/7 region that has been reduced and is complemented by residues from the pre-lariat fold. These findings provide the basis for an evolutionary mechanism that accounts for the change from group I splicing ribozyme to the branching GIR1 architecture. Such an evolutionary mechanism can be applied to other large RNAs such as the ribonuclease P

Les ribozymes siamois de Didymuim iridis et Naegleria, une étude d'un intron apparenté aux introns de groupe 1

aL’étude de l’intron Dir956-1, situé dans le gène extra-chromosomique codant pour l’ARNr 18S du myxomycète Didymium iridis a révélé une organisation originale et complexe. En effet, il est constitué d’un intron de groupe I classique (GIR2) dans lequel est inséré dans une région périphérique un gène codant pour une endonucléase mobile (HE). L’originalité de ce système provient de la présence d’un ribozyme (GIR1) situé en amont de cette phase de lecture et dont la structure semble fortement apparentée à celle des introns de groupe I défini par une architecture modulaire. Cependant, GIR1 présente des différences topologiques inhabituelles. En résumé, le cœur catalytique de ce ribozyme se distingue de celui des introns de groupe I bien que le corps de ce ribozyme présente une organisation modulaire comparable à celle des introns de groupe I. Ainsi la question sous-jacente qui se pose est l’existence d’une évolution possible d’un intron de groupe I classique vers un ribozyme de type GIR1. Une autre singularité de ce ribozyme est la réaction qu’il catalyse, qui permet la libération et la maturation en 5’ terminal de l’ARN messager codant pour l’endonucléase mobile. Ce ribozyme libère l’ARNm he de l’intron en catalysant la formation d’un lasso (« lariat ») de trois nucléotides. Ceci n’est pas sans rappeler le lasso formé lors de la première étape de l’épissage des introns de groupe II, mais dans un contexte structural dramatiquement différent. Ces caractéristiques particulières justifient l’intérêt d’étudier le repliement tridimensionnel adopté par le cœur catalytique de ce ribozyme afin de mieux comprendre les bases moléculaires à l’origine de cette activité inhabituelle.aThe twin-ribozyme introns represent a complex organization of group I introns found in the nuclear SSU rRNA gene of the myxomycete Didymium iridis and several strains of the amoebo-flagellates Naegleria. It consists of an unusual ribozyme (GIR1) and a homing endonuclease gene (HEG) both embedded in a peripheral domain of another group I ribozyme (GIR2). Interestingly, GIR1 performs a unique catalytic reaction that leads to the formation of a tiny lariat with a 3-nt loop, which caps the HE mRNA. In terms of catalysis, the GIR1 branching reaction is typical of the first step of splicing performed by group II ribozymes. However, from a structural point of view, GIR1 is clearly related to group I ribozymes. Structure probing and phylogenetic studies have in fact revealed the lack of a P1 segment playing the role of the substrate and conserved in all other known group I ribozymes. However, a P10 base paired segment as well as a novel pseudoknot P3/P15 were proposed to take place in the core region. Thus, these particular observations raise the question of why GIR1 carries out a branching reaction despite its resemblance with group I ribozyme

Exploring RNA structure by integrative molecular modelling

International audienceRNA molecular modelling is adequate to rapidly tackle the structure of RNA molecules. With new structured RNAs constituting a central class of cellular regulators discovered every year, the need for swift and reliable modelling methods is more crucial than ever. The pragmatic method based on interactive all-atom molecular modelling relies on the observation that specific structural motifs are recurrently found in RNA sequences. Once identified by a combination of comparative sequence analysis and biochemical data, the motifs composing the secondary structure of a given RNA can be extruded in three dimensions (3D) and used as building blocks assembled manually during a bioinformatic interactive process. Comparing the models to the corresponding crystal structures has validated the method as being powerful to predict the RNA topology and architecture while being less accurate regarding the prediction of base-base interactions. These aspects as well as the necessary steps towards automation will be discussed

The 23S ribosomal RNA from Pyrococcus furiosus is circularly permuted

Synthesis and assembly of ribosomal components are fundamental cellular processes and generally well-conserved within the main groups of organisms. Yet, provocative variations to the general schemes exist. We have discovered an unusual processing pathway of pre-rRNA in extreme thermophilic archaea exemplified by Pyrococcus furiosus. The large subunit (LSU) rRNA is produced as a circularly permuted form through circularization followed by excision of Helix 98. As a consequence, the terminal domain VII that comprise the binding site for the signal recognition particle is appended to the 5´ end of the LSU rRNA that instead terminates in Domain VI carrying the Sarcin-Ricin Loop, the primary interaction site with the translational GTPases. To our knowledge, this is the first example of a true post-transcriptional circular permutation of a main functional molecule and the first example of rRNA fragmentation in archaea

The GIR1 branching ribozymes

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