Figure 4
In vitro activity of P. abyssi Pab91 RNP enzyme for formation of Ψ2685 in a 143-nucleotide-long rRNA substrate. (A) The RNA substrate mimicking the peptidyl transferase center (PTC) region of the 23S rRNA. The secondary structure model of the P. abyssi PTC is represented. The U at position 2685 targeted by the Pab91 RNP is indicated. The region of RNA substrate 23S-143nt is delineated by the black line. The loop CUGA replaced a large portion of 23S rRNA corresponding to domains I, II, III, IV, VI and is used as a linker between the 5′ and 3′ parts of domain V. (B) Single-turnover activity of the P. abyssi Pab91 LCN and LCNG RNP enzymes for modification of radiolabeled 23S–143nt at 65 °C. The 32P was introduced in the phosphodiester bound preceding U2685 by a splinted ligation (detailed in Material and Methods). (C-D) Multiple-turnover activity of the LCN (C), and LCNG (D) enzymes for modification of radiolabeled 22–U substrate RNA at 65 °C. The unlabeled substrate RNA 23S-143nt was added in a 4-fold (2 μM), 10-fold (5 μM), or 20-fold (10 μM) excess over the RNP (~0.5 µM). A control was performed in absence of the unlabeled RNA (0 µM). (E) Electrophoretic mobility shift analysis (EMSA) of the binding of the radiolabeled substrate 23S–143nt with the Pab91 sRNP assembled with the L7Ae, Cbf5, and Nop10 mix. Incubation was performed at 65 °C during 10 and 60 minutes.
