Cotranslational folding of nascent proteins on Escherichia coli ribosomes.
Evidence is presented for cotranslational folding of rhodanese or ricin during its synthesis on Escherichia coli ribosomes. During transcription-translation, full-length but enzymatically inactive polypeptides accumulated as peptidyl-tRNA on the ribosomes. These polypeptides were activated and released by subsequent incubation with the bacterial chaperones and with release factor (RF-2). Coumarin was incorporated cotranslationally at the N-terminus of the nascent protein from fluorophore-S-Ac-Met-tRNAf. Changes in fluorescence indicated that DnaJ bound to the nascent proteins and to a fluorescently labeled synthetic peptide corresponding to the N-terminal 17 amino acids of bovine rhodanese. This peptide also bound to 70S ribosomes or 50S subunits but not to 30S subunits. It inhibited activation and RF-2-dependent release of the full-length ribosome-bound rhodanese. A deletion mutant of rhodanese lacking the N-terminal 23 amino acids was not accumulated on the ribosome but was synthesized very efficiently. However, the protein that was formed was enzymatically inactive. DnaJ did not bind to this deletion mutant on ribosomes. We conclude that the chaperone-mediated reactions facilitate binding of the N-terminal sequence of nascent proteins to a specific site on 50S ribosomal subunits where it blocks release. The ribosome-bound protein undergoes chaperone-mediated reactions that are required for folding into an enzymatically active conformation.
Hardesty
B
B
Department of Chemistry and Biochemistry, University of Texas of Austin, TX 78712, USA.
Kudlicki
W
W
Odom
O W
OW
Zhang
T
T
McCarthy
D
D
Kramer
G
G
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D017510:Protein Folding; D012270:Ribosomes / Q000648:ultrastructure*; D012276:Ricin / Q000235:genetics*; D013884:Thiosulfate Sulfurtransferase / Q000235:genetics*; D014158:Transcription, Genetic
D012276:Ricin; D013884:Thiosulfate Sulfurtransferase