scholarly journals Computationally Reconstructing Cotranscriptional RNA Folding Pathways from Experimental Data Reveals Rearrangement of Non-Native Folding Intermediates

2018 ◽  
Author(s):  
Angela M Yu ◽  
Paul M. Gasper ◽  
Eric J. Strobel ◽  
Kyle E. Watters ◽  
Alan A. Chen ◽  
...  
Keyword(s):  
Rna Folding ◽  
Signal Recognition Particle ◽  
Point Mutations ◽  
Structural Rearrangement ◽  
Folding Pathway ◽  
List Type ◽  
Folding Intermediates ◽  
Folding Pathways ◽  
Dynamics Simulations ◽  
Srp Rna

SummaryThe series of RNA folding events that occur during transcription, or a cotranscriptional folding pathway, can critically influence the functional roles of RNA in the cell. Here we present a method, Reconstructing RNA Dynamics from Data (R2D2), to uncover details of cotranscriptional folding pathways by predicting RNA secondary and tertiary structures from cotranscriptional SHAPE-Seq data. We applied R2D2 to the folding of the Escherichia coli Signal Recognition Particle (SRP) RNA sequence and show that this sequence undergoes folding through non-native intermediate structures that require significant structural rearrangement before reaching the functional native structure. Secondary structure folding pathway predictions and all-atom molecular dynamics simulations of folding intermediates suggest that this rearrangement can proceed through a toehold mediated strand displacement mechanism, which can be disrupted and rescued with point mutations. These results demonstrate that even RNAs with simple functional folds can undergo complex folding processes during synthesis, and that small variations in their sequence can drastically affect their cotranscriptional folding pathways.Highlights- Computational methods predict RNA structures from cotranscriptional SHAPE-Seq data- The E. coli SRP RNA folds into non-native structural intermediates cotranscriptionally- These structures rearrange dynamically to form an extended functional fold- Point mutations can disrupt and rescue cotranscriptional RNA folding pathways

scholarly journals Stabilizing Obligatory Non-native Intermediates Along Co-transcriptional Folding Trajectories of SRP RNA Affects Cell Viability

2018 ◽  
Author(s):  
Shingo Fukuda ◽  
Shannon Yan ◽  
Yusuke Komi ◽  
Mingxuan Sun ◽  
Ronen Gabizon ◽  
...  
Keyword(s):  
Cell Viability ◽  
Optical Tweezers ◽  
Protein Targeting ◽  
Signal Recognition Particle ◽  
Folding Pathway ◽  
List Type ◽  
Transcription Complexes ◽  
Native Fold ◽  
Srp Rna

SummarySignal recognition particle (SRP) inEscherichia colicomprises protein Ffh and SRP RNA. Its essential functionality—co-translational protein-targeting/delivery to cellular membranes— hinges on the RNA attaining a native long-hairpin fold that facilitates protein conformational rearrangements within the SRP complex. Since RNA folds co-transcriptionally on RNA polymerase, we use high-resolution optical tweezers to first characterize the mechanical unfolding/refolding of incrementally lengthened RNAs from stalled transcription complexes until reaching the full-length transcript. This analysis allows identification of folding intermediates adopted during the real-time co-transcriptional folding of SRP RNA. The co-transcriptional folding trajectories are surprisingly invariant to transcription rates, and involve formation of an obligatory non-native hairpin intermediate that eventually resolves into the native fold. SRP RNA variants designed to stabilize this non-native intermediate—likely sequestering the SRP ribonucleoprotein complex in an inactive form—greatly reduce cell viability, indicating that the same co-transcriptional folding mechanism operatesin vivoand possible alternative antibiotic strategies.HighlightsFolding pathway of an essential functional RNA has been resolved co-transcriptionally.The co-transcriptional folding pathway of SRP RNA is invariant to transcription rates.Nascent SRP RNA obligatorily forms a non-native intermediate before adopting the native fold.Modulating transitions from the non-native to native SRP RNA hairpin fold alters cell viability.

scholarly journals A Topological Selection of Folding Pathways from Native States of Knotted Proteins

Symmetry ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1670
Author(s):  
Agnese Barbensi ◽  
Naya Yerolemou ◽  
Oliver Vipond ◽  
Barbara I. Mahler ◽  
Pawel Dabrowski-Tumanski ◽  
...  
Keyword(s):  
Folding Pathway ◽  
Double Loop ◽  
Carbonic Anhydrases ◽  
And Topology ◽  
Folding Pathways ◽  
Local Symmetries ◽  
Knotted Proteins ◽  
Dynamics Simulations ◽  
Trefoil Knots ◽  
Selection Of

