Ribosome binding by tRNAs with fluorescent labeled 3' termini

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Transfer RNA (tRNA) is crucial for protein synthesis, carrying amino acids to the ribosome.
• Understanding tRNA-ribosome interactions is key to deciphering translation and developing antibiotics.

Purpose of the Study:

• To investigate the binding characteristics of modified yeast and E. coli tRNAPhe to E. coli 70S ribosomes.
• To determine the binding affinities and spatial arrangements of tRNAs within the ribosome.
• To explore the proximity of tRNA to erythromycin binding sites.

Main Methods:

• Oxidation and fluorescent labeling of tRNAPhe at the 3' end.
• Binding assays with poly(U)-programmed E. coli 70S tight couple ribosomes.
• Determination of binding constants for P and A sites.
• Singlet-singlet energy transfer measurements to determine distances.

Main Results:

• Yeast tRNAPhe derivatives exhibited distinct binding constants for the P (1 x 10^9 M^-1) and A (3 x 10^7 M^-1) sites.
• E. coli tRNAPhe showed similar A site affinity but a 5-fold weaker P site affinity compared to yeast tRNA.
• Distance measurements indicated erythromycin binding near the peptidyl moiety of peptidyl tRNA.
• A distance of 34 Å between the 3' ends of two simultaneously bound tRNAs was observed.

Conclusions:

• Differential binding affinities of yeast and E. coli tRNAPhe to the ribosome were characterized.
• Erythromycin's binding site is spatially close to the peptidyl-tRNA's 3' end.
• The orientation of a second bound tRNA may differ between protein synthesis and stringent response pathways.