Monitoring monomer-specific acyl-tRNA levels in cells with PARTI

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Cellular incorporation of non-natural amino acids into proteins is crucial for expanding the proteome.
• Understanding the acylation state of transfer RNA (tRNA) is key to controlling this process.
• Existing methods lack direct measurement of specific tRNA acylation states in vivo.

Purpose of the Study:

• To develop and validate a novel assay for directly assessing the acylation status of specific tRNAs within cells.
• To apply this assay to investigate the mechanisms underlying the cellular incorporation of non-canonical amino acid monomers.
• To provide insights into the fidelity and limitations of the translational machinery when using non-standard amino acids.

Main Methods:

• Development of the 3-Prime Adenosine-Retaining Aminoacyl-tRNA Isolation (PARTI) assay.
• Utilizing high-resolution mass spectrometry to identify acyl-adenosine species.
• RNase A cleavage of isolated cellular tRNA to release modified adenosine moieties.
• Application of the PARTI assay to study specific cases of non-natural amino acid incorporation.

Main Results:

• The PARTI assay was successfully developed and validated for direct measurement of tRNA acylation.
• The assay was used to investigate the selectivity of translation for β2-hydroxy acid enantiomers.
• It provided insights into the activity of PylRS variants with malonic acid derivatives.
• Direct evidence for cellular production of 2',3'-diacylated tRNA was obtained in specific instances.

Conclusions:

• The PARTI assay offers a straightforward and effective method for analyzing tRNA acylation states.
• This assay will facilitate research into the cellular incorporation of diverse non-natural amino acids.
• The findings contribute to a deeper understanding of the adaptability and constraints of the protein synthesis machinery.