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

We describe a new assay that reports directly on the acylation state of a user-chosen tRNA in cells. We call this assay 3-Prime Adenosine-Retaining Aminoacyl-tRNA Isolation (PARTI). It relies on high-resolution mass spectrometry identification of the acyl-adenosine species released upon RNase A cleavage of isolated cellular tRNA. Here we develop the PARTI workflow and apply it to understand three recent observations related to the cellular incorporation of non--amino acid monomers into protein: (1) the origins of the apparent selectivity of translation with respect to {beta}2-hydroxy acid enantiomers; (2) the activity of PylRS variants for benzyl derivatives of malonic acid; and (3) the apparent inability of N-Me amino acids to function as ribosome substrates in living cells. Using the PARTI assay, we also provide direct evidence for the cellular production of 2,3-diacylated tRNA in certain cases. The ease and simplicity of the PARTI workflow should benefit ongoing efforts to study and improve the cellular incorporation of non--amino acid monomers into proteins. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/633079v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@f3b8a0org.highwire.dtl.DTLVardef@de400dorg.highwire.dtl.DTLVardef@13f17faorg.highwire.dtl.DTLVardef@13f2c0f_HPS_FORMAT_FIGEXP M_FIG C_FIG