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In vitro mutational analysis of cis-acting RNA translational elements within the poliovirus type 2 5' untranslated
K Meerovitch1, R Nicholson, N Sonenberg
1Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Abstract:
Initiation of translation on poliovirus RNA occurs by internal binding of ribosomes to a region within the 5' untranslated region (UTR) of the mRNA. This region has been previously roughly mapped between nucleotides 140 and 631 of the 5' UTR and termed the ribosome landing pad. To identify cis-acting elements in the 5' UTR of poliovirus type 2 (Lansing strain) RNA that confer cap-independent internal initiation, we determined the in vitro translational efficiencies of a series of deletion and point mutations within the 5' UTR of the mRNA. The results demonstrate that the 3' border of the core poliovirus ribosome landing pad is located between nucleotides 556 and 585, whereas a region extending between nucleotides 585 and 612 confers enhanced translation. We studied two cis-acting elements within this region of the 5' UTR: a pyrimidine stretch which is critical for translation and an AUG (number 7 from the 5' end) that is located approximately 20 nucleotides downstream from the pyrimidine stretch and augments translation. We also show that the stem-loop structure which contains this AUG is not required for translation.
Insights
Poliovirus RNA translation initiates internally. Researchers identified key elements in the 5' untranslated region (UTR), including a pyrimidine stretch and an AUG codon, crucial for ribosome binding and enhanced translation.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Poliovirus RNA translation initiates via internal ribosome binding to the 5' untranslated region (UTR), termed the ribosome landing pad.
- Previous mapping of the ribosome landing pad was approximate, between nucleotides 140 and 631 of the 5' UTR.
Purpose of the Study:
- To identify specific cis-acting elements within the poliovirus type 2 (Lansing strain) 5' UTR responsible for cap-independent internal initiation.
- To precisely define the boundaries of the ribosome landing pad and identify elements that enhance translation.
Main Methods:
- In vitro translation assays were performed using a series of deletion and point mutations within the poliovirus 5' UTR.
- Analysis of translational efficiencies to determine the function of specific RNA sequences and structures.
Main Results:
- The 3' border of the core ribosome landing pad was localized between nucleotides 556 and 585.
- A region between nucleotides 585 and 612 was found to enhance translation.
- A critical pyrimidine stretch and an augmenting AUG codon (the 7th from the 5' end) were identified within this enhancing region.
- The stem-loop structure containing the AUG was not essential for translation.
Conclusions:
- Specific sequences within the poliovirus 5' UTR, including a pyrimidine stretch and an AUG codon, are critical for internal ribosome binding and translation initiation.
- The precise localization of the ribosome landing pad and enhancer elements provides insights into cap-independent translation mechanisms.