scholarly journals Structural dynamics of single SARS-CoV-2 pseudoknot molecules reveal topologically distinct conformers

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Krishna Neupane ◽  
Meng Zhao ◽  
Aaron Lyons ◽  
Sneha Munshi ◽  
Sandaru M. Ileperuma ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Structural Dynamics ◽  
Drug Target ◽  
The Other ◽  
Conformational Heterogeneity ◽  
Mechanical Tension ◽  
Stem Loop ◽  
Ribosomal Frameshifting ◽  
Multiple Structures ◽  
Rna Pseudoknot

AbstractThe RNA pseudoknot that stimulates programmed ribosomal frameshifting in SARS-CoV-2 is a possible drug target. To understand how it responds to mechanical tension applied by ribosomes, thought to play a key role during frameshifting, we probe its structural dynamics using optical tweezers. We find that it forms multiple structures: two pseudoknotted conformers with different stability and barriers, and alternative stem-loop structures. The pseudoknotted conformers have distinct topologies, one threading the 5′ end through a 3-helix junction to create a knot-like fold, the other with unthreaded 5′ end, consistent with structures observed via cryo-EM and simulations. Refolding of the pseudoknotted conformers starts with stem 1, followed by stem 3 and lastly stem 2; Mg2+ ions are not required, but increase pseudoknot mechanical rigidity and favor formation of the knot-like conformer. These results resolve the SARS-CoV-2 frameshift signal folding mechanism and highlight its conformational heterogeneity, with important implications for structure-based drug-discovery efforts.

scholarly journals Structural dynamics of the SARS-CoV-2 frameshift-stimulatory pseudoknot reveal topologically distinct conformers

2020 ◽  
Author(s):  
Krishna Neupane ◽  
Meng Zhao ◽  
Aaron Lyons ◽  
Sneha Munshi ◽  
Sandaru M Ileperuma ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Optical Tweezers ◽  
Structural Dynamics ◽  
Drug Target ◽  
Conformational Heterogeneity ◽  
Computational Simulations ◽  
Mechanical Tension ◽  
Stem Loop ◽  
Ribosomal Frameshifting ◽  
Rna Pseudoknot

The RNA pseudoknot that stimulates −1 programmed ribosomal frameshifting in SARS coronavirus-2 (SARS-CoV-2) is a possible drug target. To understand how this 3-stemmed pseudoknot responds to the mechanical tension applied by ribosomes during translation, which is thought to play a key role during frameshifting, we probed its structural dynamics under tension using optical tweezers. Unfolding curves revealed that the frameshift signal formed multiple different structures: at least two distinct pseudoknotted conformers with different unfolding forces and energy barriers, as well as alternative stem-loop structures. Refolding curves showed that stem 1 formed first in the pseudoknotted conformers, followed by stem 3 and then stem 2. By extending the handle holding the RNA to occlude the 5′ end of stem 1, the proportion of the different pseudoknot conformers could be altered systematically, consistent with structures observed in cryo-EM images and computational simulations that had distinct topologies: the 5′ end of the RNA threaded through the 3-helix junction to form a ring-knot, or unthreaded as in more standard H-type pseudoknots. These results resolve the folding mechanism of the frameshift signal in SARS-CoV-2 and highlight the dynamic conformational heterogeneity of this RNA, with important implications for structure-based drug-discovery efforts.

scholarly journals Complex dynamics under tension in a high-efficiency frameshift stimulatory structure

2019 ◽  
Vol 116 (39) ◽  
pp. 19500-19505 ◽  
Author(s):  
Matthew T. J. Halma ◽  
Dustin B. Ritchie ◽  
Tonia R. Cappellano ◽  
Krishna Neupane ◽  
Michael T. Woodside
Keyword(s):  
Optical Tweezers ◽  
High Efficiency ◽  
Complex Dynamics ◽  
Conformational Heterogeneity ◽  
Force Extension ◽  
Ribosomal Frameshifting ◽  
Metastable Structures ◽  
Extension Curve ◽  
Length Changes ◽  
Very High

