Bisubstrate Function of RNA Polymerases Triggered by Molecular Crowding Conditions

Area of Science:

• Biochemistry
• Origin of Life Studies
• Polymer Chemistry

Background:

• The transition from RNA to DNA as the primary genetic material is a cornerstone of life's evolution.
• Cellular environments are densely packed, potentially influencing biochemical reactions like polymerization.
• Molecular crowding effects on RNA and DNA synthesis remain incompletely understood.

Purpose of the Study:

• To investigate the impact of molecular crowding on RNA-dependent RNA and DNA polymerization.
• To examine the activity of specific enzymes (tC9Y ribozyme, T7 RNA polymerase, Klenow fragment DNA polymerase) under varying crowding conditions.
• To explore the potential for switching polymerase substrate specificity in crowded environments.

Main Methods:

• Utilized poly(ethylene glycol) (PEG) of various molecular weights as a crowding agent.
• Assessed RNA-dependent RNA and DNA polymerization catalyzed by a ribozyme and two polymerases.
• Analyzed enzyme activity under different molecular crowding conditions, including PEG with an average molecular weight of 200 (PEG200).

Main Results:

• PEG promoted both RNA and DNA polymerization catalyzed by the ribozyme.
• PEG200 reduced RNA polymerization by T7 RNA polymerase but enhanced DNA polymerization.
• PEG200 did not affect the DNA polymerase activity of Klenow fragment.

Conclusions:

• Molecular crowding can influence the preference for RNA versus DNA polymerization.
• Proteinaceous RNA polymerases may exhibit switchable bisubstrate specificity in response to environmental changes (dielectric constant, excluded volume).
• Findings support the evolutionary significance of polymerase bisubstrate activity and offer insights for developing novel biomaterials.