Simple peptides derived from the ribosomal core potentiate RNA polymerase ribozyme function - PubMed (original) (raw)
Simple peptides derived from the ribosomal core potentiate RNA polymerase ribozyme function
Shunsuke Tagami et al. Nat Chem. 2017 Apr.
Abstract
The emergence of functional interactions between nucleic acids and polypeptides was a key transition in the origin of life and remains at the heart of all biology. However, how and why simple non-coded peptides could have become critical for RNA function is unclear. Here, we show that putative ancient peptide segments from the cores of both ribosomal subunits enhance RNA polymerase ribozyme (RPR) function, as do derived homopolymeric peptides comprising lysine or the non-proteinogenic lysine analogues ornithine or, to a lesser extent, diaminobutyric acid, irrespective of chirality or chiral purity. Lysine decapeptides enhance RPR function by promoting holoenzyme assembly through primer-template docking, accelerate RPR evolution, and allow RPR-catalysed RNA synthesis at near physiological (≥1 mM) Mg2+ concentrations, enabling templated RNA synthesis within membranous protocells. Our results outline how compositionally simple, mixed-chirality peptides may have augmented the functional potential of early RNAs and promoted the emergence of the first protocells.
Conflict of interest statement
Competing financial interests
The authors declare no competing financial interests.
Figures
Figure 1. Ribosomal peptides enhance the activity of an artificial RNA polymerase ribozyme.
a, b Proteins (blue) and their peptide extensions (orange) in the structures of the T. thermophilus 30S and 50S ribosomal subunits. c, PAGE analysis of primer extensions on template TI by the RPR Z at a suboptimal [Mg2+] of 22 mM (left panel, 4°C for 22 days) or 46 mM (right panel, 4°C for 7 days), alone or in the presence of 400 μM of peptides derived from ribosomal proteins (terminating in an extra glycine residue (G) from synthesis). Ribosomal proteins comprising very long unstructured peptide segments give rise to two peptides e.g. L32-1 & L32-2. Peptide segments enhancing RPR function (red) are highlighted in bold in a & b.
Figure 2. RPR activation by homopolymeric peptides.
Top: the amino acids Lysine (K), Ornithine (Orn) and Diaminobutyric acid (Dab) (top panels) have progressively shorter amino group side chains. Below: PAGE of primer extensions on template TI by the Z RPR over 3 days at 17°C, alone or with indicated peptides of different (a) lengths, (b) concentrations and (c) side chains and chirality. d, EMSA: The fluorescently labeled Z ribozyme (Z-Fluor 647) was incubated with K10 peptide at the indicated concentrations with 22 mM [Mg2+], and resolved on a non-denaturing agarose gel.
Figure 3. K10 and [Mg2+] dependence of RPR activity by Z and the evolved 4M.
a, Outline of the TST selection scheme, showing how in cis-primer extension by a ribozyme prevents ribozyme loss during a denaturing wash on beads, allowing it to be recovered and amplified. b, Putative secondary structures of the Z and 4M ribozymes surrounding the primer (orange)/template (purple) duplex, showing 4M selected mutations in red (3’ MT = tail sequence from selection construct). c, Average primer extension per hour on template TI by the Z (left) or 4M (right) RPRs in 5 d (left) or 4 h (right) reactions at varying [Mg2+], with 6 μM K10 (filled symbols) or without it (open symbols) (error bars represent S. D., N = 3; note the change in scale indicated between the two panels (dotted red line)).
Figure 4. Robust RNA synthesis during long incubations.
PAGE of primer extensions by RPRs tC9-Y and tC9-4M tethered to the long repeat template I-6.
Figure 5. Ribozyme-catalyzed RNA synthesis in phospholipid protocells.
a, Fluorescence micrograph of POPC vesicles containing RNA and NTPs, forming across a range of conditions with and without Mg2+ and K10 peptide; the membrane is stained with Rhodamine G (red) and encapsulated RNA is labeled with fluorescein (blue). b, PAGE of extensions at 17°C upon the long repeat template I-6 encapsulated within POPC vesicles, by tC9-4M added externally or within the vesicles. Quantification (N=4) of extension per primer and full-length extension are displayed beneath.
References
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