Figure 1. Peptidyl prolyl-tRNA₂^Pro accumulates during inefficient translation termination.

(a) FLAG-(m)YbeL expression constructs. The flag-(m)ybeL message is shown schematically along with the Northern probe binding site. The 3′ coding sequences of flag-(m)ybeL-PP, -PP(uag), -PP(uga), and -EA are shown. The boxed P and A indicate the positions of the ribosomal P and A sites during translation termination. (b) Northern blot analysis of peptidyl-tRNA. Total RNA was isolated from tmRNA⁺ and ΔtmRNA cells, resolved on acid-urea polyacrylamide gels, and analyzed by Northern blot hybridization using probes specific for tRNA₂^Pro and tRNA_1B^Ala. Expression of FLAG-(m)YbeL-PP (+ IPTG) resulted in the accumulation of peptidyl prolyl-tRNA₂^Pro in both tmRNA⁺ and ΔtmRNA cells, whereas no peptidyl-tRNA was detected in cells expressing FLAG-(m)YbeL-EA. Overexpression of Pth had no significant effect on peptidyl prolyl-tRNA₂^Pro levels. (c) Treatment of peptidyl-tRNA with Pth-His₆ in vitro. Total RNA from cells expressing FLAG-(m)YbeL-PP or FLAG-SecM′ was treated with purified Pth-His₆ for the indicated time, and analyzed by Northern blot hybridization for tRNA₂^Pro or tRNA₃^Gly, respectively. (d) Fractionation of peptidyl-tRNA. Cells expressing FLAG-(m)YbeL-PP were broken by French press and fractionated by sucrose density ultracentrifugation. RNA was extracted from the lysate, pellet, and supernatant fractions, and analyzed by Northern blot for tRNA₂^Pro and tRNA_1B^Ala. Peptidyl prolyl-tRNA₂^Pro was only found in the high-speed pellet fraction.