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Project Progress

A central project goal is to develop a timeline for major events in the evolution of the ribosome and relate this history to other key events such as the origins of the genetic code and chirality. The large rRNAs are clearly much larger than their ancestral versions. Recognizing this, the Fox group (1) initially used the extent of connectivity as a marker for relative age. Separately the Williams group used an onion model (2) in which distance from the functional, core, the peptidyl transferase center (PTC), reflected relative age. Subsequently Bokov and Steinberg (3) observed that the tertiary contacts between different regions of the RNA represent potential timing events. Their analysis relied primarily on the A minor motif and one of its chief virtues is that it allowed construction of a crude phylogenetic tree of helix addition, thereby setting a new goal for the timing studies.

In order to pursue this possibility further it was essential to have much better visualization tools in order to readily access relevant information. Primary structure is used for sequence-alignments and therefore contains information on genetic variability. Secondary structure contains local organizational information and describes folding, motifs, functional and evolutionary units (helices and domains). Tertiary structure contains atomic level detail, including conformation and molecular interactions. Primary and secondary structures of rRNAs (ribosomal RNAs) and rProteins (ribosomal proteins) are available for a large number of species. Atomic-resolution three-dimensional (3D) structures of ribosomes are also available for a few species. These 3D structures are information dense, revealing tertiary structural information as well as primary and secondary structural data. Information contained in the small subset of available 3D structures, can be extrapolated to other species. However, this information was not readily accessible.

To address this, in the previous year we began to develop software that can readily and quickly transfers information between 1D (primary structure), 2D (secondary structure), and 3D (tertiary structure) spaces. Additionally, diverse information such as results of footprinting experiments, evolutionary data, and physicochemical properties can also be mapped onto any of the three spaces. The “Ribovision” (http://apollo.chemistry.gatech.edu/RiboVision/) software has now been largely completed and made available to both internal and external users (4). In order to readily compare 2D and 3D information, it was necessary to have a single model. To accomplish this, the actual secondary structure interactions were identified in the 3D structure and converted to a new 2D display which reflected the actual interactions. Although the new structure was very similar to the traditional comparative analysis structure there were some differences. In particular, one of the immediate consequences of this effort was the discovery that several core helices had been overlooked in the traditional 23S rRNA 2D structure. As a result, a new Domain, (Domain 0 on Figure 1) was discovered.

With these new tools in place, we began to examine the nature of additions to the ribosome known as expansion sequences that occur in eukaryotic cells. It was found that expansion sequences have typically been added to the rRNAs without perturbing the conformation and interactions of the pre-existing core, Figure 2. In particular, they have usually been added in one of two ways. They either elongate an existing helical element or are inserted into a pre-existing “trunk” helix. When the latter occurs the two preexisting residues surrounding the “bulge” remain essentially in their original position with the result that the addition doesn’t have any effect on the structure of the trunk helix. This is an important realization, because it represents a timing event—the bulge will be newer than the trunk helix.
It will be possible in the coming year to identify the likely order of addition of many of the local helical elements in the large rRNAs. This has already been done for example in the case of helices 90-92 in the large subunit RNA where helix 92 is likely a newer addition, Figure 3. Thus, a greatly improved phylogenetic model of helix addition in the pre-LUCA time frame may be obtainable in the coming year.

As we develop a greater understanding of the relative age of various regions of the rRNA it will also be possible to understand how the and when the dynamic aspects of the modern machinery were added. The modern ribosome is a highly dynamic molecular machine driven in large part by inter-subunit ratcheting. This motion is propagated along a series of flexible RNA elements which eventually yields forward translation. These dynamic structures can be modelled as a set of rigid fragments connected through flexible joints termed pivot points. Though many of these pivots have been identified previously, most research has been restricted to local interactions of certain functional significance, thus ignoring the global picture of rRNA motion. We have recently identified all large scale points of flexibility associated with the transition from the pre- to the post-ratcheting states in the Thermus thermophilis ribosome. This study has revealed 19 major pivot points, as many as 8 of which have previously not been recognized. Of these, 8 were observed in the 50S subunit and 11 in the 30S subunit, Figure 4. The 30S subunit head swivel, was found to be propagated along a set of at least four linked pivot points. Also, significant flexibility was observed in the B2A and B4 bridge elements, which are the major points of interaction between the two ribosomal subunits. Our study of hinge like RNA elements provides a greater mechanistic insight into the dynamics of the ratcheting process. As we understand the relative age of the various helices the order of addition of the pivot points will be clarified. It remains to be determined if the same set of pivot points are involved in the accommodation step.

