Solution structure and dynamics of the wild-type pseudoknot of human telomerase RNA.

Telomerase is a ribonucleoprotein complex that replicates the 3' ends of linear chromosomes by successive additions of telomere repeat DNA. The telomerase holoenzyme contains two essential components for catalysis, a telomerase reverse transcriptase (TERT) and telomerase RNA (TER). The TER includes a template for telomere repeat synthesis as well as other domains required for function. We report the solution structure of the wild-type minimal conserved human TER pseudoknot refined with an extensive set of RDCs, and a detailed analysis of the effect of the bulge U177 on pseudoknot structure, dynamics analyzed by RDC and 13C relaxation measurements, and base pair stability. The overall structure of PKWT is highly similar to the previously reported DeltaU177 pseudoknot (PKDU) that has a deletion of a conserved bulge U important for catalytic activity. For direct comparison to PKWT, the structure of PKDU was re-refined with a comparable set of RDCs. Both pseudoknots contain a catalytically essential triple helix at the junction of the two stems, including two stem 1-loop 2 minor groove triples, a junction loop 1-loop 2 Hoogsteen base pair, and stem 2-loop 1 major groove U.A-U Watson-Crick-Hoogsteen triples located directly above the bulge U177. However, there are significant differences in the stabilities of base pairs near the bulge and the dynamics of some nucleotides. The stability of the base pairs in stem 2 surrounding the bulge U177 is greatly decreased, with the result that the Watson-Crick pairs in the triple helix begin to unfold before the Hoogsteen pairs, which may affect telomerase assembly and activity. The bulge U is positioned in the minor groove on the face opposite the triple helical interactions, and sterically blocks the A176 2'OH, which has recently been proposed to have a role in catalysis. The bulge U may serve as a hinge providing backbone flexibility in this region.

[1]  Assignment methodology for larger RNA oligonucleotides: Application to an ATP-binding RNA aptamer , 1997, Journal of biomolecular NMR.

[2]  M. Billeter,et al.  MOLMOL: a program for display and analysis of macromolecular structures. , 1996, Journal of molecular graphics.

[3]  E. Blackburn,et al.  Comprehensive Structure-Function Analysis of the Core Domain of Human Telomerase RNA , 2003, Molecular and Cellular Biology.

[4]  P. Lansdorp,et al.  Telomeres and Aging , 2008 .

[5]  N. Young,et al.  Telomere maintenance and human bone marrow failure. , 2008, Blood.

[6]  C. Autexier,et al.  The structure and function of telomerase reverse transcriptase. , 2006, Annual review of biochemistry.

[7]  T. Veenstra,et al.  Identification of ATPases Pontin and Reptin as Telomerase Components Essential for Holoenzyme Assembly , 2008, Cell.

[8]  M. Blasco,et al.  Telomere length, stem cells and aging. , 2007, Nature chemical biology.

[9]  K. Collins.  The biogenesis and regulation of telomerase holoenzymes , 2006, Nature Reviews Molecular Cell Biology.

[10]  Jiunn-Liang Chen,et al.  Telomerase RNA structure and function: implications for dyskeratosis congenita. , 2004, Trends in biochemical sciences.

[11]  C. Greider,et al.  An emerging consensus for telomerase RNA structure. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[12]  K. Collins,et al.  Human telomerase activation requires two independent interactions between telomerase RNA and telomerase reverse transcriptase. , 2000, Molecular cell.

[13]  E. Blackburn.  Switching and Signaling at the Telomere , 2001, Cell.

[14]  Ivan Smirnov,et al.  A molecular switch underlies a human telomerase disease , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Jiunn-Liang Chen,et al.  Secondary Structure of Vertebrate Telomerase RNA , 2000, Cell.

[16]  Anthony Henras,et al.  Structural basis for recognition of the AGNN tetraloop RNA fold by the double-stranded RNA-binding domain of Rnt1p RNase III. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[17]  C. A. Theimer,et al.  Mutations linked to dyskeratosis congenita cause changes in the structural equilibrium in telomerase RNA , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[18]  K. Collins,et al.  Distinct biogenesis pathways for human telomerase RNA and H/ACA small nucleolar RNAs. , 2003, Molecular cell.

[19]  T. Cech,et al.  Purification of telomerase from Euplotes aediculatus: requirement of a primer 3' overhang. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Xiaodong Qi,et al.  The Telomerase Database , 2007, Nucleic Acids Res..

[21]  Anna Marie Pyle,et al.  Crystal Structure of a Self-Spliced Group II Intron , 2008, Science.

[22]  S. Balasubramanian,et al.  Structural analysis of the catalytic core of human telomerase RNA by FRET and molecular modeling. , 2006, Biochemistry.

[23]  Jiunn-Liang Chen,et al.  A critical stem-loop structure in the CR4-CR5 domain of mammalian telomerase RNA. , 2002, Nucleic acids research.

[24]  S. Grzesiek,et al.  Direct Observation of Hydrogen Bonds in Nucleic Acid Base Pairs by Internucleotide 2JNN Couplings , 1998 .

[25]  S. Wijmenga,et al.  Resonance assignment and structure determination for RNA. , 2001, Methods in enzymology.

