16A Mudd Hall
Department of Biology
Johns Hopkins University
3400 North Charles Street
Baltimore, MD 21218-2685
Office: (410) 516-8749
Lab: (410) 516-7316
Fax: (410) 516-5213
B.A., Middlebury College
Ph.D., Stony Brook University
Postdoc, HHMI, University of Colorado, Boulder
Telomerase, the chromosome-lengthening RNA-protein enzyme
Eukaryotic nuclear chromosomes are linear and therefore each have two ends, called telomeres. The enzyme telomerase synthesizes telomeric DNA by reverse transcription of an intrinsic RNA subunit and is required for completion of chromosome replication and genome stability. Telomerase is not normally expressed at appreciable levels in human tissues after early stages of development, and telomeres shorten with aging. However, in 90% of cancers, abnormal overproduction of telomerase permits unlimited cell proliferation. A major aim of our research is to understand the functions and coordination of the telomerase ribonucleoprotein (RNP) enzyme complex subunits as well as how telomerase activity at the ends of chromosomes is regulated.
Our previous research has shown that the budding yeast Saccharomyces telomerase RNA, TLC1, is evolving very rapidly – perhaps even more so than in other evolutionary lineages, and much faster than other essential ncRNAs in yeast. Why is this the case? Some insight into this question has also come from deletion analysis that has shown that two-thirds of S. cerevisiae telomerase RNA is dispensable for function in vivo and that protein-binding sites in the RNA (including essential ones) can even be repositioned to very unnatural locations in TLC1 with retention of function. The sum of the evidence has lead us to conclude that the large yeast telomerase RNA functions as a flexible scaffold or tether for protein subunits, thus defining a new class of RNPs.
Using phylogenetic information gleaned from alignments of Saccharomyces species' TLC1 genes and secondary structure software prediction algorithms, we determined the a model for yeast telomerase RNA (shown to right). The global folding of the RNA and its large size relative to the protein subunits begins to more clearly illustrate how TLC1 tethers, or scaffolds, telomerase protein subunits – note how the proteins decorate the three long arms in the figure above where subunits are drawn roughly to scale. Furthermore, it is satisfying to see how the core of the "Y" shaped structure brings the template nucleotides in close proximity to the binding site for telomerase reverse transcriptase (TERT, also called Est2p in yeast); see Zappulla and Cech, 2004, for the detailed model.
The most convincing evidence to date for the flexible scaffolding model for TLC1 is that the essential Est1p binding site can be repositioned to wildly different locations in the RNA with retention of function in vivo. Three of three positions tested (at nucleotide 220, 450 or 1033) all led to functional telomerase.
We also generated a miniaturized TLC1 where all of the rapidly evolving bulk of the three long arms in TLC1 were deleted and found that all of them can complement a tlc1 mutant in vivo. These "Mini-T" RNAs range in size from 500 to only 384 nts. However, the large size of wild-type TLC1 provides greater fitness in competitive growth experiments where wild-type cells are mixed with a strain expressing a miniaturized telomerase RNA called “Mini-T.” We are now determining the limits of flexible scaffolding of protein subunits by TLC1, as well as other major aspects of telomerase architecture.
The compact Mini-T RNA has afforded us the ability to reconstitute yeast telomerase activity in vitro, which has never been possible using the wild-type 1157-nt TLC1 (apparently because the big RNA misfolds in vitro, getting trapped in “alternate conformer hell”). Using this reconstituted yeast mini-telomerase in parallel with wild-type telomerase from yeast extracts and in-vivo experiments, we are investigating yeast telomerase activity. We have shown that the yeast core enzyme, comprised of the RNA and the telomerase reverse transcriptase subunit (TERT; Est2 in yeast), is intrinsically nonprocessive. However, like the human enzyme, yeast telomerase has been shown to readily add multiple telomeric repeats in vivo to a single telomere. Thus, we aim to understand what is required for significant repeat addition by yeast telomerase. One approach is to add proteins purified from heterologous expression systems into the reconstituted yeast telomerase system to test for functions of individual components. However, in addition to this "bottom-up" reductionist approach, we continue to employ "top-down" strategies by studying telomerase in yeast cells using genetic, molecular and cell biological techniques, as well as purifying telomerase regulatory factors from yeast directly.
