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Functional and structural modulation of the Tetrahymena ribozyme: activity enhancement by molecular crowding and nanostructure formation by modular engineering

ISLAM Md. Dobirul 富山大学

2022.09.28

概要

The Tetrahymena ribozyme belongs to a naturally occurring group I ribozyme composed of a common catalytic core (ΔP5) and peripheral activator elements (P5abc) that hardly promote ribozyme activity through stabilizing the non-covalent tertiary interactions. The evolution of group I ribozyme for stabilizing the catalytic core is adapted by diverse types of stimuli such as integrated external structural elements, proteins, internal sequence optimization, and environmental factors. As a part of environmental factors in evolution, synthetic molecular crowders were employed to find the rescue effect of the ribozyme in the absence of peripheral elements. The ΔP5 RNA ribozyme significantly responds to the synergetic effects of synthetic crowders, indicating that molecular crowders implicate structural stability. The degree of stability provided by polyethylene glycol is remarkably higher than the other crowders in terms of substrate affinity of the catalytic core and subsidizing the magnesium ion requirement. The crowding response of ΔP5 ribozyme also depends on the crowder size and their molecular weight, indicating the present study where PEG600 exhibited comparatively higher crowding effect than the other three different sizes of PEGs (PEG200, PEG2000, and PEG8000) molecules. The modularity of Tetrahymena ribozyme is used as a promising platform to construct diverse types of polygonal-shape nanostructures by designing a single structural unit. Rational engineering of modular interfaces between ΔP5 and P5abc has expanded the nanostructure diversity. By introducing a hairpin kissing loop as a pillar unit between two tetramers, a two-dimensional closed tetramer was expanded to a double-decker three- dimensional octamer. The octamer ribozyme assembly and catalytic activity were evaluated through electrophoresis mobility shift assay and substrate cleaved reaction, where nanostructure assembly-dependent ribozyme activities were observed.

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