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Complex RNA folding kinetics revealed by single-molecule FRET and hidden Markov models.

ABSTRACT: We have developed a hidden Markov model and optimization procedure for photon-based single-molecule FRET data, which takes into account the trace-dependent background intensities. This analysis technique reveals an unprecedented amount of detail in the folding kinetics of the Diels-Alderase ribozyme. We find a multitude of extended (low-FRET) and compact (high-FRET) states. Five states were consistently and independently identified in two FRET constructs and at three Mg(2+) concentrations. Structures generally tend to become more compact upon addition of Mg(2+). Some compact structures are observed to significantly depend on Mg(2+) concentration, suggesting a tertiary fold stabilized by Mg(2+) ions. One compact structure was observed to be Mg(2+)-independent, consistent with stabilization by tertiary Watson-Crick base pairing found in the folded Diels-Alderase structure. A hierarchy of time scales was discovered, including dynamics of 10 ms or faster, likely due to tertiary structure fluctuations, and slow dynamics on the seconds time scale, presumably associated with significant changes in secondary structure. The folding pathways proceed through a series of intermediate secondary structures. There exist both compact pathways and more complex ones, which display tertiary unfolding, then secondary refolding, and, subsequently, again tertiary refolding.

SUBMITTER: Keller BG

PROVIDER: S-EPMC3977575 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Complex RNA folding kinetics revealed by single-molecule FRET and hidden Markov models.

Keller Bettina G BG Kobitski Andrei A Jäschke Andres A Nienhaus G Ulrich GU Noé Frank F

Journal of the American Chemical Society 20140314 12

We have developed a hidden Markov model and optimization procedure for photon-based single-molecule FRET data, which takes into account the trace-dependent background intensities. This analysis technique reveals an unprecedented amount of detail in the folding kinetics of the Diels-Alderase ribozyme. We find a multitude of extended (low-FRET) and compact (high-FRET) states. Five states were consistently and independently identified in two FRET constructs and at three Mg(2+) concentrations. Struc ...[more]

PMID: 24568646

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Project description:Bacteria exploit an arsenal of antimicrobial peptides and proteins to compete with each other. Three main competition systems have been described: type six secretion systems (T6SS); contact dependent inhibition (CDI); and bacteriocins. Unlike T6SS and CDI systems, bacteriocins do not require contact between bacteria but are diffusible toxins released into the environment. Identified almost a century ago, our understanding of bacteriocin distribution and prevalence in bacterial populations remains poor. In the case of protein bacteriocins, this is because of high levels of sequence diversity and difficulties in distinguishing their killing domains from those of other competition systems. Here, we develop a robust bioinformatics pipeline exploiting Hidden Markov Models for the identification of nuclease bacteriocins (NBs) in bacteria of which, to-date, only a handful are known. NBs are large (>60 kDa) toxins that target nucleic acids (DNA, tRNA or rRNA) in the cytoplasm of susceptible bacteria, usually closely related to the producing organism. We identified >3000 NB genes located on plasmids or on the chromosome from 53 bacterial species distributed across different ecological niches, including human, animals, plants, and the environment. A newly identified NB predicted to be specific for Pseudomonas aeruginosa (pyocin Sn) was produced and shown to kill P. aeruginosa thereby validating our pipeline. Intriguingly, while the genes encoding the machinery needed for NB translocation across the cell envelope are widespread in Gram-negative bacteria, NBs are found exclusively in γ-proteobacteria. Similarity network analysis demonstrated that NBs fall into eight groups each with a distinct arrangement of protein domains involved in import. The only structural feature conserved across all groups was a sequence motif critical for cell-killing that is generally not found in bacteriocins targeting the periplasm, implying a specific role in translocating the nuclease to the cytoplasm. Finally, we demonstrate a significant association between nuclease colicins, NBs specific for Escherichia coli, and virulence factors, suggesting NBs play a role in infection processes, most likely by enabling pathogens to outcompete commensal bacteria.

Dataset Information

Complex RNA folding kinetics revealed by single-molecule FRET and hidden Markov models.

Publications

Complex RNA folding kinetics revealed by single-molecule FRET and hidden Markov models.

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