Related Experiment Video
Updated: Jun 27, 2026
11:19
Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
Published on: February 25, 2011
Crystal structure of a translation termination complex formed with release factor RF2
Andrei Korostelev1, Haruichi Asahara, Laura Lancaster
1Center for Molecular Biology of RNA and Departments of Molecular, Cell and Developmental Biology and Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA.
Summary
The crystal structure reveals how release factor RF2 interacts with stop codons during translation termination. This finding clarifies the mechanism of protein synthesis and peptide bond hydrolysis.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- Protein synthesis relies on ribosomes and release factors to terminate translation.
- Release factor 2 (RF2) recognizes specific stop codons (UAA, UAG) to end protein synthesis.
- Understanding RF2's interaction with the ribosome is crucial for deciphering translation termination.
Purpose of the Study:
- To determine the high-resolution crystal structure of the Thermus thermophilus 70S ribosome complexed with release factor RF2.
- To elucidate the molecular interactions between RF2, a stop codon, and the ribosome during translation termination.
- To provide structural insights into the catalytic mechanism of peptidyl-tRNA hydrolysis mediated by RF2.
Main Methods:
- X-ray crystallography was used to solve the structure of the ribosome-RF2-stop codon complex.
- The structure was determined at a resolution of 3 Å.
- Biochemical assays, including mutational analysis, were employed to validate functional roles.
Main Results:
- The crystal structure reveals specific recognition of the UAA stop codon by the SPF motif of RF2.
- The A3 base of the stop codon interacts with both RF2 and 16S ribosomal RNA.
- Conformational changes in the ribosome and RF2 facilitate the docking of the GGQ motif into the peptidyl transferase center (PTC).
- The main-chain amide nitrogen of the GGQ motif's glutamine is positioned for catalysis of peptidyl-tRNA hydrolysis.
- Eliminating the H-bonding capability of the GGQ glutamine's N-H group abolishes peptidyl-tRNA esterase activity.
Conclusions:
- The structure provides a detailed molecular basis for stop codon recognition by RF2.
- Ribosome conformational changes are essential for activating RF2's catalytic function.
- The conserved GGQ motif directly participates in the hydrolysis of the peptidyl-tRNA ester bond.
- This work supports a model where RF2 utilizes ribosome-induced conformational changes to achieve catalysis.
Related Concept Videos
01:44Termination of Translation
The large ribosomal subunit has several important structures essential to translation. These include the peptidyl transferase center (PTC) - which is the site where the peptide bond is formed - and a large, internal, water-filled tube through which the nascent polypeptide moves. This latter structure is called the Peptide Exit Tunnel, and it begins at the PTC and spans the body of the large ribosomal subunit. During translation, as the nascent polypeptide chain is synthesized, it passes through...
01:44Termination of Translation
The large ribosomal subunit has several important structures essential to translation. These include the peptidyl transferase center (PTC) - which is the site where the peptide bond is formed - and a large, internal, water-filled tube through which the nascent polypeptide moves. This latter structure is called the Peptide Exit Tunnel, and it begins at the PTC and spans the body of the large ribosomal subunit. During translation, as the nascent polypeptide chain is synthesized, it passes through...
01:29Translation in Prokaryotes
Prokaryote translation is a complex, highly coordinated process that converts genetic information from mRNA into functional proteins. It involves three stages: initiation, elongation, and termination, each facilitated by specific molecular components.Initiation of TranslationThe process begins with the assembly of the ribosomal subunits and initiation factors on the mRNA. In bacteria, the 30S ribosomal subunit recognizes the Shine-Dalgarno sequence in the mRNA, a conserved region upstream of...
01:34Directing Proteins to the Rough Endoplasmic Reticulum
The organelle-specific signaling sequences direct proteins synthesized in the cytosol to their final destination like ER, mitochondria, peroxisomes, etc. Some of the proteins directed to ER are then trafficked via vesicles to other organelles within the cell or the extracellular environment through the Golgi complex. For example, the rough ER synthesizes soluble proteins for transportation to the lysosomes or secretion out of the cell. It can also synthesize transmembrane proteins that can...
02:42Transcription Attenuation in Prokaryotes
Transcriptional attenuation occurs when RNA transcription is prematurely terminated due to the formation of a terminator mRNA hairpin structure. Bacteria use these hairpins to regulate the transcription process and control the synthesis of several amino acids including histidine, lysine, threonine, and phenylalanine. Transcription attenuation takes place in the non-coding regions of mRNA.
There are several different mechanisms used to attenuate transcription. In ribosome mediated...
There are several different mechanisms used to attenuate transcription. In ribosome mediated...
01:23Transcriptional Regulation: Riboswitches
Riboswitches are RNA elements that regulate gene expression by altering their secondary structures in response to specific effector molecules. These elements, located in the leader regions of certain mRNAs, act as transcriptional regulators by toggling between alternative conformations to control downstream gene expression. Riboswitch-mediated regulation is a precise mechanism for modulating biosynthetic pathways, as exemplified by the riboflavin biosynthesis pathway in Bacillus...