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Coding sequence-dependent ribosomal arrest at termination of translation
1Division of Molecular Medicine and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104, USA.
Abstract:
A remarkably high percentage of proto-oncogene, growth factor, cellular receptor, and viral transcript leaders contain short upstream open reading frames (uORFs), yet the significance and regulatory effects of these uORFs have not been well characterized. In the case of the human cytomegalovirus gpUL4 (gp48) transcript, the second of three uORFs (uORF2) inhibits translation of the downstream cistron by a process that depends on the uORF2 amino acid coding information. To investigate the mechanism underlying this unusual regulatory element, we adapted the toeprinting (or reverse transcriptase extension inhibition) assay for use in detecting positions of ribosomal stalling on gp48 transcripts. Using a cell-free translation system, we demonstrate that ribosomes arrest at the termination codon of uORF2 by a uORF2 coding sequence-dependent mechanism. Further, the sequence requirements for ribosomal stalling are the same as for inhibition of downstream translation. We also provide evidence for ribosomal stalling in vivo, on the natural viral mRNA. These data support the hypothesis that the inhibition of downstream translation results from uORF2 peptide-dependent ribosomal arrest at termination and suggest that translation termination may be a regulatory step in expression of some eukaryotic genes.
Insights
Short upstream open reading frames (uORFs) regulate gene expression. In human cytomegalovirus, a specific uORF (uORF2) peptide causes ribosomal stalling at its termination codon, inhibiting downstream protein production.
Area of Science:
- Molecular Biology
- Virology
- Gene Regulation
Background:
- Short upstream open reading frames (uORFs) are common in viral and cellular transcripts but their regulatory roles are not fully understood.
- The human cytomegalovirus gpUL4 (gp48) transcript features three uORFs, with uORF2 known to inhibit downstream translation.
Purpose of the Study:
- To investigate the mechanism by which uORF2 inhibits translation of the downstream cistron.
- To determine if ribosomal stalling at uORF2 is responsible for translational inhibition.
Main Methods:
- Adaptation of the toeprinting assay (ribosomal stalling detection) for use with the gp48 transcript.
- Utilizing a cell-free translation system to analyze ribosomal behavior on the mRNA.
- In vivo validation of ribosomal stalling on the natural viral mRNA.
Main Results:
- Ribosomes were observed to stall at the termination codon of uORF2 in a cell-free system.
- This stalling was dependent on the amino acid coding sequence of uORF2.
- The sequence requirements for stalling matched those for downstream translation inhibition.
- Evidence of ribosomal stalling at uORF2 was also found in vivo.
Conclusions:
- uORF2 peptide-dependent ribosomal arrest at termination is the mechanism inhibiting downstream translation.
- Translation termination can serve as a regulatory step in eukaryotic gene expression.
- This finding provides insight into the complex regulation of viral gene expression.