By replenishing the DNA at the 3' end of each chromosome, the enzyme telomerase is one key to cellular immortality. Functionally, telomerase is a remarkable nucleic acid polymerase that uses its integral RNA subunit as the template for the synthesis of the DNA located at the 3' end of eukaryotic chromosomes. The product of telomerase, telomeric DNA, is a single strand of highly repetitive G-rich DNA that is part of the specialized nucleoprotein complex called the telomere. Telomere maintenance is essential because the telomere guards the chromosome ends from mistaken recognition by the DNA repair machinery and consequent nucleolytic degradation. Several features of the catalytic mechanism of telomerase remain mysterious, and the research conducted in this project should illuminate the structural basis for telomerase function. Specifically, using Tetrahymena thermophila telomerase RNA (tTR) as a model, the investigator will elucidate the role of telomerase RNA through the use of a variety of high resolution biochemical techniques. In the first specific aim the structure of tTR will be studied both in solution and in the catalytically active complex using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). In the second aim important functional groups throughout tTR will be identified using nucleotide analog interference mapping, a high throughput approach that allows simultaneous interrogation of functional groups of each nucleotide of an RNA. In the final aim, site-specific mutants of tTR, designed based on data from aims 1 and 2, will be used to corroborate those findings.
This research will be conducted by students, including members of groups underrepresented in science. Furthermore, students who participate in a minority summer research program are expected to also participate in this project. Funding for the project will provide students with supplies and research stipends. Furthermore, telomerase is an important enzyme that affects many biological phenomena, including that of aging, and thus its understanding can benefit society in many ways.
". Telomerase is an unusual cellular machine, or enzyme, that contains both proteins and an RNA. Biologically, telomerase activity is essential for continual proliferation of the vast majority of eukaryotic cells including human cells because it ensure the faithful replication of the terminal fragments of chromosome DNA. When we initiated our studies, there was very little data reporting on the structure of telomerase RNA both within the telomerase complex. This prevented proper models of telomerase activity to be developed. In our studies, we explored the structure of both human telomerase RNA and the model telomerase RNA from the ciliate Tetrahymena thermophile. Specifically, we sought to establish the structure of telomerase RNA in the telomerase complex and at discrete steps of the catalytic cycle. We first did this be investigating the structure of Tetrahymena thermophila telomerase RNA using a technique that allowed us to investigate the conformation and dynamics of individual nucleotides. The result are helping us develop the highest resolution picture of telomerase RNA to date. We combined high resolution biochemical assays, biophysical data, and computational approaches to generate all atom models of Tetrahymena telomerase RNA both free of protein in solution and bound to the telomerase catalytic subunit. We discovered that Tetrahymena telomerase RNA undergoes a dramatic conformation change during the assembly process. This model for the conformational changes of telomerase RNA were confirmed using a series of biochemical and structure probing assays using carefully designed single point mutations. In our studies with human telomerase RNA, we found that it to undergoes a structural rearrangement during assembly. In addition, we characterized several disease causing mutations of human telomerase RNA and are developing a better understanding of how these mutations cause the misfolding of human telomerase RNA and therefore cause telomerase dysfunction and the associated disease phenotypes. Based on the fact that both RNAs undergo substantial conformational change during assemble, we hypothesize that these conformational changes protect specific nucleotides in templating domain of telomerase RNA from hydrolysis. These nucleotides code for the telomeric DNA found at the ends of chromosomes and damage from hydrolysis would lead to loss of fidelity in telomeric DNA sequences, which is known to cause cell death. These protein free structures of telomerase RNA had not been previously characterized owing to the lack of appropriate techniques. Characterizing this conformational change highlights one of the major intellectual merit of our studies. The broader impacts of the proposal are highlighted by the training the funding provided, the reporting of the discoveries to the scientific community, and the collaborations the funding fostered. During the funding period, four graduate students earned their PhD while working on aspects of the studies. In addition, one to two undergraduate students per year participated on the research projects and these students have all matriculated to post graduate studies either in professional MD and PharmD programs or in PhD granting programs.