Understanding how knotted proteins fold is a challenging problem in biology. Researchers have proposed several models for their folding pathways, based on theory, simulations and experiments. The geometry of proteins with the same knot type can vary substantially and recent simulations reveal different folding behaviour for deeply and shallow knotted proteins. We analyse proteins forming open-ended trefoil knots by introducing a topologically inspired statistical metric that measures their entanglement. By looking directly at the geometry and topology of their native states, we are able to probe different folding pathways for such proteins. In particular, the folding pathway of shallow knotted carbonic anhydrases involves the creation of a double-looped structure, contrary to what has been observed for other knotted trefoil proteins. We validate this with Molecular Dynamics simulations. By leveraging the geometry and local symmetries of knotted proteins’ native states, we provide the first numerical evidence of a double-loop folding mechanism in trefoil proteins.

scholarly journals DUETT quantitatively identifies known and novel events in nascent RNA structural dynamics from chemical probing data

2018 ◽  
Author(s):  
Albert Y. Xue ◽  
Angela M Yu ◽  
Julius B. Lucks ◽  
Neda Bagheri
Keyword(s):  
Structural Dynamics ◽  
Rna Folding ◽  
Signal Recognition Particle ◽  
Bias Error ◽  
Structural Transitions ◽  
Chemical Probing ◽  
Wide Range ◽  
Nascent Rna ◽  
Nucleotide Resolution ◽  
Srp Rna

AbstractMotivationRNA molecules can undergo complex structural dynamics, especially during transcription, which influence their biological functions. Recently developed high-throughput chemical probing experiments study RNA cotranscriptional folding to generate nucleotide-resolution ‘reactivities’ for each length of a growing nascent RNA and reflect structural dynamics. However, the manual annotation and qualitative interpretation of reactivity across these large datasets can be nuanced, laborious, and difficult for new practitioners. We developed a quantitative and systematic approach to automatically detect RNA folding events from these datasets to reduce human bias/error, standardize event discovery, and generate hypotheses about RNA folding trajectories for further analysis and experimental validation.ResultsDetection ofUnknownEvents withTunableThresholds (DUETT) identifies RNA structural transitions in cotranscriptional RNA chemical probing datasets. DUETT employs a feedback control-inspired method and a linear regression approach and relies on interpretable and independently tunable parameter thresholds to match qualitative user expectations with quantitatively identified folding events. We validate the approach by identifying known RNA structural transitions within the cotranscriptional folding pathways of theEscherichia colisignal recognition particle (SRP) RNA and theBacillus cereus crcBfluoride riboswitch. We identify previously overlooked features of these datasets such as heightened reactivity patterns in the SRP RNA about 12 nucleotide lengths before base pair rearrangement. We then apply a sensitivity analysis to identify tradeoffs when choosing parameter thresholds. Finally, we show that DUETT is tunable across a wide range of contexts, enabling flexible application to study broad classes of RNA folding mechanisms.Availabilityhttps://github.com/BagheriLab/[email protected],[email protected]

scholarly journals Parallel G-triplexes and G-hairpins as potential transitory ensembles in the folding of parallel-stranded DNA G-Quadruplexes

2019 ◽  
Vol 47 (14) ◽  
pp. 7276-7293 ◽  
Author(s):  
Petr Stadlbauer ◽  
Petra Kührová ◽  
Lukáš Vicherek ◽  
Pavel Banáš ◽  
Michal Otyepka ◽  
...  
Keyword(s):  
Single Molecule ◽  
Local Equilibrium ◽  
Enhanced Sampling ◽  
Loop Formation ◽  
Folding Intermediates ◽  
G4 Dna ◽  
Folding Pathways ◽  
Dynamics Simulations ◽  
Genomic Regions

