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2013 Annual Science Report

Georgia Institute of Technology Reporting  |  SEP 2012 – AUG 2013

Resurrection of an Ancestral Peptidyl Transferase

Project Summary

Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro, and in vivo. The resulting model of the ancestral ribosome incorporates about 20% of the extant 23S rRNA and fragments of four ribosomal proteins. We confirmed that the ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure, and (iii) form native complexes with ribosomal protein fragments. We call the assembled a-RNA and rPeptide fragments the aPTC. We are currently focusing on characterizing the catalytic activity of the a-PTC.

4 Institutions
3 Teams
3 Publications
0 Field Sites
Field Sites

Project Progress

Several groups, including Fox [1], Noller [2], Steinberg [3], Williams [4], Yonath [5] and Gutell and Harvey [6], have proposed molecular-level events in early ribosomal evolution or have determined universally conserved ribosomal components. We previously used a consensus of proposed evolutionary information to design and construct a molecular-level model, in silico, in vitro and in vivo, of an ancestral PTC [7]. The a-PTC incorporates fragments of the 23S rRNA, fragments of ribosomal proteins and divalent cations.

Using computation, in vitro and yeast-three-hybrid experiments, we tested the hypotheses that: (i) a-rRNA adopts LSU-like secondary structure, (ii) a-rRNA in association with Mg2+ adopts LSU-like tertiary interactions, and (iii) a-rRNA forms specific LSU-like complexes with a-rPeptides. Components of the a-PTC were assembled in silico, in vitro, and in vivo and these hypotheses tested by various modeling, footprinting, and binding assays.

During the last year we have worked to determine if a-rRNA in association with a-rPeptides can form a functional a-PTC, as defined by catalytic activity. We have used the fragment assay [8] along with Mass Spectrometry to assay the a-PTC for activity under various conditions. The a-PTC does show weak activity. We are working to optimize the reaction and validated the reaction products. We hope to have sufficient data to publish in the near future..

References
1. Hury J, Nagaswamy U, Larios-Sanz M, Fox GE (2006) Ribosome origins: The relative age of 23S rRNA domains. Orig Life Evol Biosph 36: 421-429.
2. Noller HF (2010) Evolution of protein synthesis from an RNA world. Cold Spring Harb Perspect Biol 7: 7.
3. Bokov K, Steinberg SV (2009) A hierarchical model for evolution of 23S ribosomal RNA. Nature 457: 977-980.
4. Hsiao C, Mohan S, Kalahar BK, Williams LD (2009) Peeling the onion: Ribosomes are ancient molecular fossils. Mol Biol Evol 26: 2415-2425.
5. Krupkin M, Matzov D, Tang H, Metz M, Kalaora R, Belousoff MJ, Zimmerman E, Bashan A, Yonath A (2011) A vestige of a prebiotic bonding machine is functioning within the contemporary ribosome. Philos Trans R Soc Lond B Biol Sci 366: 2972-2978.
6. Mears JA, Cannone JJ, Stagg SM, Gutell RR, Agrawal RK, Harvey SC (2002) Modeling a minimal ribosome based on comparative sequence analysis. J Mol Biol 321: 215-234.
7. Hsiao C, Lenz TK, Peters JK, Fang PY, Schneider DM, Anderson EJ, Preeprem T, Bowman JC, O’Neill EB, Lie L, Athavale SS, Gossett JJ, Trippe C, Murray J, Petrov AS, Wartell RM, Harvey SC, Hud NV, Williams LD (2013) Molecular paleontology: A biochemical model of the ancestral ribosome. Nucleic Acids Res 41: 3373-3385.
8. Anderson RM, Kwon M, Strobel SA (2007) Toward ribosomal RNA catalytic activity in the absence of protein. J Mol Evol 64: 472-483.

Publications

  • Hsiao, C., Lenz, T. K., Peters, J. K., Fang, P-Y., Schneider, D. M., Anderson, E. J., … Dean Williams, L. (2013). Molecular paleontology: a biochemical model of the ancestral ribosome. Nucleic Acids Research, 41(5), 3373–3385. doi:10.1093/nar/gkt023

  • Petrov, A. S., Bernier, C. R., Gulen, B., Waterbury, C. C., Hershkovits, E., Hsiao, C., … Williams, L. D. (2014). Secondary Structures of rRNAs from All Three Domains of Life. PLoS ONE, 9(2), e88222. doi:10.1371/journal.pone.0088222

  • Petrov, A. S., Bernier, C. R., Hershkovits, E., Xue, Y., Waterbury, C. C., Hsiao, C., … Williams, L. D. (2013). Secondary structure and domain architecture of the 23S and 5S rRNAs. Nucleic Acids Research, 41(15), 7522–7535. doi:10.1093/nar/gkt513