B. Hardesty et al., COTRANSLATIONAL FOLDING OF NASCENT PROTEINS ON ESCHERICHIA-COLI RIBOSOMES, Biochemistry and cell biology, 73(11-12), 1995, pp. 1199-1207
Evidence is presented for cotranslational folding of rhodanese or rici
n during its synthesis on Escherichia coli ribosomes. During transcrip
tion-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 ch
aperones and with release factor (RF-2). Coumarin was incorporated cot
ranslationally at the N-terminus of the nascent protein from fluoropho
re-S-Ac-Met-tRNA(f) Changes in fluorescence indicated that DnaJ bound
to the nascent proteins and to a fluorescently labeled synthetic pepti
de corresponding to the N-terminal 17 amino acids of bovine rhodanese.
This peptide also bound to 70S ribosomes or 50S subunits but not to 3
0S 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 ribos
ome but was synthesized very efficiently. However, the protein that wa
s formed was enzymatically inactive. DnaJ did not bind to this deletio
n mutant on ribosomes. We conclude that the chaperone-mediated reactio
ns facilitate binding of the N-terminal sequence of nascent proteins t
o a specific site on 50S ribosomal subunits where it blocks release. T
he ribosome-bound protein undergoes chaperone-mediated reactions that
are required for folding into an enzymatically active conformation.