Resurrection of an Ancestral Peptidyl Transferase
The ribosome is a molecular machine responsible for synthesis of coded protein in all living systems. The ribosome is made of a small subunit (SSU) that decodes the messenger RNA and a large subunit (LSU) that catalyzes peptide bond formation.
Some of the RNA and protein components of the ribosome are highly conserved everywhere in extant life, and are considered to be very ancient (1-6). The peptidyl transferase center (PTC), in particular, is thought to be even older than coded protein (7-10). In this view, the PTC emerged from some type of ‘RNA World’ (11-15) participating in the transformation to current biology in which information is transduced from nucleic acids to proteins. In essentially any reasonable model of origin of life and evolution of the ribosome, the PTC appears as one of our most direct biochemical links to the distant evolutionary past and to early life. Understanding the PTC is key to understanding ancient biology.
Here we create and test both in silico and in vitro models of an ancestral PTC. Our models incorporate recent proposals (7, 8, 10) that point to portions of Domains V, IV and II of the modern 23S rRNA as elements inherited from an ancestral PTC (Figure 1A). Our in silico and in vitro models contain a significantly reduced 23S rRNA (called a-rRNA-g, Figure 1B), retraining the rRNA that forms and surrounds the PTC. Using the 3D structure of the T. thermophilus LSU (16) as a template, the 23S rRNA was ‘shaved’ to a rough sphere of around 30 Å in radius (8), centered on the site of peptidyl transfer. Cuts were performed selectively in A-form helical regions, which were capped with stable stem-loops. The result is a single RNA polymer, which is predicted by several proposals of PTC evolution (summarized in Figure 1A), and contains around 20% of the 23S rRNA linked by 11 stem-loops. As illustrated in Figure 1, a-rRNA-g contains rRNA segments that are (i) are universally conserved in all organisms and organelles (17), (ii) extremely ancient (6, 7) (iii) tightly networked (6), (iv) highly coordinated by magnesium ions (18), and (v) in the central shells of the ribosomal ‘onion’ (8).
To complete the in silico and in vitro models of the ancestral PTC (a-PTC-g in silico and a-PTC-g in vitro), we have combined a-rRNA-g (above) with peptides derived from the ribosomal proteins. Ribosomal proteins do not directly engage in catalysis (19, 20) but do interact extensively with the rRNA that lines the catalytic center. The peptide components of a-PTC-g are the ‘tail’ segments of ribosomal proteins. Ribosomal proteins L2, L3, L4, L15 and L22 were shaved at the boundary of the sphere defined by a-rRNA-g. The shaving process did not disrupt protein secondary or tertiary interactions: none of the ribosomal protein tails within a-PTC-g are globular structures in the intact LSU. The peptide components of a-PTC-g are called a-rPeptide-L2, a-rPeptide-L3, a-rPeptide-L4, a-rPeptide-L15, and a-rPeptide-L22 (Table 1).
Here, using computation and solution experiments, we test hypotheses that: (i) a-rRNA-g can assume the canonical LSU secondary structure as shown in Figure 1B, (ii) a-rRNA-g in association with magnesium can assume LSU-like 3D structure and tertiary interactions as shown in Figure 1C, (iii) a-rRNA-g forms LSU-like complexes with shaved ribosomal protein segments as shown in Figure 1C, and (iv) a-rRNA-g in association with these ribosomal protein segments form a functional a-PTC-g, which is defined by peptidyl transferase catalytic activity. Here a-rPTC-g was assembled in vitro and in silico, and these hypotheses are tested by various modeling, footprinting, binding and catalytic assays.
Our current models of a-PTC- are steps in an iterative process that will allow us to test hypotheses related to evolution of translation, including models of RNA folding, assembly, and catalysis. The ultimate goal is to develop biochemically functional ancestral ribosomes. Our current models contain about 500 nucleotides of RNA that thought to predate the coding functionality but not the peptidyl transferase activity of the ribosome. Fragments of ancestral RNA are joined together by stem-loops to form a single RNA polymer called a-rRNA-γ. To assemble a-PTC-, magnesium ions and six peptides are annealed with a-rRNA-. The six peptides in a-PTC-are non-globular fragments of ribosomal proteins representing Fox’s molecular fossils of non-coded precursors of modern proteins (6).
Our fundamental prediction here is that a-PTC-γ in vitro will form the same basic structure, with the same RNA conformation, secondary and tertiary interactions, and the same RNA-protein and RNA-ion interactions, as a-PTC-γ in silico. The 3D structure of a-PTC-γ in silico (Figure 1C) in turn is based on the 3D structure of the extant ribosome (1jj2, T. Thermophilus). The interior of the a-PTC-γ in silico model is very similar to the corresponding region of the extant ribosome. The surface region of a-PTC-γ in silico, in particular the stitching stem-loops, obviously diverge from the extant ribosome.
