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Related Experiment Video

Updated: Jun 12, 2026

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

Published on: July 6, 2021

Time-dependent increase in ribosome processivity.

Jennifer M Bonderoff¹, Richard E Lloyd

¹Department of Molecular Virology and Microbiology and Interdepartmental Program in Cell and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

Nucleic Acids Research

|June 24, 2010

Summary

This summary is machine-generated.

This study introduces a new reporter RNA to measure translation at both ends of long open reading frames (ORFs). Findings show poly(A) tail and PABP enhance ribosome processivity on long ORFs.

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Area of Science:

Molecular Biology
Biochemistry
Genetics

Background:

Ribosome translation efficiency is crucial for gene expression.
Understanding ribosome processivity on long open reading frames (ORFs) is complex.
Existing methods struggle to assess translation dynamics at both ends of long ORFs simultaneously.

Purpose of the Study:

To develop a novel tripartite reporter RNA to simultaneously measure ribosome translation rates at the 5' and 3' ends of a large ORF.
To investigate the factors influencing ribosome processivity and translation rates in vitro.
To elucidate the roles of poly(A) tail, poly(A)-binding protein (PABP), and eIF4F in regulating translation of long ORFs.

Main Methods:

Creation of a tripartite reporter RNA containing Renilla luciferase (RLuc), β-galactosidase, and firefly luciferase (FLuc) ORFs.
In vitro translation assays using HeLa cell lysates.
Analysis of FLuc production rates, dependence on poly(A) tail and PABP, and independence from eIF4F.
Investigation of eIF4G cleavage effects on translation.
Electron microscopy of polysome structures to assess ribosome packing and contact.

Main Results:

ORF length significantly impacts ribosome processivity, reducing the completion rate of translation.
FLuc production rate increased over time, dependent on the poly(A) tail and PABP, but not eIF4F.
Shorter RNA templates showed earlier stimulation of FLuc production.
Cleavage of eIF4G did not immediately halt 5'-RLuc translation, with a delay that decreased on shorter templates.
Electron microscopy revealed increased ribosome packing and contact on the FLuc ORF, correlating with enhanced processivity.

Conclusions:

ORF transit and PABP function synergistically promote ribosome interactions that enhance or maintain processivity on long ORFs.
The developed tripartite reporter RNA is a valuable tool for studying translation dynamics of long ORFs.
Ribosome processivity on long ORFs is a regulated process influenced by RNA structure and protein factors.