Abstract A human telomere ends in a single-stranded 3′ tail, composed of repeats of T2AG3. G-quadruplexes (GQs) formed from four consecutive repeats have been shown to possess high-structural and mechanical diversity. In principle, a GQ can form from any four repeats that are not necessarily consecutive. To understand the dynamics of GQs with positional multiplicity, we studied five and six repeats human telomeric sequence using a combination of single molecule FRET and optical tweezers. Our results suggest preferential formation of GQs at the 3′ end both in K+ and Na+ solutions, with minor populations of 5′-GQ or long-loop GQs. A vectorial folding assay which mimics the directional nature of telomere extension showed that the 3′ preference holds even when folding is allowed to begin from the 5′ side. In 100 mM K+, the unassociated T2AG3 segment has a streamlining effect in that one or two mechanically distinct species was observed at a single position instead of six or more observed without an unassociated repeat. We did not observe such streamlining effect in 100 mM Na+. Location of GQ and reduction in conformational diversity in the presence of extra repeats have implications in telomerase inhibition, T-loop formation and telomere end protection.
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Vectorial folding of telomere overhang promotes higher accessibility
Human telomere overhang composed of tandem repeats of TTAGGG folds into G-quadruplex (G4). Unlike in an experimental setting in the test tube in which the entire length is allowed to fold at once, inside the cell, the overhang is expected to fold as it is synthesized directionally (5′ to 3′) and released segmentally by a specialized enzyme, the telomerase. To mimic such vectorial G4 folding process, we employed a superhelicase, Rep-X which can unwind DNA to release the TTAGGG repeats in 5′ to 3′ direction. We demonstrate that the folded conformation achieved by the refolding of full sequence is significantly different from that of the vectorial folding for two to eight TTAGGG repeats. Strikingly, the vectorially folded state leads to a remarkably higher accessibility to complementary C-rich strand and the telomere binding protein POT1, reflecting a less stably folded state resulting from the vectorial folding. Importantly, our study points to an inherent difference between the co-polymerizing and post-polymerized folding of telomere overhang that can impact telomere architecture and downstream processes.
more » « less- NSF-PAR ID:
- 10368122
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 50
- Issue:
- 11
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- p. 6271-6283
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Telomeres terminate with a 50–300 bases long single-stranded G-rich overhang, which can be misrecognized as a DNA damage repair site. Shelterin plays critical roles in maintaining and protecting telomere ends by regulating access of various physiological agents to telomeric DNA, but the underlying mechanism is not well understood. Here, we measure how shelterin affects the accessibility of long telomeric overhangs by monitoring transient binding events of a short complementary peptide nucleic acid (PNA) probe using FRET-PAINT in vitro. We observed that the POT1 subunit of shelterin reduces the accessibility of the PNA probe by ∼2.5-fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin stabilizes POT1 binding to the overhang by tethering POT1 to the double-stranded telomeric DNA and reduces the accessibility of telomeric overhangs by ∼5-fold. This enhanced protection suggests shelterin restructures the junction between single and double-stranded telomere, which is otherwise the most accessible part of the telomeric overhang.
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This data set for the manuscript entitled "Design of Peptides that Fold and Self-Assemble on Graphite" includes all files needed to run and analyze the simulations described in the this manuscript in the molecular dynamics software NAMD, as well as the output of the simulations. The files are organized into directories corresponding to the figures of the main text and supporting information. They include molecular model structure files (NAMD psf or Amber prmtop format), force field parameter files (in CHARMM format), initial atomic coordinates (pdb format), NAMD configuration files, Colvars configuration files, NAMD log files, and NAMD output including restart files (in binary NAMD format) and trajectories in dcd format (downsampled to 10 ns per frame). Analysis is controlled by shell scripts (Bash-compatible) that call VMD Tcl scripts or python scripts. These scripts and their output are also included.
Version: 2.0
Changes versus version 1.0 are the addition of the free energy of folding, adsorption, and pairing calculations (Sim_Figure-7) and shifting of the figure numbers to accommodate this addition.
