Evidence for a Conformational Change in the Exit Site of the Escherichia coli Ribosome upon tRNA Binding

J. Stephen Lodmell, William E Tapprich, Walter E. Hill

Research output: Contribution to journalArticle

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Abstract

The exit (E) site of the Escherichia coli ribosome was investigated using oligodeoxyribonucleotides complementary to single-stranded regions of ribosomal RNA suggested to be involved in tRNA binding in the E site [Moazed, D., & Noller, H. (1989) Cell 57,585–597]. Radiolabeled DNA oligomers (probes) were hybridized in situ to complementary sites on the ribosomal RNA of ribosomes or ribosomal subunits, and the effects of simultaneous tRNA or antibiotic binding on probe binding were measured using a nitrocellulose filtration binding assay. Site specificity of probe binding was assured using ribonuclease H to cleave the ribosomal RNA at the site of probe binding. When 50S subunits were hybridized with a probe spanning bases 2109–2119 and deacylated tRNA was added incrementally, probe binding decreased, suggesting that the probe and tRNA competed for the same binding site or that tRNA was allosterically affecting the probe binding site. When 70S ribosomes were substituted for 50S subunits, probe binding to this site initially increased and then decreased at higher concentrations of deacylated tRNA. Titrating probe–ribosome complexes with acylated tRNA, N-acetyl-acylated tRNA, tetracycline, or chloramphenicol had no effect on probe binding. The data presented provide evidence for tRNA/rRNA interaction at or near the E site of the E. coli ribosome and suggest that a conformational change occurs in the E site when deacylated tRNA is bound to the P site. The data suggest that deacylated tRNA in the P site serves as a translocational trigger by causing the E site to change conformations, making it more available for tRNA (and probe) binding and therefore promoting translocation. Upon saturation of the P site, deacylated tRNA then binds to the E site, competing with the probe bound there.

Original languageEnglish (US)
Pages (from-to)4067-4072
Number of pages6
JournalBiochemistry
Volume32
Issue number15
DOIs
StatePublished - Apr 1 1993

Fingerprint

Transfer RNA
Ribosomes
Escherichia coli
Ribosomal RNA
Binding Sites
Ribosome Subunits
Ribonuclease H
Collodion
Oligodeoxyribonucleotides
DNA Probes
Chloramphenicol
Tetracycline
Oligomers
Conformations
Assays
Anti-Bacterial Agents

ASJC Scopus subject areas

  • Biochemistry

Cite this

Evidence for a Conformational Change in the Exit Site of the Escherichia coli Ribosome upon tRNA Binding. / Stephen Lodmell, J.; Tapprich, William E; Hill, Walter E.

In: Biochemistry, Vol. 32, No. 15, 01.04.1993, p. 4067-4072.

Research output: Contribution to journalArticle

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