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.
