Figure 3. 5'-end repair substrates based on mt-tRNAIleGAU(1) from D. discoideum.
To evaluate the 5'-repair activity of TLPs, two model tRNA substrates were based on the sequence of mt-tRNAIle. In vitro transcripts initiating with either C+4 (C+4-start) or U+3 (U+3-start) were generated and either 5'-32P-labeled or internally labeled with [α-32P]-ATP, as indicated. The U+3 and C+4 nucleotides are highlighted in blue and nucleotides expected to be added by TLPs to repair the 5'-aminoacyl-acceptor stem according to the 3'-template sequence are marked in red. The position of G15, where RNase T1 cleaves to yield a labeled 5'-fragment to be resolved by PAGE, is indicated.