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Biomedical subjects

B Hardesty

Publications and source records attributed to B Hardesty.

141 records · Page 8Linked to original sources
PubMed1969

Deacylated tRNA-phe binding to a reticulocyte ribosomal site for the initiation of polyphenylalanine synthesis.

The initiation of polyphenylalanine synthesis at low MgCl(2) concentration in the reticulocyte transfer system has been found to have a stringent requirement for deacylated tRNA(Phe). An initial poly-U-directed complex between deacylated tRNA(Phe) and ribosomes is formed. The onset of polyphenylalanine synthesis causes the rapid release of tRNA(Phe) from the complex. Thermal dissociation studies indicate that deacylated tRNA(Phe) and phenylalanyl-tRNA are bound to the same ribosomes. A total of three ribosomal tRNA binding sites is indicated.

Binding Sites↗
PubMed1969

The mechanism of messenger RNA translocation through ribosomes.

The two recognized enzymatic steps involved in the extension of peptides on ribosomes of the 80S type have been studied in a highly purified transfer system derived from rabbit reticulocytes. Data presented are interpreted to reflect three ribosomal binding sites through which transfer RNA is moved in two independent enzymatic reactions each of which requires guanosine 5'-triphosphate hydrolysis. The binding enzyme facilitates translocation between the entry and acceptor ribosomal sites. Transferase II is involved in translocation between the acceptor and donor ribosomal sites. A model is proposed to account for movement of transfer RNA and messenger RNA between the ribosomal sites.

Animals↗
PubMedDate not supplied

The use of synthetic tRNAs as probes for examining nascent peptides on Escherichia coli ribosomes.

The polyuridylic acid-dependent syntheses of polycysteine and polyserine were carried out on Escherichia coli ribosomes using two new synthetic tRNA species. The peptides were initiated with N-acetyl or N-acyl coumarin derivatives of either Ser-tRNA or Phe-tRNA. The properties of the resulting nascent peptides were compared to those of nascent polyphenylalanine chains synthesized under similar conditions. This was accomplished by following changes in the fluorescence properties of the probes covalently linked to the amino-terminus of each of the nascent polypeptides as they were formed on the ribosomes. Nascent polycysteine and polyserine peptides appeared quite different from those of polyphenylalanine, as indicated by the anisotropy of fluorescence from the amino terminal probe. In contrast to serine and cysteine peptides, the synthesis of all the polyphenylalanine peptides was insensitive to inhibition by erythromycin, even though these peptides were initiated with N-acyl serine. The results support the hypothesis that nascent polyphenylalanine peptides have atypical physical and chemical properties and demonstrate the utility of using modified tRNAs to study ribosome function and the synthesis of proteins.

Base Sequence↗
PubMedDate not supplied

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.

Amino Acid Sequence↗