A GTP synthase ribozyme with increased GTP turnover.

Area of Science:

• Origin of life studies
• Biochemistry
• Molecular biology

Background:

• Early life likely used catalytic RNAs (ribozymes) before protein synthesis.
• RNA self-replication requires nucleotide polymerization, powered by nucleoside triphosphates (NTPs).
• Previous work created a ribozyme (GTR1) for guanosine triphosphate (GTP) synthesis from guanosine and cyclic trimetaphosphate (cTmp), but with low turnover.

Purpose of the Study:

• To enhance the efficiency of a ribozyme responsible for synthesizing GTP.
• To develop a more robust system for RNA polymerization, mimicking early life processes.
• To advance the laboratory modeling of RNA-based life.

Main Methods:

• In vitro selection in emulsion was used to evolve the guanosine triphosphorylation ribozyme (GTR1).
• A doped library of GTR1 was metabolically coupled to a polymerase ribozyme for selection.
• High-throughput sequencing and biochemical analyses identified and characterized improved ribozyme variants.

Main Results:

• An evolved variant, GTR1e, with 19 mutations showed a ~7.6-fold increase in GTP turnover number (~13).
• GTR1e exhibited biphasic reaction kinetics with a K_M^APP of approximately 11 mM for cTmp.
• When coupled with an RNA polymerase ribozyme, up to five guanosines were incorporated into an RNA polymer.

Conclusions:

• The enhanced ribozyme GTR1e represents a significant improvement in GTP synthesis efficiency.
• This work demonstrates progress toward creating a self-replicating RNA system in vitro.
• The findings contribute to understanding the potential biochemical pathways of early life.