Our current timelines for ribosome evolution indicate that the peptidyl transferase center (PTC) and parts of the 50S subunit emerged before any aspects of the 30S subunit. In particular, the PTC is by necessity present at the very beginning of translation as we know it. It was initially observed that the PTC is more than just a catalytic center. It also includes the entrance to an exit tunnel that allows the growing peptide to leave the ribosome. This feature is actually a nanopore and is likely an essential feature of the primitive ribosome because it allowed the newly formed peptide chain to continue to grow without obstruction from what had already been made (5). This raises the question of whether such RNA nanopores are common or rare. A search of the large RNAs revealed 11 additional pores of similar size (6). However, they are dramatically different in that the usual pores are lined with the phosphorous backbone, whereas in the PTC pore several of the nitrogenous bases line the pore making it more promising as a catalytic center, Figure 5.

Why is the PTC pore different? It was previously observed that the PTC was comprised of a sizeable symmetrical region (7). This suggests that the PTC pore was initially formed not by folding of the RNA but rather by dimerization of two smaller RNAS of similar structure. In contrast, the other RNA pores are clearly formed by local folding of the RNA. In many cases, effectively larger RNAs can be formed by hybridization. For example, although it is a distinct RNA, the eukaryotic 5.8S rRNA actually anneals to the large subunit RNA to form effectively one molecule. This occurs because there are three segments of Watson-Crick base pairs holding the two RNAs together. In the case of the hypothetical PTC dimerization there are no such interactions. Instead there are several A-minor interactions and magnesium interactions. Would this be sufficient to hold the two RNAs together? Possibly. In the case of the L1 ligase the functional unit is a dimer. An examination of the structure of the dimer revealed that in fact it was held together by A-minor interactions.

Presentations

Fox GE. “The origins of the translation machinery may provide insight to the emergence of the last universal common ancestor” SPIE Conference: Instruments, Methods, and Missions for Astrobiology XVI San Diego, CA 8/17-8/20, 2014 (forthcoming).

Williams LD. “The origin of the ribosome,” Origins of Life Gordon Conference, Hotel Galvez, Galveston, TX 1/12-1/17, 2014 (forthcoming).

Paci M. & Fox GE. “Centers of motion in the bacterial ribosome,” 5th Annual Graduate Student Symposium, University of Houston, 12/7/2013

Fox GE, Tran Q, Rivas M. & Stepanov V. “Origins and Evolution of the Translation Machinery,” Albany 2013: The 18th Conversation, Albany, NY, June 11-15, 2013.
Abstract: J Biomol Struct Dyn. 31” (sup1), 2-2

Fox GE. “5S ribosomal RNA studies,” Invited seminar, Texas Southern University, March 17, 2013.

Fox GE. “Origins and evolution of the ribosome,” Origin of Life Workshop; Princeton Center for Theoretical Science, Princeton, NJ, 1/20-1/25, 2013.

References
1. 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.
2. Hsiao C, Mohan S, Kalahar BK, Williams LD (2009) Peeling the Onion: Ribosomes Are Ancient Molecular Fossils. Mol Biol Evol 26:2415-2425.
3. Bokov K, Steinberg SV (2009) A Hierarchical Model for Evolution of 23S Ribosomal RNA. Nature 457:977-980.
4. Petrov AS, Bernier CR, Hershkovits E, Xue Y, Waterbury CC, Hsiao C, Stepanov VG, Grover MA, Harvey SC, Hud NV, Wartell RM, Fox GE, & Williams LD. “Secondary Structure and Domain Architecture of the 23S rRNA, Nucl Acids Res. 41: 7512-7521 (2013).
5. Fox GE, Tran Q, & Yonath A. “An Exit Cavity was Crucial to the Polymerase Activity of the Early Ribosome”, Astrobiology, 12: 57-60 (2012).
6. Rivas M, Tran Q, & Fox GE. “Nanometer Scale Pores Similar in Size to the Entrance to
the Ribosomal Exit Cavity are a Common Feature of Large RNAs”, RNA, 19: 1-6 (2013).
7. Agmon I, Bashan A, Zarivach R, Yonath A (2005) Biol Chem 386:833-844