For those interested in graduate study: see the Cellular, Molecular and Developmental Biology Graduate Program website.

 

My postdoctoral research: 

Reconstitution of yeast telomerase activity via modeling and miniaturizing yeast telomerase RNA 

During the beginning of my postdoc with Tom Cech (HHMI, CU Boulder), I derived a secondary structure model for the 1157-nt Saccharomyces cerevisiae telomerase RNA, TLC1, based on phylogenetics and RNA folding software predictions (Zappulla and Cech, 2004). Using this model as a guide, I have since designed yeast telomerase RNAs that are as little as one-third the size of wild type, even smaller than human telomerase RNA (451 nt), yet retain essential function in vivo (Zappulla et al., 2005). Furthermore, these miniaturized (Mini-T) telomerase RNAs have allowed me to reconstitute yeast telomerase activity in vitro, something that has been  impossible for the past 10 years because of the massive size, and related RNA folding problems, of wild-type TLC1. 

The identification of reconstituted yeast telomerase activity now allows telomerase and telomere protein functions to be individually assessed for the first time. In collaboration with Debbie Wuttke's lab, we have already deduced that purified telomeric DNA end-binding protein Cdc13p inhibits reconstituted telomerase activity (manuscript in preparation), suggesting a pivotal role for this protein in regulating telomerase access in vivo.  The telomerase subunit Est1p, which binds Cdc13p and is also essential for telomerase function in vivo, is now also being introduced into the system, as well as Cdc13p binding partners Stn1p and Ten1p and the Pif1p helicase (provided by Virginia Zakian's lab) that is required for yeast telomerase dissociation from DNA. I hope to reconstitute the system that exists in vivo by adding in these protein factors and learn the mechanistic features of yeast telomerase, such as the requirements for telomere repeat addition in vitro. 

FIGURE: Yeast telomerase RNA secondary structure and its reduction to create "Mini-T." The model on the left is my model for the wild-type 1157-nt TLC1 RNA and I designed the 500-nt Mini-T RNA by assembling predicted functional domains (boxed), discarding more than half of the RNA while retaining function in vivo and allowing reconstituted activity for the first time in vitro.

Yeast telomerase RNA as a flexible scaffold

In addition to demonstrating that >70% of TLC1 RNA is dispensable for function in vivo and that the bulk of the sequence is also evolving rapidly (even among species of Saccharomyces), I have shown that the essential Est1 protein binding site in TLC1 RNA can be dramatically repositioned in the RNA with retention of function (Zappulla and Cech, 2004). Together, these results provide evidence that TLC1 RNA serves as a flexible scaffold for proteins in this RNP enzyme. Thus, the telomerase RNP is apparently quite different from, for example, the ribosome and is potentially better fit to a "beads" (proteins) on a "string" (RNA) model for its global architecture (for review, see Zappulla and Cech, 2006). 

 

 

FIGURE: Schematic of yeast telomerase RNA TLC1 secondary structure model bound to proteins (drawn approximately to scale). 
TLC1 RNA nucleotides (circles) are shown in rainbow spectrum colors (red ––> violet) correlating with the predicted accuracy of their modeling by Mfold RNA secondary structure prediction software (red = best-determined; Zuker and Jacobson, 1998). Telomerase RNA-binding proteins Est2p (TERT), Est1p, Ku heterodimer and Sm7 heteroheptamer are drawn to scale with the RNA. 

Of course, the discrete catalytic center of the RNP, where TLC1 binds the protein reverse transcriptase subunit (Est2p) and telomeric DNA, is almost certainly structurally ordered for coordinating reverse transcription. Scaffold roles for RNAs may be quite prevalent in biology, applying to other RNPs in addition to yeast telomerase. Examples of other RNA scaffolds may include Xist RNA, certain viral RNAs (e.g. some IRES's), tmRNA, pre-mRNA/protein complexes, and others (Zappulla and Cech, 2006).  

 

I am now collaborating with Peter Baumann (Stowers Institute for Medical Research) on the fission yeast telomerase RNA secondary structure. This RNA, which is evolutionarily very distant, also has many hallmarks of being a flexible scaffold: it is very large (1213 nts), is evolving very rapidly, and preliminary modeling shows that it has long quasi-helical arms, much like TLC1. The flexibility even extends into the catalytic action of this RNP, as we have already discovered (Box et al, 2008).

My PhD research: 

Transcriptional silencing, DNA replication and the nuclear periphery
While pursuing my PhD in the laboratory of Rolf Sternglanz, at Stony Brook University, I studied transcriptional silencing in yeast, as well as its relationship to timing of DNA replication origin firing. I demonstrated that an early origin of DNA replication in a euchromatic region of the genome could be reprogrammed to fire late by introducing a transcriptional silencing element, the HMR-E silencer, nearby (Zappulla et al., 2002). Tethering an early origin to the nuclear periphery, where silenced late-replicating chromatin resides, was insufficient to delay replication timing in the absence of forming silent chromatin at the locus, suggesting that perinuclear localization per se is insufficient for late replication timing. Strikingly, simply targeting of the silencing protein Sir4p to the early origin reprogrammed it to fire late while also repressing transcription of a nearby gene, further arguing that chromatin state was important for origin firing.  

