Fig. 2: Production and acylation of split tRNAs expressed from split and circularly permuted genes.
From: Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism
a, Schematic for producing split tRNAs in trans. The tRNA gene is split at the anticodon loop and the anticodon stem sequence is extended for optimal assembly of the transcribed RNA in vivo; this creates two genes: one for the 5′ half and one for the 3′ half of the split tRNA. These genes are transcribed and the split tRNA is assembled, matured and acylated in cells. b, Schematic for producing split tRNAs in cis from a single gene. The tRNA sequence is circularly permutated by connecting the 3′ half, via an intervening loop sequence, to the 5′ half, splitting the sequence at the anticodon and extending the anticodon stem. Transcription, assembly in cis and maturation leads to a functional split tRNA. c, in vivo transcription, assembly, maturation and acylation of split tRNAPyl produced from genes for the 5′ half and 3′ half. Cells were grown in the presence of PylRS. Only the expression of both tRNAPyl halves led to a BocK (1)-dependent acylation signal, as judged by fluoro-tREX. Note that under the purification conditions used to isolate these stRNAs, we do not observe the Cy3 probe. d, Circularly permutated split tRNAPyl with different loop sequences were assayed by fluoro-tREX. For the argY–argZ and leuP–leuV loops derived from the intergenic regions of pairs of tRNA genes in E. coli, the fluoro-tREX signal for split tRNA production (Cy3) and acylation (Cy5) was comparable to the corresponding signal for intact tRNAPyl (Supplementary Fig. 6). Experiments in c,d were repeated three times with similar results.
