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. 2017 Jul 7;36(16):2435–2454. doi: 10.15252/embj.201796808

Figure 5. Reconstitution of KPAF3‐stimulated mRNA adenylation in vitro .

Figure 5

Reactions were performed for 20 min, and products were resolved on 10% polyacrylamide/8 M urea gel.
  1. KPAP1 and RET1 were incubated with 5′‐labeled 66‐mer RNA, ATP, and UTP, as indicated, in the presence of increasing KPAF3 amounts. Protein inclusion in combinatorial reactions is indicated by brackets.
  2. KPAP1 was incubated with 5′‐labeled 66‐mer RNA and ATP in the presence of increasing KPAF1‐2 and KPAF3 amounts.
  3. RET1 was incubated with 5′‐labeled 66‐mer RNA and UTP in the presence of increasing KPAF1‐2 and KPAF3 amounts for 20 min.
  4. KPAP1 was incubated with 5′‐labeled 24‐mer RNAs and ATP in the presence of increasing KPAF3 amounts.
  5. KPAP1 was incubated with 5′‐labeled 24‐mer [6A] RNA, ATP, and increasing concentrations of indicated homopolymers in the absence (left panel) or presence (right panel) of fixed KPAF3 amount.
  6. KPAP1 was incubated with 5′‐labeled 24‐mer RNA terminating with 6As, various NTPs, and fixed KPAF3 amount, as indicated.