Three-dimensional structure-guided evolution of a ribosome with tethered subunits

Area of Science:

• Synthetic biology
• Molecular biology
• RNA engineering

Background:

• RNA-based macromolecular machines, like ribosomes, feature complex structures with interactions between distant sequences.
• These long-range interactions pose challenges for traditional methods in exploring mutations and designing novel structures.
• Understanding and manipulating these interactions is crucial for advancing synthetic biology applications.

Purpose of the Study:

• To develop a novel method, Evolink, for high-throughput evolution of sequence-distant regions in large RNA machines.
• To enable the design of structurally stable RNA molecules using computational modeling.
• To enhance the functionality of macromolecular machines for synthetic biology.

Main Methods:

• Evolink (evolution and linkage) method for high-throughput evolution of sequence-distant regions.
• Computational RNA modeling for guiding library design and exploring stable structures.
• Application to a tethered ribosome system for functional enhancement.

Main Results:

• Achieved a 58% increase in orthogonal protein translation activity in an evolved tethered ribosome.
• Demonstrated a 97% improvement in cellular doubling times using the engineered ribosome.
• Identified new permissible sequences within specific ribosomal helices, expanding knowledge of RNA structure-function relationships.

Conclusions:

• Evolink facilitates the engineering of large RNA machines with improved functions.
• The method overcomes limitations in exploring sequence-distant interactions, crucial for RNA design.
• This approach holds significant potential for advancing synthetic biology through enhanced macromolecular machines.