Abstract Guanine quadruplexes (G4s) are non-canonical nucleic acids structures common in important genomic regions. Parallel-stranded G4 folds are the most abundant, but their folding mechanism is not fully understood. Recent research highlighted that G4 DNA molecules fold via kinetic partitioning mechanism dominated by competition amongst diverse long-living G4 folds. The role of other intermediate species such as parallel G-triplexes and G-hairpins in the folding process has been a matter of debate. Here, we use standard and enhanced-sampling molecular dynamics simulations (total length of ∼0.9 ms) to study these potential folding intermediates. We suggest that parallel G-triplex per se is rather an unstable species that is in local equilibrium with a broad ensemble of triplex-like structures. The equilibrium is shifted to well-structured G-triplex by stacked aromatic ligand and to a lesser extent by flanking duplexes or nucleotides. Next, we study propeller loop formation in GGGAGGGAGGG, GGGAGGG and GGGTTAGGG sequences. We identify multiple folding pathways from different unfolded and misfolded structures leading towards an ensemble of intermediates called cross-like structures (cross-hairpins), thus providing atomistic level of description of the single-molecule folding events. In summary, the parallel G-triplex is a possible, but not mandatory short-living (transitory) intermediate in the folding of parallel-stranded G4.

Reshaping the Protein Folding Pathway by Osmolyte via its Effects on the Folding Intermediates

2015 ◽  
Vol 16 (6) ◽  
pp. 513-520 ◽  
Author(s):  
Gurumayum Sharma ◽  
Tanveer Dar ◽  
Laishram Singh
Keyword(s):  
Protein Folding ◽  
Folding Pathway ◽  
Folding Intermediates

scholarly journals Comparative Assessment of NMR Probes for the Experimental Description of Protein Folding Pathways with High-Pressure NMR

Biology ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 656
Author(s):  
Vincent Van Deuren ◽  
Yin-Shan Yang ◽  
Karine de Guillen ◽  
Cécile Dubois ◽  
Catherine Anne Royer ◽  
...  
Keyword(s):  
High Pressure ◽  
Staphylococcal Nuclease ◽  
Comparative Assessment ◽  
Side Chain ◽  
Folding Pathway ◽  
Hydrophobic Core ◽  
Folding Pathways ◽  
Partial Unfolding ◽  
Protein Folding Pathways ◽  
Similar Description

Multidimensional NMR intrinsically provides multiple probes that can be used for deciphering the folding pathways of proteins: NH amide and CH groups are strategically located on the backbone of the protein, while CH3 groups, on the side-chain of methylated residues, are involved in important stabilizing interactions in the hydrophobic core. Combined with high hydrostatic pressure, these observables provide a powerful tool to explore the conformational landscapes of proteins. In the present study, we made a comparative assessment of the NH, CH, and CH3 groups for analyzing the unfolding pathway of ∆+PHS Staphylococcal Nuclease. These probes yield a similar description of the folding pathway, with virtually identical thermodynamic parameters for the unfolding reaction, despite some notable differences. Thus, if partial unfolding begins at identical pressure for these observables (especially in the case of backbone probes) and concerns similar regions of the molecule, the residues involved in contact losses are not necessarily the same. In addition, an unexpected slight shift toward higher pressure was observed in the sequence of the scenario of unfolding with CH when compared to amide groups.

SRP RNA Remodeling by SRP68 Explains Its Role in Protein Translocation

Science ◽  
2014 ◽  
Vol 344 (6179) ◽  
pp. 101-104 ◽  
Author(s):  
Jan Timo Grotwinkel ◽  
Klemens Wild ◽  
Bernd Segnitz ◽  
Irmgard Sinning
Keyword(s):  
Ribosomal Rna ◽  
Protein Targeting ◽  
Rna Binding ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Structural Basis ◽  
Signal Recognition ◽  
Rna Remodeling ◽  
Srp Rna ◽  
Arginine Rich Motif

The signal recognition particle (SRP) is central to membrane protein targeting; SRP RNA is essential for SRP assembly, elongation arrest, and activation of SRP guanosine triphosphatases. In eukaryotes, SRP function relies on the SRP68-SRP72 heterodimer. We present the crystal structures of the RNA-binding domain of SRP68 (SRP68-RBD) alone and in complex with SRP RNA and SRP19. SRP68-RBD is a tetratricopeptide-like module that binds to a RNA three-way junction, bends the RNA, and inserts an α-helical arginine-rich motif (ARM) into the major groove. The ARM opens the conserved 5f RNA loop, which in ribosome-bound SRP establishes a contact to ribosomal RNA. Our data provide the structural basis for eukaryote-specific, SRP68-driven RNA remodeling required for protein translocation.

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