Microbiology JoVE (Open Access) Citable · DOI

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Name: Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
Uploaded: 2026-06-23
Duration: 11 min 19 s
Description: Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

DOI: 10.3791/2498-v

What you'll learn

✓ Prepare cell-free extracts and biotin-labeled mRNA templates for ribosome studies
✓ Isolate ribosomes containing specific peptidyl-tRNAs using affinity purification
✓ Analyze and characterize isolated translating ribosomes biochemically

Protocol

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

Difficulty

advanced

Total time

~4–6 hours per sample (excluding overnight incubations)

Biosafety

BSL-1

Steps

Prepare cell-free extracts depleted of RF2

Generate bacterial cell-free translation systems with release factor 2 removed to prevent premature translation termination. This enables controlled ribosome stalling at desired mRNA positions.

▶ 02:59

Prepare DNA template for mRNA synthesis

Generate or clone a DNA template containing the target mRNA sequence with a built-in ribosome stalling site. This serves as the blueprint for in vitro mRNA production.

▶ 04:28

Synthesize and label mRNA with biotin

Transcribe mRNA from the DNA template and incorporate biotin labels for downstream affinity-based isolation of ribosomes bound to the specific mRNA sequence.

▶ 05:18

Isolate translating ribosomes containing peptidyl-tRNA

Perform in vitro translation, then use streptavidin-coated magnetic beads to capture biotin-labeled mRNA and associated ribosomes stalled with peptidyl-tRNA bound. Purify the complex away from non-specific ribosomes.

▶ 06:19

Analyze isolated ribosomes by biochemical methods

Perform functional and structural characterization of the purified ribosome–peptidyl-tRNA complexes using techniques such as toe-printing, mass spectrometry, or cryo-EM sample preparation.

▶ 07:56

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