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. 2023 Mar;29(3):263-272.
doi: 10.1261/rna.079417.122. Epub 2023 Jan 5.

How to build a protoribosome: structural insights from the first protoribosome constructs that have proven to be catalytically active

Mario Rivas  1 , George E Fox  2
Affiliations

How to build a protoribosome: structural insights from the first protoribosome constructs that have proven to be catalytically active

Mario Rivas et al. RNA. 2023 Mar.

Abstract

The modern ribosome catalyzes all coded protein synthesis in extant organisms. It is likely that its core structure is a direct descendant from the ribosome present in the last common ancestor (LCA). Hence, its earliest origins likely predate the LCA and therefore date further back in time. Of special interest is the pseudosymmetrical region (SymR) that lies deep within the large subunit (LSU) where the peptidyl transfer reaction takes place. It was previously proposed that two RNA oligomers, representing the P- and A-regions of extant ribosomes dimerized to create a pore-like structure, which hosted the necessary properties that facilitate peptide bond formation. However, recent experimental studies show that this may not be the case. Instead, several RNA constructs derived exclusively from the P-region were shown to form a homodimer capable of peptide bond synthesis. Of special interest will be the origin issues because the homodimer would have allowed a pre-LCA ribosome that was significantly smaller than previously proposed. For the A-region, the immediate issue will likely be its origin and whether it enhances ribosome performance. Here, we reanalyze the RNA/RNA interaction regions that most likely lead to SymR formation in light of these recent findings. Further, it has been suggested that the ability of these RNA constructs to dimerize and enhance peptide bond formation is sequence-dependent. We have analyzed the implications of sequence variations as parts of functional and nonfunctional constructs.

Keywords: RNA World; SymR; origin of life; protoribosome.

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Figures

FIGURE 1.
FIGURE 1.
Secondary structure representation of the pseudosymmetrical region (SymR) and the tt_A1P1 construct of the protoribosome plotted over the Thermus thermophilus secondary structure of Domain V from the LSU. Secondary structure was derived from crystallographic structure data (Petrov et al. 2013, 2014a). Filled circles mark ribonucleotides that comprise the six regions of RNA/RNA interactions that have been proposed to be the driving force behind the hybridization of the P- and A-regions (Supplemental Table S1; Rivas and Fox 2020). (A) Secondary structure representation for the original proposal of the SymR (Agmon et al. 2005). P-region comprises 89 ribonucleotides and A-region is equally made from 89 for a total of 178. No proposal was originally made for the possible sequences that should be used at the tips of H75, H89, and H91 nor for the connection at the end of H80 and the beginning of H74. Hence, they are represented by gray arrows. (B) Secondary structure representation of the protoribosome construct tt_A1P1 (Bose et al. 2021, 2022). It starts with G2058 (red font) instead of A2058 due to experimental protocol requirements. P-region comprises 71 ribonucleotides while A-region is made from 64 for a total of 135. Sequence 5′-CUUCGG-3′ was used to close helixes 74, 89, and 90 (red font). C2463 and A2518 in proposed region 2 were left out in the tt_A1P1 construct (Supplemental Table S1).
FIGURE 2.
FIGURE 2.
Proposed pore-like structure of the pseudosymmetrical region (Agmon et al. 2005; Agmon 2009; Rivas and Fox 2020) and the tt_P1 construct (Bose et al. 2021, 2022). Both were modeled over the crystallographic structure of T. thermophilus (PDB ID 4WPO, Lin et al. 2015). (A) P- and A-regions (green and blue, respectively) hybridize together to form a heterodimer with a pore-like structure that enhances peptide bond formation, as it occurs in the extant ribosomal region called the peptidyl transferase center or PTC. (B) Space-filled structural model of two tt_P1 constructs highlighted in two tonalities of green, hypothetically dimerized into a similar pore-like structure that is also able to enhance peptide bond formation. Structure was obtained by structural superposition of a second tt_P1 element over the original A-region RNA as described in the Materials and Methods section.
FIGURE 3.
FIGURE 3.
Proposed secondary structures of the P-region constructs from Thermus thermophilus (tt_P1, tt_P1c) and Escherichia coli (ec_P1c). Structures were drawn based on sequences reported by Bose et al. (2022) and secondary structure predictions (Supplemental Fig. S5–S10). Each structure is depicted as a pair of P-region and P′-region, meaning two independent but identical entities, which are necessary to form a pore-like structure capable of catalyzing peptide bond formation. The T. thermophilus ribosome numbering system is used. Initial guanine is highlighted using red font. The 5′ and 3′ ends of each RNA oligomer are labeled. 5′-CUUCGG-3′ and 5′-GUGA-3′ sequences at the tips of helices 74 and 89 are also highlighted by using red font. Proposed regions of long-range RNA/RNA interactions are surrounded by blue-dotted boxes and connected by blue-dotted lines. (A) tt_P1 construct with 5′-CUUCGG-3′ sequence at the tip of both H74 and H89. This construct is made from 71 ribonucleotides. It has been reported as able to dimerize and be catalytically active. (B) tt_P1c construct with 5′-CUUCGG-3′ sequence at the tip of H74 and the 5′-GUGA-3′ sequence at the tip of H89. It is made from 67 ribonucleotides. It is also reported as able to dimerize and be catalytically active. (C) ec_P1c construct with 5′-CUUCGG-3′ sequence at the tip of H74 and the 5′-GUGA-3′ sequence at the tip of H89. It is also made from 67 ribonucleotides. Reported as neither able to dimerize nor catalytically active. Seven sequence differences are found. The initial G2058 being restored to the conserved A2058. In H74, the pair G2070:C2441 was replaced by A2070:U2441, and the pair G2072:U2438 was replaced by C2072:G2438. In H89, the pair C2461:G2489 was replaced by A2461:U2489. Change in the base pair pattern of H74, as the consequence of the change in pair G2070:C2441 while replaced by A2070:U2441, seems to be negatively affecting this construct's capacity to dimerize and consequently to show catalytic activity.
Mario Rivas
Mario Rivas
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