Related Experiment Video
Updated: Jan 1, 2026
Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA
Published on: July 6, 2012
Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts
Petr Tesina1, Laura N Lessen2,3, Robert Buschauer1
1Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.
Abstract:
Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of in vitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an in vitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base-pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall- and collision-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.
Insights
Novel mRNA structures cause ribosome stalling, reducing protein production. Researchers used biochemistry, ribosome profiling, and cryo-EM to uncover these mechanisms, revealing how mRNA sequences directly impede translation.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- Inhibitory codon pairs and poly(A) tracts in mRNA can stall ribosomes, decreasing protein synthesis.
- The precise molecular mechanisms underlying these translational stalling events remain largely undefined.
Purpose of the Study:
- To elucidate the molecular mechanisms responsible for ribosome stalling induced by specific mRNA sequences.
- To provide structural insights into how mRNA conformation leads to translation inhibition.
Main Methods:
- In vitro reconstituted yeast translation system
- Ribosome profiling
- Cryo-electron microscopy (cryo-EM)
Main Results:
- Inhibitory codon pairs were shown to slow elongation rates, with partial rescue by increased tRNA or artificial tRNAs.
- Ribosome profiling identified paused ribosomes with empty A sites on these inhibitory sequences.
- Cryo-EM structures revealed decoding-incompatible mRNA conformations, including nucleotide stacking in poly(A) tracts, within the ribosome's A site.
Conclusions:
- Translational stalling is driven by novel, mRNA-induced mechanisms in eukaryotic ribosomes.
- Specific mRNA sequences, through conformational changes, directly impede the ribosome's decoding process.
- These findings offer a new understanding of translational regulation and its impact on protein output.
Related Concept Videos
02:07Improving Translational Accuracy
02:27Nonsense-mediated mRNA Decay
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
02:27Nonsense-mediated mRNA Decay
02:42Transcription Attenuation in Prokaryotes
There are several different mechanisms used to attenuate transcription. In ribosome mediated...
02:33Initiation of Translation
First, the initiator tRNA must be selected from the pool of elongator tRNAs by eukaryotic initiation factor 2 (eIF2). The initiator tRNA (Met-tRNAi) has conserved sequence elements including modified bases at...
02:33Initiation of Translation