2010 Annual Science Report
Georgia Institute of Technology Reporting | SEP 2009 – AUG 2010
Molecular Resurrection of the Ancestral Peptidyl Transferase Center
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
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.
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PROJECT INVESTIGATORS:Loren Williams
Project InvestigatorChristine Dunham
PROJECT MEMBERS:Chiaolong Hsiao
RELATED OBJECTIVES:Objective 3.2
Origins and evolution of functional biomolecules