Specific structures in mRNA can stimulate programmed ribosomal frameshifting (PRF). PRF efficiency can vary enormously between different stimulatory structures, but the features that lead to efficient PRF stimulation remain uncertain. To address this question, we studied the structural dynamics of the frameshift signal from West Nile virus (WNV), which stimulates −1 PRF at very high levels and has been proposed to form several different structures, including mutually incompatible pseudoknots and a double hairpin. Using optical tweezers to apply tension to single mRNA molecules, mimicking the tension applied by the ribosome during PRF, we found that the WNV frameshift signal formed an unusually large number of different metastable structures, including all of those previously proposed. From force-extension curve measurements, we mapped 2 mutually exclusive pathways for the folding, each encompassing multiple intermediates. We identified the intermediates in each pathway from length changes and the effects of antisense oligomers blocking formation of specific contacts. Intriguingly, the number of transitions between the different conformers of the WNV frameshift signal was maximal in the range of forces applied by the ribosome during −1 PRF. Furthermore, the occupancy of the pseudoknotted conformations was far too low for static pseudoknots to account for the high levels of −1 PRF. These results support the hypothesis that conformational heterogeneity plays a key role in frameshifting and suggest that transitions between different conformers under tension are linked to efficient PRF stimulation.

scholarly journals Stem-loop structures can effectively substitute for an RNA pseudoknot in -1 ribosomal frameshifting

2011 ◽  
Vol 39 (20) ◽  
pp. 8952-8959 ◽  
Author(s):  
C.-H. Yu ◽  
M. H. Noteborn ◽  
C. W. A. Pleij ◽  
R. C. L. Olsthoorn
Keyword(s):  
Stem Loop ◽  
Ribosomal Frameshifting ◽  
Rna Pseudoknot

scholarly journals Regulators of Viral Frameshifting: More Than RNA Influences Translation Events

2020 ◽  
Vol 7 (1) ◽  
pp. 219-238
Author(s):  
Wesley D. Penn ◽  
Haley R. Harrington ◽  
Jonathan P. Schlebach ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Messenger Rna ◽  
Protein Production ◽  
Productive Infection ◽  
Cis Elements ◽  
Stem Loop ◽  
Viral Translation ◽  
Ribosomal Frameshifting ◽  
Evolutionarily Conserved ◽  
Rna Pseudoknot ◽  
New Protein

Programmed ribosomal frameshifting (PRF) is a conserved translational recoding mechanism found in all branches of life and viruses. In bacteria, archaea, and eukaryotes PRF is used to downregulate protein production by inducing a premature termination of translation, which triggers messenger RNA (mRNA) decay. In viruses, PRF is used to drive the production of a new protein while downregulating the production of another protein, thus maintaining a stoichiometry optimal for productive infection. Traditionally, PRF motifs have been defined by the characteristics of two cis elements: a slippery heptanucleotide sequence followed by an RNA pseudoknot or stem-loop within the mRNA. Recently, additional cis and new trans elements have been identified that regulate PRF in both host and viral translation. These additional factors suggest PRF is an evolutionarily conserved process whose function and regulation we are just beginning to understand.

scholarly journals MorphOT: transport-based interpolation between EM maps with UCSF ChimeraX

2020 ◽  
Author(s):  
Arthur Ecoffet ◽  
Frédéric Poitevin ◽  
Khanh Dao Duc
Keyword(s):  
Optimal Transport ◽  
Three Dimensional ◽  
Linear Interpolation ◽  
The Other ◽  
Supplementary Information ◽  
Conformational Heterogeneity ◽  
Supplementary Data ◽  
Image Dataset ◽  
Standard Linear ◽  
Unique Potential

Abstract Motivation Cryogenic electron microscopy (cryo-EM) offers the unique potential to capture conformational heterogeneity, by solving multiple three-dimensional classes that co-exist within a single cryo-EM image dataset. To investigate the extent and implications of such heterogeneity, we propose to use an optimal-transport-based metric to interpolate barycenters between EM maps and produce morphing trajectories. Results While standard linear interpolation mostly fails to produce realistic transitions, our method yields continuous trajectories that displace densities to morph one map into the other, instead of blending them. Availability and implementation Our method is implemented as a plug-in for ChimeraX called MorphOT, which allows the use of both CPU or GPU resources. The code is publicly available on GitHub (https://github.com/kdd-ubc/MorphOT.git), with documentation containing tutorial and datasets. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Stem–loop formation drives RNA folding in mechanical unzipping experiments

2022 ◽  
Vol 119 (3) ◽  
pp. e2025575119
Author(s):  
Paolo Rissone ◽  
Cristiano V. Bizarro ◽  
Felix Ritort
Keyword(s):  
Optical Tweezers ◽  
Rna Structure ◽  
Nearest Neighbor ◽  
Rna Folding ◽  
General Mechanism ◽  
Loop Formation ◽  
Stem Loop ◽  
Wide Range ◽  
Irreversible Effects ◽  
And Function

