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2010 Annual Science Report

Georgia Institute of Technology Reporting  |  SEP 2009 – AUG 2010

Molecular Resurrection of the Ancestral Peptidyl Transferase Center

Project Summary

We have resurrected, reconstructed, and are currently reconstituting a model of the a-PTC (ancestral Peptidyl Transferase Center), which we believe to have evolved around 4 billion years ago. The proposed a-PTC contains 644 nucleotides of ancestral ribosomal RNA (a-rRNA), five ancestral ribosomal peptides (a-rPeptides), and inorganic cations. Here we show data of the a-rRNA folding with Mg2+ and a-rPeptides

4 Institutions
3 Teams
9 Publications
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Project Progress

We have previously shown the successful resurrection and reconstruction of a model of the native ancestral rRNA (a-rRNA), which has 644 nucleotides, in silico and in vitro. Our current efforts are to (i) characterize the a-rRNA folding and assembly with five ancestral ribosomal peptides (a-rPeptides) and Mg2+ to form the ancestral Peptidyl Transferase Center (a-PTC) by gel mobility shift assay, (ii) characterize the enzymatic activity of the a-PTC by the “fragment assay” 1,2, and ultimately, (iii) determine the 3D structure of the a-PTC by X-ray diffraction.

The a-rRNA with magnesium ions. Four recurrent rRNA-Mg2+ complexes (Mg2+-microclusters D1- D4) 3 are found to provide the framework for the PTC in the large subunit (LSU) of H. marismortui 4 and T. thermophilus 5. Three Mg2+-microclusters (D1, D2, and D4) along with other highly coordinated Mg2+ ions are considered to be critical for the a-rRNA folding6,7. Here we study the a-rRNA folding with different Mg2+ concentrations by gel mobility shift assay (Figure 1). The result shows that in the absence of Mg2+, the a-rRNA has three different conformations at equilibrium. By increasing the Mg2+ concentration, the bottom band begins to shift (at 100 μM Mg2+) toward the middle band, then to the top band at higher Mg2+ concentrations. At the highest Mg2+ concentration (5 mM, in this study), most of the conformations are shifted to the top, showing the a-rRNA folding is affected by the Mg2+.

The a-rRNA with the five a-rPeptides. Several ribosomal proteins within the 50S ribosome are considered to be essential to maintain the enzymatic activity of the ribosome 8,9. We have identified five ribosomal peptides (a-rPeptides L2, L3, L4, L15, and L22) that we think are ancient and important to a-PTC folding and function. We studied the a-rRNA folding in the absence of Mg2+ ions for each a-rPeptide by gel mobility shift assay. We see the mobility shift on the a-rRNA with each a-rPeptide, except for L2. Band shifting of the a-rRNA with the L2 takes place only in the presence of Mg2+ ions (Figure 2A). This suggests the a-rRNA folding with the a-rPeptide L2 requires Mg2+ ions where we see the L2 tail is intimately complexed with the ribosomal RNA and the Mg2+-microcluster in the three-dimensional structures of the ribosomes (Figure 2B) 3. Overall, the band shifting patterns of the a-rRNA with each a-rPeptide are different than the a-rRNA with Mg2+ ions and the a-rRNA with spermine (data not shown). These results indicate all the a-rPeptides affect the a-rRNA folding.

(1) Schmeing, T. M.; Seila, A. C.; Hansen, J. L.; Freeborn, B.; Soukup, J. K.; Scaringe, S. A.; Strobel, S. A.; Moore, P. B.; Steitz, T. A. Nat Struct Biol. 2002, 9, 225.
(2) Anderson, R. M.; Kwon, M.; Strobel, S. A. J Mol Evol. 2007, 64, 472.
(3) Hsiao, C.; Williams, L. D. Nucleic Acids Res. 2009, 37, 3134.
(4) Ban, N.; Nissen, P.; Hansen, J.; Moore, P. B.; Steitz, T. A. Science 2000, 289, 905.
(5) Selmer, M.; Dunham, C. M.; Murphy, F. V.; Weixlbaumer, A.; Petry, S.; Kelley, A. C.; Weir, J. R.; Ramakrishnan, V. Science 2006, 313, 1935.
(6) Hsiao, C.; Tannenbaum, M.; VanDeusen, H.; Hershkovitz, E.; Perng, G.; Tannenbaum, A.; Williams, L. D. In Nucleic Acid Metal Ion Interactions; Hud, N., Ed.; The Royal Society of Chemistry: London, 2008, p in press.
(7) Hsiao, C.; Mohan, S.; Kalahar, B. K.; Williams, L. D. Mol Biol Evol 2009.
(8) Schulze, H.; Nierhaus, K. H. EMBO J. 1982, 1, 609.
(9) Khaitovich, P.; Mankin, A. S.; Green, R.; Lancaster, L.; Noller, H. F. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 85.

Figure 1. The a-rRNA folding with Mg2+. Shown is the a-rRNA folding with different magnesium concentrations on a 5% acrylamide gel. The Mg2+ concentrations in each lane are: Lane 1 (no Mg2+); Lane 2: 1 μM; Lane 3: 50 μM; Lane 4: 100 μM, Lane 5: 500 μM, Lane 6: 1 mM, Lane 7: 5 mM.

Figure 2A. The L2-Mg2+-rRNA complex and folding. Shown is the mobility shift of the a-RNA with the a-rPeptide L2 and Mg2+ ions on a 5% acrylamide gel. Lane 1 is a-rRNA only. Lane 2 contains 10 μM of Mg2+. Lanes 3 through 6 are the a-rRNA folding with the a-rPeptide L2 at the molar ratio of 1, 5, 20, and 40 in the absence of Mg2+. Lanes 7 through 10 are the a-rRNA folding with the a-rPeptide L2 at the molar ratio of 1, 5, 20, and 40 in the presence of 10 μM Mg2+.

Figure 2B. An ancestral scaffold for the PTC of archaea and bacteria where it shows the a-rPeptide L2 is complexed with the Mg2+ and the a-rRNA. The positions of the a-rRNA, the magnesium ions, and the a-rPeptide have been conserved over billions of years of evolution.