A Concept for Selection of Codon-Suppressor tRNAs Based on Read-Through Ribosome Display in an In Vitro Compartmentalized Cell-Free Translation System
Figure 3
(a) Summary of selection/amplification of anticodon-randomized or anticodon-matched/anticodon-loop-randomized tRNAs for Ser. The numbers of clones that possess the sequence shown are indicated in parentheses. (b) Western blotting analysis of the translation of nonfused (tRNA-lacking) mRNA templates under normal (noncompartmentalized) conditions with (lanes 9–15) or without (lane 8) tRNA; (lane9), (natural tRNA for Ser, lane 10), anticodon-loop-randomized after 0, 3, or 5 rounds of selection/amplification (lanes 11, 12, or 13, respectively), or anticodon-matched but singly loop-mutated (lanes 14 and 15). Lane 7 represents a control translation using amber-free mRNA with no use of tRNA as a positive control and lanes 1–7 are a calibration set.
