Assembly of recently translated full-length and C-terminal truncated human γ-globin chains with a pool of α-globin chains to form Hb F in a cell-free system

Assembly of α-globin with translated, full-length and C-terminal truncated human γ-globin to form Hb F was assessed in a cell-free transcription/translation system. Polysome profiles showed two amino acid C-terminal-truncated γ-chains retained on polysomes can assemble with unlabeled holo α-chains only after puromycin-induced chain release. Two amino acid C-terminal truncated γ-chains encoded from vectors containing a stop codon at the translation termination site were released from polysomes and assembled with α-chains in the absence of puromycin addition, while removal of 11 or more amino acids from the γ-chain carboxy-terminus inhibited assembly with α-chains. These results suggest that amino acids in the HC- and H-helix γ-chain regions including amino acids 135–144 at the C-terminus in the translated γ-chains play a key role in assembly with α-chains, and that assembly occurs soon after exit of translated γ-chains from the ribosome tunnel and release from polysomes thereby preventing stable γ2 homo-dimer formation.