[3'-32P]-labeling tRNA with nucleotidyltransferase for assaying aminoacylation and peptide bond formation

Abstract

The analysis of reactions involving amino acids esterified to tRNAs traditionally uses radiolabeled amino acids. We describe here an alternative assay involving [3'-32P]-labeled tRNA followed by nuclease digestion and TLC analysis that permits aminoacylation to be monitored in an efficient, quantitative manner while circumventing many of the problems faced when using radiolabeled amino acids. We also describe a similar assay using [3'-32P]-labeled aa-tRNAs to determine the rate of peptide bond formation on the ribosome. This type of assay can also potentially be adapted to study other reactions involving an amino acid or peptide esterified to tRNA.

Figure 1

[3’-³²P] labeling tRNA by nucleotide exchange. (A) Two step reaction scheme for nucleotide exchange by tRNA nucleotidyltransferase. (B) PEI cellulose TLC plate showing [α-³²P] ATP before (left) and after (right) incorporation into tRNA with a yield of 76%. (C) 10% denaturing polyacrylamide gel showing 20 μM tRNA^fMet after treatment different concentrations of tRNA nucleotidyltransferase: Lane 1: 6 μM; Lane 2: 2 μM; Lane 3: 0.67 μM; Lane 4: 0.22 μM.

Figure 2

Aminoacylation analysis of [3’-³²P] tRNA^Ala by AlaRS. S1 nuclease digestion of Ala-tRNA^Ala results in free [³²P] AMP and [³²P] Ala-AMP that can be separated on a PEI cellulose TLC plate in glacial acetic acid/1 M NH₄Cl/water (5:10:85). Reaction yields as a function of AlaRS concentration: Lane 1: 100 pM; Lane 2: 1 nM; Lane 3: 10 nM; Lane 4: 100 nM, Lane 5: 1 μM.

Figure 3

Analysis of k_pep with fMet-tRNA^fMet in the P site and [3’-³²P] Ala-tRNA^Ala in the A site. S1 nuclease digestion of reaction mix results in free [³²P] AMP, [³²P] Ala-AMP, and [³²P] fMet-Ala-AMP. Kinetic data is shown as digestion products developed on a PEI cellulose TLC plate in glacial acetic acid/1 M NH₄Cl/water (5:10:85) and graphically to give k_pep of 2.3 s⁻¹.