The third ribosomal tRNA-binding site, the E site, is occupied in native polysomes

Area of Science:

• Molecular Biology
• Biochemistry
• Microbiology

Background:

• Ribosome function is central to protein synthesis.
• Understanding the elongation cycle is crucial for molecular biology.
• Existing models for ribosome function include two-site and three-site mechanisms.

Purpose of the Study:

• To investigate the occupancy of the ribosome's E site with tRNA during the elongation cycle.
• To determine if ribosomes in the post-translocational state carry tRNA in the E site.
• To provide experimental evidence supporting or refuting existing models of translation elongation.

Main Methods:

• Uniformly labeling Escherichia coli nucleic acids with 32P.
• Labeling nascent peptidyl chains with 3H-amino acids.
• Isolating polysomes and assessing the post-translocational state via puromycin reaction.
• Quantifying tRNA content per 70S ribosome using 5S rRNA as a reference after gel electrophoresis.

Main Results:

• At least 75% of post-translocational ribosomes within isolated native polysomes were found to carry tRNA in their E site.
• The quantification of tRNA content per ribosome was performed accurately.
• The presence of tRNA in the E site was confirmed through biochemical assays.

Conclusions:

• The experimental data strongly support the allosteric three-site model for the ribosome's elongation cycle.
• The findings contradict predictions of the two-site model for translation.
• This study provides key insights into the dynamic state of ribosomes during protein synthesis.