New models of tetrahymena telomerase RNA from experimentally derived constraints and modeling
Daud I. Cole, Jason D. Legassie, Laura N. Bonifacio, Vijay G. Sekaran, Feng Ding, Nikolay V. Dokholyan, Michael B. Jarstfer
The telomerase ribonucleoprotein complex ensures complete replication of eukaryotic chromosomes. Telomerase RNA (TER) provides the template for replicating the G-rich strand of telomeric DNA, provides an anchor site for telomerase-associated proteins, and participates in catalysis through several incompletely characterized mechanisms. A major impediment toward understanding its nontemplating roles is the absence of high content structural information for TER within the telomerase complex. Here, we used selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) to examine the structure of Tetrahymena TER free in solution and bound to tTERT in the minimal telomerase RNP. We discovered a striking difference in the two conformations and established direct evidence for base triples in the tTER pseudoknot. We then used SHAPE data, previously published FRET data, and biochemical inference to model the structure of tTER using discrete molecular dynamics simulations. The resulting tTER structure was docked with a homology model of the Tetrahymena telomerase reverse transcriptase (tTERT) to characterize the conformational changes of tTER telomerase assembly. Free in solution, tTER appears to contain four pairing regions: stems I, II, and IV, which are present in the commonly accepted structure, and stem III, a large paired region that encompasses the template and pseudoknot domains. Our interpretation of the data and subsequent modeling affords a molecular model for telomerase assemblage in which a large stem III of tTER unwinds to allow proper association of the template with the tTERT active site and formation of the pseudoknot. Additionally, analysis of our SHAPE data and previous enzymatic footprinting allow us to propose a model for stem-loop IV function in which tTERT is activated by binding stem IV in the major groove of the helix-capping loop.
https://doi.org/10.1021/ja305636u
Published - Dec 12 2012
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telomerase RNA Medicine & Life Sciences
Tetrahymena Medicine & Life Sciences
Acylation Engineering & Materials Science
RNA Engineering & Materials Science
Telomerase Medicine & Life Sciences
Catalysis Engineering & Materials Science
Hydroxyl Radical Medicine & Life Sciences
Cole, D. I., Legassie, J. D., Bonifacio, L. N., Sekaran, V. G., Ding, F., Dokholyan, N. V., & Jarstfer, M. B. (2012). New models of tetrahymena telomerase RNA from experimentally derived constraints and modeling. Journal of the American Chemical Society, 134(49), 20070-20080. https://doi.org/10.1021/ja305636u
Cole, Daud I. ; Legassie, Jason D. ; Bonifacio, Laura N. ; Sekaran, Vijay G. ; Ding, Feng ; Dokholyan, Nikolay V. ; Jarstfer, Michael B. / New models of tetrahymena telomerase RNA from experimentally derived constraints and modeling. In: Journal of the American Chemical Society. 2012 ; Vol. 134, No. 49. pp. 20070-20080.
@article{743845c8c89545a6a2555fddba2df685,
title = "New models of tetrahymena telomerase RNA from experimentally derived constraints and modeling",
abstract = "The telomerase ribonucleoprotein complex ensures complete replication of eukaryotic chromosomes. Telomerase RNA (TER) provides the template for replicating the G-rich strand of telomeric DNA, provides an anchor site for telomerase-associated proteins, and participates in catalysis through several incompletely characterized mechanisms. A major impediment toward understanding its nontemplating roles is the absence of high content structural information for TER within the telomerase complex. Here, we used selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) to examine the structure of Tetrahymena TER free in solution and bound to tTERT in the minimal telomerase RNP. We discovered a striking difference in the two conformations and established direct evidence for base triples in the tTER pseudoknot. We then used SHAPE data, previously published FRET data, and biochemical inference to model the structure of tTER using discrete molecular dynamics simulations. The resulting tTER structure was docked with a homology model of the Tetrahymena telomerase reverse transcriptase (tTERT) to characterize the conformational changes of tTER telomerase assembly. Free in solution, tTER appears to contain four pairing regions: stems I, II, and IV, which are present in the commonly accepted structure, and stem III, a large paired region that encompasses the template and pseudoknot domains. Our interpretation of the data and subsequent modeling affords a molecular model for telomerase assemblage in which a large stem III of tTER unwinds to allow proper association of the template with the tTERT active site and formation of the pseudoknot. Additionally, analysis of our SHAPE data and previous enzymatic footprinting allow us to propose a model for stem-loop IV function in which tTERT is activated by binding stem IV in the major groove of the helix-capping loop.",
author = "Cole, {Daud I.} and Legassie, {Jason D.} and Bonifacio, {Laura N.} and Sekaran, {Vijay G.} and Feng Ding and Dokholyan, {Nikolay V.} and Jarstfer, {Michael B.}",
doi = "10.1021/ja305636u",
journal = "Journal of the American Chemical Society",
Cole, DI, Legassie, JD, Bonifacio, LN, Sekaran, VG, Ding, F, Dokholyan, NV & Jarstfer, MB 2012, 'New models of tetrahymena telomerase RNA from experimentally derived constraints and modeling', Journal of the American Chemical Society, vol. 134, no. 49, pp. 20070-20080. https://doi.org/10.1021/ja305636u
New models of tetrahymena telomerase RNA from experimentally derived constraints and modeling. / Cole, Daud I.; Legassie, Jason D.; Bonifacio, Laura N.; Sekaran, Vijay G.; Ding, Feng; Dokholyan, Nikolay V.; Jarstfer, Michael B.
