Matching Items (3)
Description
Telomerase enzyme is a truly remarkable enzyme specialized for the addition of short, highly repetitive DNA sequences onto linear eukaryotic chromosome ends. The telomerase enzyme functions as a ribonucleoprotein, minimally composed of the highly conserved catalytic telomerase reverse transcriptase and essential telomerase RNA component containing an internalized short template region within the vastly larger non-coding RNA. Even among closely related groups of species, telomerase RNA is astonishingly divergent in sequence, length, and secondary structure. This massive disparity is highly prohibitive for telomerase RNA identification from previously unexplored groups of species, which is fundamental for secondary structure determination. Combined biochemical enrichment and computational screening methods were employed for the discovery of numerous telomerase RNAs from the poorly characterized echinoderm lineage. This resulted in the revelation that--while closely related to the vertebrate lineage and grossly resembling vertebrate telomerase RNA--the echinoderm telomerase RNA central domain varies extensively in structure and sequence, diverging even within echinoderms amongst sea urchins and brittle stars. Furthermore, the origins of telomerase RNA within the eukaryotic lineage have remained a persistent mystery. The ancient Trypanosoma telomerase RNA was previously identified, however, a functionally verified secondary structure remained elusive. Synthetic Trypanosoma telomerase was generated for molecular dissection of Trypanosoma telomerase RNA revealing two RNA domains functionally equivalent to those found in known telomerase RNAs, yet structurally distinct. This work demonstrates that telomerase RNA is uncommonly divergent in gross architecture, while retaining critical universal elements.
ContributorsPodlevsky, Joshua (Author) / Chen, Julian (Thesis advisor) / Mangone, Marco (Committee member) / Kusumi, Kenro (Committee member) / Wilson-Rawls, Norma (Committee member) / Arizona State University (Publisher)
Created2015
Description
Telomerase is a specialized enzyme that adds telomeric DNA repeats to the chromosome ends to counterbalance the progressive telomere shortening over cell divisions. It has two essential core components, a catalytic telomerase reverse transcriptase protein (TERT), and a telomerase RNA (TR). TERT synthesizes telomeric DNA by reverse transcribing a short template sequence in TR. Unlike TERT, TR is extremely divergent in size, sequence and structure and has only been identified in three evolutionarily distant groups. The lack of knowledge on TR from important model organisms has been a roadblock for vigorous studies on telomerase regulation. To address this issue, a novel in vitro system combining deep-sequencing and bioinformatics search was developed to discover TR from new phylogenetic groups. The system has been validated by the successful identification of TR from echinoderm purple sea urchin Strongylocentrotus purpuratus. The sea urchin TR (spTR) is the first invertebrate TR that has been identified and can serve as a model for understanding how the vertebrate TR evolved with vertebrate-specific traits. By using phylogenetic comparative analysis, the secondary structure of spTR was determined. The spTR secondary structure reveals unique sea urchin specific structure elements as well as homologous structural features shared by TR from other organisms. This study enhanced the understanding of telomerase mechanism and the evolution of telomerase RNP. The system that was used to identity telomerase RNA can be employed for the discovery of other TR as well as the discovery of novel RNA from other RNP complex.
ContributorsLi, Yang (Author) / Chen, Julian Jl (Thesis advisor) / Yan, Hao (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
Telomerase is a reverse transcriptase that is responsible for the addition of telomeric repeats on to the ends of eukaryotic chromosomes. The purple sea urchin, Strongylocentrotus purpuratus, telomerase enzyme is unique in that its telomerase RNA does not contain the ancestrally conserved CR4/5 domain and instead contains the functionally equivalent eCR4/5 domain. Binding between the purple sea urchin TRBD and eCR4/5 domain is currently poorly understood due to eCR4/5's unique structure. In this work the telomerase RNA binding domain, TRBD, of the purple sea urchin telomerase reverse transcriptase, TERT, was fused to maltose binding protein (MBP) using several different short amino acid linkers and purified via amylose column purification. Short amino acid linkers were cloned into the MBP sea urchin TRBD constructs to facilitate better crystallization of the fusion protein. Future work of this project includes testing telomerase RNA binding affinity to the TRBD constructs and determining the crystal structure of the sea urchin TRBD with bound eCR4/5. Elucidating how eCR4/5 binds to the sea urchin TRBD will provide insights into the evolutionary relationship between eCR4/5 and the pseudoknot/template domain of sea urchin telomerase RNA.
ContributorsKing, Robert (Author) / Chen, Julian (Thesis director) / Li, Yang (Committee member) / Barrett, The Honors College (Contributor)
Created2018-05