Conventions Used in These Files
===============================Structure Files
----------------
- graph_*.psf or sol_*.psf (original NAMD (XPLOR?) format psf file including atom details (type, charge, mass), as well as definitions of bonds, angles, dihedrals, and impropers for each dipeptide.)- graph_*.pdb or sol_*.pdb (initial coordinates before equilibration)
- repart_*.psf (same as the above psf files, but the masses of non-water hydrogen atoms have been repartitioned by VMD script repartitionMass.tcl)
- freeTop_*.pdb (same as the above pdb files, but the carbons of the lower graphene layer have been placed at a single z value and marked for restraints in NAMD)
- amber_*.prmtop (combined topology and parameter files for Amber force field simulations)
- repart_amber_*.prmtop (same as the above prmtop files, but the masses of non-water hydrogen atoms have been repartitioned by ParmEd)Force Field Parameters
----------------------
CHARMM format parameter files:
- par_all36m_prot.prm (CHARMM36m FF for proteins)
- par_all36_cgenff_no_nbfix.prm (CGenFF v4.4 for graphene) The NBFIX parameters are commented out since they are only needed for aromatic halogens and we use only the CG2R61 type for graphene.
- toppar_water_ions_prot_cgenff.str (CHARMM water and ions with NBFIX parameters needed for protein and CGenFF included and others commented out)Template NAMD Configuration Files
---------------------------------
These contain the most commonly used simulation parameters. They are called by the other NAMD configuration files (which are in the namd/ subdirectory):
- template_min.namd (minimization)
- template_eq.namd (NPT equilibration with lower graphene fixed)
- template_abf.namd (for adaptive biasing force)Minimization
-------------
- namd/min_*.0.namdEquilibration
-------------
- namd/eq_*.0.namdAdaptive biasing force calculations
-----------------------------------
- namd/eabfZRest7_graph_chp1404.0.namd
- namd/eabfZRest7_graph_chp1404.1.namd (continuation of eabfZRest7_graph_chp1404.0.namd)Log Files
---------
For each NAMD configuration file given in the last two sections, there is a log file with the same prefix, which gives the text output of NAMD. For instance, the output of namd/eabfZRest7_graph_chp1404.0.namd is eabfZRest7_graph_chp1404.0.log.Simulation Output
-----------------
The simulation output files (which match the names of the NAMD configuration files) are in the output/ directory. Files with the extensions .coor, .vel, and .xsc are coordinates in NAMD binary format, velocities in NAMD binary format, and extended system information (including cell size) in text format. Files with the extension .dcd give the trajectory of the atomic coorinates over time (and also include system cell information). Due to storage limitations, large DCD files have been omitted or replaced with new DCD files having the prefix stride50_ including only every 50 frames. The time between frames in these files is 50 * 50000 steps/frame * 4 fs/step = 10 ns. The system cell trajectory is also included for the NPT runs are output/eq_*.xst.Scripts
-------
Files with the .sh extension can be found throughout. These usually provide the highest level control for submission of simulations and analysis. Look to these as a guide to what is happening. If there are scripts with step1_*.sh and step2_*.sh, they are intended to be run in order, with step1_*.sh first.
CONTENTS
========The directory contents are as follows. The directories Sim_Figure-1 and Sim_Figure-8 include README.txt files that describe the files and naming conventions used throughout this data set.
Sim_Figure-1: Simulations of N-acetylated C-amidated amino acids (Ac-X-NHMe) at the graphite–water interface.
Sim_Figure-2: Simulations of different peptide designs (including acyclic, disulfide cyclized, and N-to-C cyclized) at the graphite–water interface.
Sim_Figure-3: MM-GBSA calculations of different peptide sequences for a folded conformation and 5 misfolded/unfolded conformations.
Sim_Figure-4: Simulation of four peptide molecules with the sequence cyc(GTGSGTG-GPGG-GCGTGTG-SGPG) at the graphite–water interface at 370 K.
Sim_Figure-5: Simulation of four peptide molecules with the sequence cyc(GTGSGTG-GPGG-GCGTGTG-SGPG) at the graphite–water interface at 295 K.
Sim_Figure-5_replica: Temperature replica exchange molecular dynamics simulations for the peptide cyc(GTGSGTG-GPGG-GCGTGTG-SGPG) with 20 replicas for temperatures from 295 to 454 K.
Sim_Figure-6: Simulation of the peptide molecule cyc(GTGSGTG-GPGG-GCGTGTG-SGPG) in free solution (no graphite).
