Optical Control of Transcription: Genetically Encoded Photoswitchable Variants of T7 RNA Polymerase

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Light-sensing protein domains, particularly Light-Oxygen-Voltage (LOV) photoreceptors, are valuable tools for controlling enzyme activity with light.
• LOV domains enable the engineering of novel regulatory mechanisms and the study of dynamic intracellular processes.
• Traditional terminal fusion strategies for integrating LOV domains are limited by enzyme structural constraints, such as those in T7 RNA polymerase.

Purpose of the Study:

• To develop a novel method for integrating LOV domains into enzymes where terminal fusion is not feasible.
• To create a light-responsive T7 RNA polymerase by covalently inserting LOV domains.
• To demonstrate that internal insertion preserves enzyme activity and introduces light-induced allosteric control.

Main Methods:

• Covalent insertion of LOV domains into the T7 RNA polymerase protein structure.
• Characterization of the engineered polymerase's enzymatic activity.
• Assessment of light-induced allosteric coupling and responsivity.

Main Results:

• Successful internal insertion of LOV domains into T7 RNA polymerase was achieved.
• The engineered T7 RNA polymerase retained its catalytic activity.
• The insertion generated a novel light-responsive allosteric coupling, allowing external control via blue light.

Conclusions:

• Internal covalent insertion of LOV domains is a viable strategy for engineering light-responsive enzymes, even when terminal fusions are not possible.
• This approach expands the toolkit for optogenetic control of biological processes.
• The light-controlled T7 RNA polymerase offers new possibilities for studying and manipulating gene expression with temporal precision.