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

Accumulation of stable full-length circular group i intron RNAs during heat-shock

Group I introns in nuclear ribosomal RNA of eukaryotic microorganisms are processed by splicing or circularization. The latter results in formation of full-length circular introns without ligation of the exons and has been proposed to be active in intron mobility. We applied qRT-PCR to estimate the copy number of circular intron RNA from the myxomycete Didymium iridis . In exponentially growing amoebae, the circular introns are nuclear and found in 70 copies per cell. During heat-shock, the circular form is up-regulated to more than 500 copies per cell. The intron harbours two ribozymes that have the potential to linearize the circle. To understand the structural features that maintain circle integrity, we performed chemical and enzymatic probing of the splicing ribozyme combined with molecular modeling to arrive at models of the inactive circular form and its active linear counterpart. We show that the two forms have the same overall structure but differ in key parts, including the catalytic core element P7 and the junctions at which reactions take place. These differences explain the relative stability of the circular species, demonstrate how it is prone to react with a target molecule for circle integration and thus supports the notion that the circular form is a biologically significant molecule possibly with a role in intron mobility

The Mechanisms of Action of Ribosome-Targeting Peptide Antibiotics

The ribosome is one of the major targets in the cell for clinically used antibiotics. However, the increase in multidrug resistant bacteria is rapidly reducing the effectiveness of our current arsenal of ribosome-targeting antibiotics, highlighting the need for the discovery of compounds with new scaffolds that bind to novel sites on the ribosome. One possible avenue for the development of new antimicrobial agents is by characterization and optimization of ribosome-targeting peptide antibiotics. Biochemical and structural data on ribosome-targeting peptide antibiotics illustrates the large diversity of scaffolds, binding interactions with the ribosome as well as mechanism of action to inhibit translation. The availability of high-resolution structures of ribosomes in complex with peptide antibiotics opens the way to structure-based design of these compounds as novel antimicrobial agents