ELONGATION AND FOLDING OF NASCENT RICIN CHAINS AS PEPTIDYL-TRANSFER-RNA ON RIBOSOMES - THE EFFECT OF AMINO-ACID DELETIONS ON THESE PROCESSES

Ricin A-chain was used as a test protein to study the effects of delet ion of codons on the ribosomal synthesis, release and chaperone-mediat ed folding of the proteins. Synthesis of wild-type ricin and five muta nt proteins was carried out in an Escherichia coli cell-free coupled t ranscription/translation system from otherwise identical non-linearize d plasmids. The deletions involved small numbers of contiguous amino a cid residues at different points from the N terminus to the C terminus of the wild-type protein. Deletion of the N-terminal 20 amino acid re sidues caused a 45 % reduction in total protein synthesis whereas dele tion of the next three amino acid residues caused a 1.5-fold increase in synthesis compared with wild-type with an accumulation of full-leng th polypeptides as peptidyl-tRNA in the ribosomal P site. Intermediate levels of synthesis and release were seen with the other three mutant s. Enzymatic activity was detected only with wild-type protein and a m utant lacking the C-terminal five amino acid residues. These were the only ricin species in which chaperone-dependent reactions could be det ected by fluorescence from coumarin incorporated with methionine at th e N terminus of the proteins. By using sparsomycin to block terminatio n of full-length peptidyl-tRNA, it was demonstrated that the chaperone -mediated reactions detected by fluorescence occur on the ribosomes an d involve folding of the nascent protein as peptidyl-tRNA. The results presented provide a direct demonstration of two points of fundamental importance: folding of nascent proteins involving chaperone-mediated reactions can occur on ribosomes and is directly related to the confor mation of the native enzyme. Deletion of amino acid residues at differ ent points from the N terminus to the C terminus affects the reactions of elongation, chaperone-mediated folding and release of full-length protein. (C) 1995 Academic Press Limited