NAI
2010 Annual Science Report
Georgia Institute of Technology
Reporting | SEP 2009 – AUG 2010
Reverse-Evolution of an RNA-based RNA Polymerase
Project Summary
The RNA World Hypothesis suggests an RNA molecule is capable both of encoding information and replicating it. In essence, the RNA World Hypothesis predicts an RNA polymerase ribozyme. Since there are no extant RNA-based RNA polymerases, we must instead search the evolutionary fossil record for hints. Our primary goal is to test the hypothesis of Poole that the Small Subunit (SSU) of the ribosome may have evolved from an RNA-dependent RNA polymerase ribozyme.1 We will test the plausibility of an RNA polymerase origin of the SSU by using in vitro reverse evolution; If we can reverse-evolve the SSU into an RNA polymerase, we can demonstrate the a possible evolutionary pathway between a putative primordial ribozyme polymerase and modern ribosomes.
Project Progress
We have pursued two lines of inquiry. The first involves an in vitro evolved RNA ligase ribozyme, whose structure was determined in our laboratory. This ribozyme mimics the chemical step required of an RNA-dependent RNA polymerase. We have “reverse-evolved” the ribozyme to utilize 2’,3’-cyclic phosphate substrates and have obtained crystals and a dataset of the new ribozyme for purposes of structural determination. Obtaining the structure has proven to be difficult (which is not completely surprising, given that the first ligase (1, 2) required six years of effort and the invention of a new technique.
The second line of inquiry involves applying the same technique of in vitro “reverse evolution” to the ribosome small subunit. Our initial experiments have not yet demonstrated that this is feasible. Hence we are adopting a second approach, based upon a hypothesis recently proposed by Harry Noller involving “duplicator” tRNA precursors (3).
1. Robertson MP & Ellington AD (2000) Design and optimization of effector-activated ribozyme ligases. (Translated from eng) Nucleic Acids Res. 28(8):1751-1759 (in eng).
2. Robertson MP & Scott WG (2007) The structural basis of ribozyme-catalyzed RNA assembly. (Translated from eng) Science 315(5818):1549-1553 (in eng).
3. Noller HF (2010) Evolution of Protein Synthesis from an RNA World. (Translated from Eng) Cold Spring Harb Perspect Biol 7:7 (in Eng).
Publications
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Noller, H. F. (2010). Evolution of Protein Synthesis from an RNA World. Cold Spring Harbor Perspectives in Biology, 4(4), a003681–a003681. doi:10.1101/cshperspect.a003681
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Robertson, M. P., & Scott, W. G. (2007). The Structural Basis of Ribozyme-Catalyzed RNA Assembly. Science, 315(5818), 1549–1553. doi:10.1126/science.1136231
- Robertson, M.P. & Ellington, A.D. (2000). Design and optimization of effector-activated ribozyme ligases. Nucleic Acids Res, 28(8): 1751-9.
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PROJECT INVESTIGATORS:
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PROJECT MEMBERS:
Michael Robertson
Postdoc
Eric Schultz
Graduate Student
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RELATED OBJECTIVES:
Objective 3.2
Origins and evolution of functional biomolecules
