1 research outputs found
Directed Evolution of Heterologous tRNAs Leads to Reduced Dependence on Post-transcriptional Modifications
Heterologous tRNA:aminoacyl tRNA
synthetase pairs are often employed
for noncanonical amino acid incorporation in the quest for an expanded
genetic code. In this work, we investigated one possible mechanism
by which directed evolution can improve orthogonal behavior for a
suite of <i>Methanocaldococcus jannaschii</i> (<i>Mj</i>) tRNA<sup>Tyr</sup>-derived amber suppressor tRNAs. Northern blotting
demonstrated that reduced expression of heterologous tRNA variants
correlated with improved orthogonality. We suspected that reduced
expression likely minimized nonorthogonal interactions with host cell
machinery. Despite the known abundance of post-transcriptional modifications
in tRNAs across all domains of life, few studies have investigated
how host enzymes may affect behavior of heterologous tRNAs. Therefore,
we measured tRNA orthogonality using a fluorescent reporter assay
in several modification-deficient strains, demonstrating that heterologous
tRNAs with high expression are strongly affected by some native <i>E. coli</i> RNA-modifying enzymes, whereas low abundance
evolved heterologous tRNAs are less affected by these same enzymes.
We employed mass spectrometry to map ms<sup>2</sup>i<sup>6</sup>A37
and Ψ39 in the anticodon arm of two high abundance tRNAs (Nap1
and tRNA<sup>Opt</sup><sub>CUA</sub>), which provides (to our knowledge)
the first direct evidence that MiaA and TruA post-transcriptionally
modify evolved heterologous amber suppressor tRNAs. Changes in total
tRNA modification profiles were observed by mass spectrometry in cells
hosting these and other evolved suppressor tRNAs, suggesting that
the demonstrated interactions with host enzymes might disturb native
tRNA modification networks. Together, these results suggest that heterologous
tRNAs engineered for specialized amber suppression can evolve highly
efficient suppression capacity within the native post-transcriptional
modification landscape of host RNA processing machinery