Reengineering a tRNA Methyltransferase To Covalently Capture New RNA Substrates

Covalent RNA modifications can alter
the function and metabolism
of RNA transcripts. Altering the RNA substrate specificities of the
enzymes that install these modifications can provide powerful tools
to study and manipulate RNA. To develop new tools and probe the plasticity
of the substrate specificity of one of these enzymes, we examined
the engineerability of the uridine-54 tRNA methyltransferase, TrmA.
Starting from a mutant that remains covalently bound to its substrate
RNA (E358Q, TrmA*), we were able to use both rational design and a
high-throughput sequencing assay to examine the RNA substrates of
TrmA*. Although rational engineering substantially changed TrmA* specificity,
the rationally designed substrate mutants we developed still retained
activity with the wild-type protein. Using high-throughput substrate
screening of additional TrmA* mutants, we identified a triple mutant
of the substrate RNA (C56A;A58G;C60U) that is efficiently trapped
by a TrmA* double mutant (E49R;R51E) but not by the wild-type TrmA*.
This work establishes a foundation for using protein engineering to
reconfigure substrate specificities of RNA-modifying enzymes and covalently
trap RNAs with engineered proteins.