Elongation and folding of nascent ricin chains as peptidyl-tRNA 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 deletion of codons on the ribosomal synthesis, release and chaperone-mediated folding of the proteins. Synthesis of wild-type ricin and five mutant proteins was carried out in an Escherichia coli cell-free coupled transcription/translation system from otherwise identical non-linearized plasmids. The deletions involved small numbers of contiguous amino acid 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 residues caused a 45% reduction in total protein synthesis whereas deletion of the next three amino acid residues caused a 1.5-fold increase in synthesis compared with wild-type with an accumulation of full-length polypeptides as peptidyl-tRNA in the ribosomal P site. Intermediate levels of synthesis and release were seen with the other three mutants. Enzymatic activity was detected only with wild-type protein and a mutant lacking the C-terminal five amino acid residues. These were the only ricin species in which chaperone-dependent reactions could be detected by fluorescence from coumarin incorporated with methionine at the N terminus of the proteins. By using sparsomycin to block termination of full-length peptidyl-tRNA, it was demonstrated that the chaperone-mediated reactions detected by fluorescence occur on the ribosomes and 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 conformation of the native enzyme. Deletion of amino acid residues at different points from the N terminus to the C terminus affects the reactions of elongation, chaperone-mediated folding and release of full-length protein.
Kudlicki
W
W
Department of Chemistry and Biochemistry, University of Texas at Austin 78712, USA.
Kitaoka
Y
Y
Odom
O W
OW
Kramer
G
G
Hardesty
B
B
D002474:Cell-Free System; D003062:Codon / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D018832:Molecular Chaperones / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010441:Peptide Chain Elongation, Translational; D010443:Peptide Chain Termination, Translational; D010458:Peptidyl Transferases / Q000378:metabolism; D014176:Protein Biosynthesis; D017510:Protein Folding; D011691:Puromycin / Q000494:pharmacology; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D012276:Ricin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D014158:Transcription, Genetic
D003062:Codon; D018832:Molecular Chaperones; D012346:RNA, Transfer, Amino Acyl; C019037:tRNA, peptidyl-; D011691:Puromycin; D012276:Ricin; D010458:Peptidyl Transferases