W. Kudlicki et al., ELONGATION AND FOLDING OF NASCENT RICIN CHAINS AS PEPTIDYL-TRANSFER-RNA ON RIBOSOMES - THE EFFECT OF AMINO-ACID DELETIONS ON THESE PROCESSES, Journal of Molecular Biology, 252(2), 1995, pp. 203-212
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