Sim_Figure-7: Free energy calculations for folding, adsorption, and pairing for the peptide CHP1404 (sequence: cyc(GTGSGTG-GPGG-GCGTGTG-SGPG)). For folding, we calculate the PMF as function of RMSD by replica-exchange umbrella sampling (in the subdirectory Folding_CHP1404_Graphene/). We make the same calculation in solution, which required 3 seperate replica-exchange umbrella sampling calculations (in the subdirectory Folding_CHP1404_Solution/). Both PMF of RMSD calculations for the scrambled peptide are in Folding_scram1404/. For adsorption, calculation of the PMF for the orientational restraints and the calculation of the PMF along z (the distance between the graphene sheet and the center of mass of the peptide) are in Adsorption_CHP1404/ and Adsorption_scram1404/. The actual calculation of the free energy is done by a shell script ("doRestraintEnergyError.sh") in the 1_free_energy/ subsubdirectory. Processing of the PMFs must be done first in the 0_pmf/ subsubdirectory. Finally, files for free energy calculations of pair formation for CHP1404 are found in the Pair/ subdirectory.
Sim_Figure-8: Simulation of four peptide molecules with the sequence cyc(GTGSGTG-GPGG-GCGTGTG-SGPG) where the peptides are far above the graphene–water interface in the initial configuration.
Sim_Figure-9: Two replicates of a simulation of nine peptide molecules with the sequence cyc(GTGSGTG-GPGG-GCGTGTG-SGPG) at the graphite–water interface at 370 K.
Sim_Figure-9_scrambled: Two replicates of a simulation of nine peptide molecules with the control sequence cyc(GGTPTTGGGGGGSGGPSGTGGC) at the graphite–water interface at 370 K.
Sim_Figure-10: Adaptive biasing for calculation of the free energy of the folded peptide as a function of the angle between its long axis and the zigzag directions of the underlying graphene sheet.
This material is based upon work supported by the US National Science Foundation under grant no. DMR-1945589. A majority of the computing for this project was performed on the Beocat Research Cluster at Kansas State University, which is funded in part by NSF grants CHE-1726332, CNS-1006860, EPS-1006860, and EPS-0919443. This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI-1548562, through allocation BIO200030. -
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Abstract RNAs begin to fold and function during transcription. Riboswitches undergo cotranscriptional switching in the context of transcription elongation, RNA folding, and ligand binding. To investigate how these processes jointly modulate the function of the folate stress-sensing
Fusobacterium ulcerans ZTP riboswitch, we apply a single-molecule vectorial folding (VF) assay in which an engineered superhelicase Rep-X sequentially releases fluorescently labeled riboswitch RNA from a heteroduplex in a 5′-to-3′ direction, at ~60 nt s−1[comparable to the speed of bacterial RNA polymerase (RNAP)]. We demonstrate that the ZTP riboswitch is kinetically controlled and that its activation is favored by slower unwinding, strategic pausing between but not before key folding elements, or a weakened transcription terminator. Real-time single-molecule monitoring captures folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These results show how individual nascent RNAs occupy distinct channels within the folding landscape that controls the fate of the riboswitch. -
Abstract Ring origami has emerged as a robust strategy for designing foldable and deployable structures due to its impressive packing abilities achieved from the snap-folding mechanism. In general, polygonal rings with rationally designed geometric parameters can fold into compacted three-loop configurations with curved segments, which result from the internal bending moment in the folded state. Inspired by the internal bending moment-induced curvature in the folded state, we explore how this curvature can be tuned by introducing initial natural curvature to the segments of the polygonal rings in their deployed stress-free state, and study how this initial curvature affects their folded configurations. Taking a clue from straight-segmented polygonal rings that fold into overlapping curved loops, we find it is possible to reverse the process by introducing curvature into the ring segments in the stress-free initial state such that the rings fold into a straight-line looped pattern with “zero” area. This realizes extreme packing. In this work, by a combination of experimental observation, finite element analysis, and theoretical modeling, we systematically study the effect of segment curvature on folding behavior, folded configurations, and packing of curved ring origami with different geometries. It is anticipated that curved ring origami can open a new avenue for the design of foldable and deployable structures with simple folded configurations and high packing efficiency.more » « less