We synthesized the macromolecular components of a-PTC-γ. The gene for a-rRNA- was assembled by recursive PCR and used as a template in in vitro transcription. The six ancestral peptides were made by solid phase synthesis. The structure and assembly of a-PTC-γ in vitro was probed by a variety of well-established biophysical methods. The results suggest that a-PTC-γ in silico is an excellent predictor of the folding and assembly of a-PTC-γ in vitro. This conclusion is based on (i) RNase H digestion and SHAPE profiles, which support the predicted secondary structure of a-rRNA-γ, (ii) the magnesium-dependence of the SHAPE profile and the magnesium-dependence of gel mobility, which indicate specific magnesium interactions within a-rRNA-, and magnesium-induced formation of tertiary interactions and (iii) specific binding of ancestral peptides with a-rRNA-, which is consistent with folding and assembly of a-rRNA- and the peptides to form a-PTC-.
The combined results here indicated that the ribosome and its components are highly robust in folding and assembly. We have shaved around 2500 nucleotides from the 23S rRNA and the vast majority of amino acids from the protein components, excising the globular domains in toto. Yet, the remaining rRNA and peptides retain the ability to fold and specifically assemble. It is particularly striking that the ribosomal protein tails, lacking a-helices, b-sheets, and globular domains, retain the ability to interact specifically with a-rRNA-. This robustness is consistent with the premise that the ribosome is an ancient assembly that evolved in a primitive biological environment, and has survived billions of years of evolution, growing in size and complexity, without major changes in core structure or function. Although the modern LSU has the appearance of a massive monolithic assembly (19), the results here, and elsewhere (21), indicate that it is in fact composed of small RNA and protein elements that retain ancient abilities to fold and assembly independently.
1. Woese CR (2000) Interpreting the Universal Phylogenetic Tree. Proc Natl Acad Sci U S A 97:8392-8396.
2. Woese CR (2001) Translation: In Retrospect and Prospect. RNA 7:1055-1067.
3. Fournier GP, Neumann JE, Gogarten JP (2010) Inferring the Ancient History of the Translation Machinery and Genetic Code Via Recapitulation of Ribosomal Subunit Assembly Orders. PLoS One 5:e9437.
4. Williams D, Fournier GP, Lapierre P, Swithers KS, Green AG, Andam CP, Gogarten JP (2011) A Rooted Net of Life. Biol Direct 6:45.
5. Wolf YI, Koonin EV (2007) On the Origin of the Translation System and the Genetic Code in the RNA World by Means of Natural Selection, Exaptation, and Subfunctionalization. Biol Direct 2:14.
6. Fox GE (2010) Origin and Evolution of the Ribosome. Cold Spring Harb Perspect Biol 2:a003483.
7. Bokov K, Steinberg SV (2009) A Hierarchical Model for Evolution of 23S Ribosomal RNA. Nature 457:977-980.
8. Hsiao C, Mohan S, Kalahar BK, Williams LD (2009) Peeling the Onion: Ribosomes Are Ancient Molecular Fossils. Mol Biol Evol 26:2415-2425.
9. Belousoff MJ, Davidovich C, Zimmerman E, Caspi Y, Wekselman I, Rozenszajn L, Shapira T, Sade-Falk O, Taha L, Bashan A, Weiss MS, Yonath A (2010) Ancient Machinery Embedded in the Contemporary Ribosome. Biochem Soc Trans 38:422-427.
10. 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.
11. Rich A (1962) On the Problems of Evolution and Biochemical Information Transfer. Horizons in Biochemistry, eds Kasha M & Pullman B (New York: Academic), pp 103–126.
12. Woese CR (1967) The Genetic Code: The Molecular Basis for Genetic Expression (Harper & Row, N.Y.).
13. Gilbert W (1986) Origin of Life: The RNA World. Nature 319:618-618.
14. Crick FH (1968) The Origin of the Genetic Code. J Mol Biol 38:367-379.
15. Orgel LE (1968) Evolution of the Genetic Apparatus. J Mol Biol 38:381-393.
16. Selmer M, Dunham CM, Murphy FV, Weixlbaumer A, Petry S, Kelley AC, Weir JR, Ramakrishnan V (2006) Structure of the 70S Ribosome Complexed with mRNA and tRNA. Science 313:1935-1942.
17. 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.
18. Hsiao C, Williams LD (2009) A Recurrent Magnesium-Binding Motif Provides a Framework for the Ribosomal Peptidyl Transferase Center. Nucleic Acids Res 37:3134-3142.
19. Ban N, Nissen P, Hansen J, Moore PB, Steitz TA (2000) The Complete Atomic Structure of the Large Ribosomal Subunit at 2.4 Å Resolution. Science 289:905-920.
20. Noller HF, Hoffarth V, Zimniak L (1992) Unusual Resistance of Peptidyl Transferase to Protein Extraction Procedures. Science 256:1416-1419.
21. Athavale SS, Gossett JJ, Hsiao C, Bowman JC, O’Neill E, E H, Preeprema T, Hud NV, Wartell R, Williams LD (2012) Domain III of the T. Thermophilus 23S rRNA Folds Independently to a near-Native State. RNA:in press.