In: Journal of the American Chemical Society, Vol. 134, No. 49, 12.12.2012, p. 20070-20080.
T1 - New models of tetrahymena telomerase RNA from experimentally derived constraints and modeling
AU - Cole, Daud I.
AU - Legassie, Jason D.
AU - Bonifacio, Laura N.
AU - Sekaran, Vijay G.
AU - Jarstfer, Michael B.
N2 - The telomerase ribonucleoprotein complex ensures complete replication of eukaryotic chromosomes. Telomerase RNA (TER) provides the template for replicating the G-rich strand of telomeric DNA, provides an anchor site for telomerase-associated proteins, and participates in catalysis through several incompletely characterized mechanisms. A major impediment toward understanding its nontemplating roles is the absence of high content structural information for TER within the telomerase complex. Here, we used selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) to examine the structure of Tetrahymena TER free in solution and bound to tTERT in the minimal telomerase RNP. We discovered a striking difference in the two conformations and established direct evidence for base triples in the tTER pseudoknot. We then used SHAPE data, previously published FRET data, and biochemical inference to model the structure of tTER using discrete molecular dynamics simulations. The resulting tTER structure was docked with a homology model of the Tetrahymena telomerase reverse transcriptase (tTERT) to characterize the conformational changes of tTER telomerase assembly. Free in solution, tTER appears to contain four pairing regions: stems I, II, and IV, which are present in the commonly accepted structure, and stem III, a large paired region that encompasses the template and pseudoknot domains. Our interpretation of the data and subsequent modeling affords a molecular model for telomerase assemblage in which a large stem III of tTER unwinds to allow proper association of the template with the tTERT active site and formation of the pseudoknot. Additionally, analysis of our SHAPE data and previous enzymatic footprinting allow us to propose a model for stem-loop IV function in which tTERT is activated by binding stem IV in the major groove of the helix-capping loop.
AB - The telomerase ribonucleoprotein complex ensures complete replication of eukaryotic chromosomes. Telomerase RNA (TER) provides the template for replicating the G-rich strand of telomeric DNA, provides an anchor site for telomerase-associated proteins, and participates in catalysis through several incompletely characterized mechanisms. A major impediment toward understanding its nontemplating roles is the absence of high content structural information for TER within the telomerase complex. Here, we used selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) to examine the structure of Tetrahymena TER free in solution and bound to tTERT in the minimal telomerase RNP. We discovered a striking difference in the two conformations and established direct evidence for base triples in the tTER pseudoknot. We then used SHAPE data, previously published FRET data, and biochemical inference to model the structure of tTER using discrete molecular dynamics simulations. The resulting tTER structure was docked with a homology model of the Tetrahymena telomerase reverse transcriptase (tTERT) to characterize the conformational changes of tTER telomerase assembly. Free in solution, tTER appears to contain four pairing regions: stems I, II, and IV, which are present in the commonly accepted structure, and stem III, a large paired region that encompasses the template and pseudoknot domains. Our interpretation of the data and subsequent modeling affords a molecular model for telomerase assemblage in which a large stem III of tTER unwinds to allow proper association of the template with the tTERT active site and formation of the pseudoknot. Additionally, analysis of our SHAPE data and previous enzymatic footprinting allow us to propose a model for stem-loop IV function in which tTERT is activated by binding stem IV in the major groove of the helix-capping loop.
U2 - 10.1021/ja305636u
DO - 10.1021/ja305636u
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
Cole DI, Legassie JD, Bonifacio LN, Sekaran VG, Ding F, Dokholyan NV et al. New models of tetrahymena telomerase RNA from experimentally derived constraints and modeling. Journal of the American Chemical Society. 2012 Dec 12;134(49):20070-20080. https://doi.org/10.1021/ja305636u