Senescence and aging
With our knowledge of telomerase, we are now beginning to explore the relationship between telomerase, senescence and aging. Budding yeast is an excellent model to examine such relationships because of its tractability and because many of the basic factors involved have been identified. We are developing experimental conditions that allow us to ask new questions about the role of telomerase in both senescence and aging. These studies will certainly also teach us a great deal about the molecular biological contributions of these conserved processes to human cancer.
Lebo, K.J., and Zappulla, D.C. (2012) Stiffened yeast telomerase RNA supports RNP function in vitro and in vivo. RNA 18:1666–78.
Zappulla, D.C. (corresponding author), Goodrich, K.J., Arthur, J.R., Gurski, L.A., Denham, E.M., Stellwagen, A.E. and Cech, T.R. (2011) Ku can contribute to telomere lengthening in yeast at multiple positions in the telomerase RNP. RNA. 17:298–311.
Zappulla, D.C.*1, Roberts, J.N.1, Goodrich, K.J., Cech, T.R. and Wuttke, D.S.* (2009) (*corresponding authors, 1first authors) Inhibition of yeast telomerase action by the telomeric ssDNA-binding protein, Cdc13p. Nucleic Acids Research. 37:354–367.
Box, J.A., Bunch, J.T., Zappulla, D.C., Glynn, E.F., and Baumann, P. (2008) A flexible template boundary element in the RNA subunit of fission yeast telomerase. Journal of Biological Chemistry. 283:24224–33.
Zappulla, D.C. and Cech, T.R. (2006) RNA as a flexible scaffold for proteins: yeast telomerase and beyond. Cold Spring Harbor Symposia on Quantitative Biology. 71:217–224.
Zappulla, D.C., Maharaj, A.M., Connelly, J.J., Jockusch, R., and Sternglanz, R. (2006) Rtt107/Esc4 binds silent chromatin and DNA repair proteins using different BRCT motifs. BMC Molecular Biology. 4:40–52.
Zappulla, D.C., Goodrich, K., and Cech, T.R. (2005) A miniature yeast telomerase RNA functions in vivo and reconstitutes activity in vitro. Nature Structural and Molecular Biology. 12:1072–1077.
Zappulla, D.C. and Cech, T.R. (2004) Yeast telomerase RNA: a flexible scaffold for protein subunits. Proceedings of the National Academy of Sciences. 101:10024–10029.
Andrulis, E.D., Zappulla, D.C., Alexieva-Botcheva, K., Evangelista, C. and Sternglanz, R. (2004) One-hybrid screens at the Saccharomyces cerevisiae HMR locus identify novel transcriptional silencing factors. Genetics. 166:631–635.
Zappulla, D.C., Sternglanz, R., and Leatherwood, J. (2002) Control of DNA replication timing by a transcriptional silencer. Current Biology 12:869–875.
Andrulis, E.D.*, Zappulla, D.C.*, Ansari, A.*, Perrod, S., Laiosa, C.V., Gartenberg, M.R., and Sternglanz, R. (* equal contribution). (2002) Esc1, a nuclear periphery protein required for Sir4-based plasmid anchoring and partitioning. Molecular and Cellular Biology. 22:8292–8301.
Andrulis, E.D., Neiman, A.M., Zappulla, D.C., and Sternglanz, R. (1998) Perinuclear localization of chromatin facilitates transcriptional silencing. Nature. 394:592–595.
Tufarelli, C., Fujiwara, Y., Zappulla, D.C., and Neufeld, E.J. (1998) Hair defects and pup loss in mice with targeted deletion of the first cut repeat domain of the Cux/CDP homeoprotein gene. Developmental Biology. 200:69–81.
Yandava, C.N., Zappulla, D.C., Korf, B.R., Neufeld, E.J. (1996) ARMS test for diagnosis of factor VLeiden mutation, a common cause of inherited thrombotic tendency. Journal of Clinical Laboratory Analysis. 10:414-7.