Accurate knowledge of RNA hybridization is essential for understanding RNA structure and function. Here we mechanically unzip and rezip a 2-kbp RNA hairpin and derive the 10 nearest-neighbor base pair (NNBP) RNA free energies in sodium and magnesium with 0.1 kcal/mol precision using optical tweezers. Notably, force–distance curves (FDCs) exhibit strong irreversible effects with hysteresis and several intermediates, precluding the extraction of the NNBP energies with currently available methods. The combination of a suitable RNA synthesis with a tailored pulling protocol allowed us to obtain the fully reversible FDCs necessary to derive the NNBP energies. We demonstrate the equivalence of sodium and magnesium free-energy salt corrections at the level of individual NNBP. To characterize the irreversibility of the unzipping–rezipping process, we introduce a barrier energy landscape of the stem–loop structures forming along the complementary strands, which compete against the formation of the native hairpin. This landscape correlates with the hysteresis observed along the FDCs. RNA sequence analysis shows that base stacking and base pairing stabilize the stem–loops that kinetically trap the long-lived intermediates observed in the FDC. Stem–loops formation appears as a general mechanism to explain a wide range of behaviors observed in RNA folding.

scholarly journals Single-Stranded DNA Structure and Positional Context of the Target Cytidine Determine the Enzymatic Efficiency of AID

2007 ◽  
Vol 27 (23) ◽  
pp. 8038-8048 ◽  
Author(s):  
Mani Larijani ◽  
Alberto Martin
Keyword(s):  
Target Genes ◽  
Cytidine Deaminase ◽  
Single Stranded Dna ◽  
Stem Loop ◽  
Double Stranded Dna ◽  
Antibody Diversification ◽  
Multiple Structures ◽  
Optimal Target

ABSTRACT Activation-induced cytidine deaminase (AID) initiates antibody diversification processes by deaminating immunoglobulin sequences. Since transcription of target genes is required for deamination in vivo and AID exclusively mutates single-stranded DNA (ssDNA) in vitro, AID has been postulated to mutate transcription bubbles. However, since ssDNA generated by transcription can assume multiple structures, it is unknown which of these are targeted in vivo. Here we examine the enzymatic and binding properties of AID for different DNA structures. We report that AID has minimal activity on stem-loop structures and preferentially deaminates five-nucleotide bubbles. We compared AID activity on cytidines placed at various distances from the single-stranded/double-stranded DNA junction of bubble substrates and found that the optimal target consists of a single-stranded NWRCN motif. We also show that high-affinity binding is required for but does not necessarily lead to efficient deamination. Using nucleotide analogues, we show that AID's WRC preference (W = A or T; R = A or G) involves the recognition of a purine in the R position and that the carbonyl or amino side chains of guanosine negatively influence specificity at the W position. Our results indicate that AID is likely to target short-tract regions of ssDNA produced by transcription elongation and that it requires a fully single-stranded WRC motif.

Myosin VI: a multifunctional motor

2004 ◽  
Vol 32 (5) ◽  
pp. 685-688 ◽  
Author(s):  
I. Lister ◽  
R. Roberts ◽  
S. Schmitz ◽  
M. Walker ◽  
J. Trinick ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Actin Filaments ◽  
Biochemical Characterization ◽  
Force Transducer ◽  
The Other ◽  
Myosin Vi ◽  
Coated Pits ◽  
Binding Partners ◽  
Alternative Approach

Myosin VI moves towards the minus end of actin filaments unlike all the other myosins so far studied, suggesting that it has unique properties and functions. Myosin VI is present in clathrin-coated pits and vesicles, in membrane ruffles and in the Golgi complex, indicating that it has a wide variety of functions in the cell. To investigate the cellular roles of myosin VI, we have identified a variety of myosin VI-binding partners and characterized their interactions. As an alternative approach, we have studied the in vitro properties of intact myosin VI. Previous studies assumed that myosin VI existed as a dimer but our biochemical characterization and electron microscopy studies reveal that myosin VI is a monomer. Using an optical tweezers force transducer, we showed that monomeric myosin VI is a non-processive motor with a large working stroke of 18 nm. Potential roles for myosin VI in cells are discussed.

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