[26]  Y. Tzfati,et al.  A Triple Helix within a Pseudoknot Is a Conserved and Essential Element of Telomerase RNA , 2007, Molecular and Cellular Biology.

[27]  V. Lundblad,et al.  Structural Elements Required for Association of the Saccharomyces cerevisiae Telomerase RNA with the Est2 Reverse Transcriptase , 2004, Molecular and Cellular Biology.

[28]  E. Blackburn,et al.  A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis , 1989, Nature.

[29]  K. Collins,et al.  Telomerase in the human organism , 2002, Oncogene.

[30]  I. Dokal,et al.  Dyskeratosis congenita: a genetic disorder of many faces , 2007, Clinical genetics.

[31]  S. Grzesiek,et al.  Direct detection of N−H⋯O=C hydrogen bonds in biomolecules by NMR spectroscopy , 2008, Nature Protocols.

[32]  C. A. Theimer,et al.  Structure and function of telomerase RNA. , 2006, Current opinion in structural biology.

[33]  Sherif Abou Elela,et al.  A Phylogenetically Based Secondary Structure for the Yeast Telomerase RNA , 2004, Current Biology.

[34]  H. Al‐Hashimi,et al.  Dynamics of large elongated RNA by NMR carbon relaxation. , 2007, Journal of the American Chemical Society.

[35]  Bryan Frank,et al.  Two Inactive Fragments of the Integral RNA Cooperate To Assemble Active Telomerase with the Human Protein Catalytic Subunit (hTERT) In Vitro , 1999, Molecular and Cellular Biology.

[36]  Sean R. Eddy,et al.  Rfam: annotating non-coding RNAs in complete genomes , 2004, Nucleic Acids Res..

[37]  Feng Qiao,et al.  Triple-helix structure in telomerase RNA contributes to catalysis , 2008, Nature Structural &Molecular Biology.

[38]  Mira Abraham,et al.  A universal telomerase RNA core structure includes structured motifs required for binding the telomerase reverse transcriptase protein. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[39]  J. Shay,et al.  Telomerase therapeutics for cancer: challenges and new directions , 2006, Nature Reviews Drug Discovery.

[40]  S. Grzesiek,et al.  Direct detection of N−H⋯N hydrogen bonds in biomolecules by NMR spectroscopy , 2008, Nature Protocols.

[41]  C. A. Theimer,et al.  New applications of 2D filtered/edited NOESY for assignment and structure elucidation of RNA and RNA-protein complexes , 2004, Journal of biomolecular NMR.

[42]  Jiunn-Liang Chen,et al.  Functional analysis of the pseudoknot structure in human telomerase RNA. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[43]  J. Feigon,et al.  A novel family of RNA tetraloop structure forms the recognition site for Saccharomyces cerevisiae RNase III , 2001, The EMBO journal.

[44]  T. Cech.  Beginning to Understand the End of the Chromosome , 2004, Cell.

[45]  S. Grzesiek,et al.  Internucleotide Scalar Couplings Across Hydrogen Bonds in Watson-Crick and Hoogsteen Base Pairs of a DNA Triplex , 1999 .

[46]  C. Autexier,et al.  Reconstitution of human telomerase activity and identification of a minimal functional region of the human telomerase RNA. , 1996, The EMBO journal.

[47]  J H Prestegard,et al.  NMR structures of biomolecules using field oriented media and residual dipolar couplings , 2000, Quarterly Reviews of Biophysics.

[48]  Bruce A Shapiro,et al.  The prediction of the wild-type telomerase RNA pseudoknot structure and the pivotal role of the bulge in its formation. , 2006, Journal of molecular graphics & modelling.

[49]  T. Cech,et al.  The POT1–TPP1 telomere complex is a telomerase processivity factor , 2007, Nature.

[50]  R. Batey,et al.  Structure of the SAM-II riboswitch bound to S-adenosylmethionine , 2008, Nature Structural &Molecular Biology.

[51]  R. Lavery,et al.  Defining the structure of irregular nucleic acids: conventions and principles. , 1989, Journal of biomolecular structure & dynamics.

[52]  A. Bax,et al.  Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. , 1997, Science.

[53]  L. Mueller,et al.  Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions , 1998, Nature Structural Biology.

[54]  Tamás Kiss,et al.  Structural and functional characterization of human telomerase RNA processing and cajal body localization signals. , 2007, Molecular cell.

[55]  K. Collins.  Physiological assembly and activity of human telomerase complexes , 2008, Mechanisms of Ageing and Development.

[56]  Axel Mosig,et al.  Structure and Function of the Smallest Vertebrate Telomerase RNA from Teleost Fish* , 2008, Journal of Biological Chemistry.

[57]  C. A. Theimer,et al.  Structure of the human telomerase RNA pseudoknot reveals conserved tertiary interactions essential for function. , 2005, Molecular cell.

[58]  M. Dreyfus,et al.  A mutation in T7 RNA polymerase that facilitates promoter clearance , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[59]  Ioan Andricioaei,et al.  Characterizing complex dynamics in the transactivation response element apical loop and motional correlations with the bulge by NMR, molecular dynamics, and mutagenesis. , 2008, Biophysical journal.