I also investigated the biological function of Esc1 and Esc4/Rtt107 proteins, both of which were identified in the Sternglanz lab as having the ability to recruit the Sir2/3/4 silencing protein complex when artificially tethered to a chromosome (using the Gal4p system). I discovered that Esc1p localizes to the nuclear periphery and is required for membrane protein targeted silencing (see Andrulis et al., 1998 for identification of membrane protein silencing), and together we found that Esc1p binds Sir4p and is required for Sir4p-based plasmid partitioining and anchoring in the nucleus (Andrulis/Zappulla/Ansari et al., 2002 -- N.B. co-first authorship).  Esc1p appears to serve nuclear lamina-type functions in budding yeast. As for Esc4p, I did phylogenetic work to demonstrate that this protein has six BRCT motifs (not just four), binds Slx4p with the N-terminal four BRCTs, is important for DNA repair and that it also binds to Sir3p (using C-terminal two BRCTs), explaining why it causes targeted silencing (Zappulla et al., 2006). 

 

Grants:

National Institutes of Health “Pathway to Independence” K99/R00 grant awardee

 

Publications:

Zappulla, D.C.*, Arthur, J.R., Goodrich, K., Gurski, L.A., Cech, T.R., and Stellwagen, A.E.*  Positional requirements of Ku binding to telomerase for extension of native and broken chromosome ends. (*co-corresponding author) (In preparation)

Zappulla, D.C.*, Roberts, J.N., Goodrich, K., Cech, T.R. and Wuttke, D.S.* Inhibition of yeast telomerase action by the telomeric ssDNA-binding protein, Cdc13p. (*co-corresponding author) (In press)

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. J. of Biological Chemistry 283(35):24224-33

Zappulla, D.C. and Cech, T.R.  RNA as a flexible scaffold for proteins: yeast telomerase and beyond. (2006) Cold Spring Harbor Symposia on Quantitative Biology (Symposium 71: Regulatory RNAs), 71:217–224.

Zappulla, D.C., Maharaj, A.M., Connelly, J., Jockusch, R., and Sternglanz, R.  Rtt107/Esc4 binds silent chromatin and DNA repair proteins using different BRCT motifs.  (2006)  BMC Molecular Biology, 4: 40-72.

Zappulla, D.C., Goodrich, K., and Cech, T.R. A miniature yeast telomerase RNA functions in vivo and reconstitutes activity in vitro. (2005) Nature Structural and Molecular Biology 12(12):1072-1077. 101(27):10024-10029. 21(19): 6606-6614.

Zappulla, D.C. and Cech, T.R.  Yeast telomerase RNA: a flexible scaffold for protein subunits. (2004) Proceedings of the National Academy of Sciences 101(27): 10024-10029.

Andrulis, E.D., Zappulla, D.C., Alexieva-Botcheva, K., Evangelista, C. and Sternglanz, R. (2004) Targeted silencing screens at HMR 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(23): 8292-8301.

Xie, W., Gai, X., Zhu, Y., Zappulla, D.C., Sternglanz, R., and Voytas, D. (2001) Targeting of the yeast Ty5 retrotransposon to silent chromatin is mediated by interactions between integrase and Sir4p. Molecular and Cellular Biology

Andrulis, E.D., Neiman, A.M., Zappulla, D.C., and Sternglanz, R. (1998) Perinuclear localization of chromatin facilitates transcriptional silencing. Nature 394: 592-595.

Ong B.C., Zimmerman A.A., Zappulla D.C., Neufeld E.J., Burrows, F.A. (1998) Prevalence of factor VLeiden in a population of patients with congenital heart disease. Canadian Journal of Anesthesia 45(12): 1176-1180.

Tufarelli C., Fujiwara Y., Zappulla D.C., 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(1): 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(6): 414-417.
 

 

Other links:

Johns Hopkins University

Local seminars: Department of Biology, Johns Hopkins Scientific Calendar (JHMI), Carnegie Institute Seminars (on the JHU Campus),

Yeast-related: Saccharomyces Genome Database (SGD), 

NIH Pathway to Independence K99/R00 Award Website

Tom Cech's website at University of Colorado at Boulder in Department of Chemistry and Biochemistry

    President and Investigator, Howard Hughes Medical Insititute (HHMI)

My PhD thesis advisor, Rolf Sternglanz, at Stony Brook University

Ellis J. Neufeld's laboratory at Harvard Medical School (Children's Hospital) where I was a technician for two years after graduating from Middlebury College with a B.A. in Biology in 1995. 

Protocols from: the Gottschling lab, Botstein lab,

Mfold RNA folding prediction server  

RNAalifold RNA sequence alignment-based folding server

Other places where I have studied: 

    Middlebury College, Middlebury, Vermont

    Sea Education Assocation, Woods Hole, Massachusetts.  

    Rocky Mountain Biological Laboratory, Gothic, Colorado.

    The Center for Northern Studies, Wolcott, Vermont. 

Extracurricular 

Ultimate frisbee, golf, squash, sailing