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Project Progress

We are developing systems to deconstruct the extant LSU so that we can study the molecular interactions within it. Noller, ¹ Nierhaus, ² and others showed that extant LSU ribosomal assembly requires only ribosomal proteins L2, L3 and L4, the 23S rRNA, along with inorganic ions such as Mg²⁺, K⁺, and Na⁺. Our model of the a-PTC contains a-rRNA and three a-rPeptides (a-P^L2, a-P^L3, and a-P^L4), with sequences corresponding to the L2, L3, and L4 tails that drill deeply into the LSU. The a-PTC contains six highly coordinated Mg²⁺ ions, which are in the form of three “magnesium microclusters” (Mg²⁺-μc’s). The sequences and molecular interactions of these ancestral components are conserved over vast evolutionary timescales, predating the last common universal ancestor of life. ^{3, 4} Our structural analysis and published biochemical results5 indicated that one of the Mg²⁺-μc’s and coordinated a-rRNA is self-assembling.

Exclusive of our model a-PTC, a Mg²⁺-μc in Domain III (Figure 1) is unique in its autonomy within LSU. Consistent with the Bokov assembly model ⁶, the Domain III rRNA:Mg²⁺-μc complex appears to fold independent of other LSU rRNA, but integrates closely with rPeptide P^L39 in the H. marismortui crystal structure and rPeptide P^L23 in the T. thermophilus and H. marismortui structures. Most of the P^L23 structure in complex with the T.thermophilus Domain III aligns closely with P^L23 in H. marismortui when the rRNA and Mg²⁺-μc’s are superimposed. As coordination of the Mg²⁺-μc in Domain III is contained entirely within this domain, an independent fold and distinct evolutionary origin seems possible, if not likely. However, the “tail” region of P^L23 in T.thermophilus extends into and creates part of the PTC exit tunnel, whereas the “tail” region of H. marismortui’s P^L23 is much shorter. Unlike T.thermophilus, a second protein (rPeptide P^L39) associates with Domain III of H. marismortui and extends into the PTC exit tunnel, possibly replacing P^L23. Our team’s sequence mining and consensus analyses on P^L23 and P^L39 suggest that the “tail” region of P^L23 observed to create part of the PTC exit tunnel in T.thermophilus appears characteristic of all bacteria and absent in archaea and eukaryotes. Additionally, P^L39 is fairly conserved among eukaryotes and archaea, and entirely absent in bacteria.

Though independent of the model a-PTC, Domain III in the large subunit of the extant ribosome is a promising experimental small model system. One of two initial model systems of the Domain III rRNA has been synthesized (in DNA form) and replicated in preparation for transcription. Our goals for this model system are to characterize its ability to fold and assemble as a small model system, which will facilitate an integrated experimental and computational investigation of its interactions and assembly, and later to evaluate its interactions with the model a-PTC and 23S lacking Domain III.

1. Khaitovich, P.; Mankin, A. S.; Green, R.; Lancaster, L.; Noller, H. F., Characterization of functionally active subribosomal particles from Thermus aquaticus. PNAS 1999, 96 (1), 85-90.
2. Schulze, H.; Nierhaus, K. H., Minimal set of ribosomal components for reconstitution of the peptidyltransferase activity. EMBO J. 1982, 1, 609-613.
3. Hsiao, C.; Mohan, S.; Kalahar, B. K.; Williams, L. D., Peeling the onion: Ribosomes are ancient molecular fossils. Molecular Biology and Evolution 2009, 26 (11), 2415-2425.
4. Hsiao, C.; Tannenbaum, M.; VanDeusen, H.; Hershkovitz, E.; Perng, G.; Tannenbaum, A.; Williams, L. D., Complexes of Nucleic Acids with Group I and II Cations. In Nucleic Acid Metal Ion Interactions, Hud, N., Ed. The Royal Society of Chemistry: London, 2008; pp 1-35.
5. Kitahara, K.; Kajiura, A.; Sato, N. S.; Suzuki, T., Functional genetic selection of helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2. Nucleic Acids Research 2007, 35 (12), 4018-4029.
6. Bokov, K.; Steinberg, S. V., A hierarchical model for evolution of 23S ribosomal RNA. Nature 2009, 457 (7232), 977-980.