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Effect of eukaryotic initiation factor 4F on AUG selection in a bicistronic mRNA
S M Tahara1, T A Dietlin, T E Dever
1Department of Microbiology, University of Southern California School of Medicine, Los Angeles 90033-1054.
The Journal of Biological Chemistry
|February 25, 1991
Summary
Mouse cell extracts show a preference for internal translation initiation over the first start codon in bicistronic mRNAs. Eukaryotic initiation factor 4F (eIF4F) addition promotes translation from the 5'-proximal start site.
Area of Science:
- Molecular Biology
- Gene Expression Regulation
Background:
- Bicistronic and multicistronic mRNAs allow for the expression of multiple proteins from a single transcript.
- Understanding the mechanisms governing the preferential translation of specific open reading frames (ORFs) is crucial for controlling protein synthesis.
Purpose of the Study:
- To investigate the translation initiation mechanisms of artificial bicistronic mRNAs in a cell-free system.
- To determine the role of cap-dependent and cap-independent initiation in controlling the expression of proximal and distal ORFs.
Main Methods:
- Construction of artificial bicistronic mRNAs containing rabbit beta-globin and chloramphenicol acetyltransferase sequences.
- Translation assays using a mouse astrocytoma cell-free extract.
- Addition of exogenous reticulocyte initiation factors, including eukaryotic initiation factor 4F (eIF4F).
Main Results:
- The mouse cell-free extract preferentially initiated translation at internal or 5"-distal start codons over the 5"-proximal start codon.
- Internal initiation was cap-independent, as indicated by lack of inhibition by cap analogs and insensitivity to the 5"-cap structure.
- Addition of eIF4F, a cap-binding protein, promoted 5"-proximal initiation, increasing the relative expression of the proximal ORF product.
Conclusions:
- Translation initiation in this cell-free system favors nonstandard, cap-independent mechanisms.
- The relative translation efficiency of proximal versus distal ORFs in bicistronic mRNAs is influenced by the availability of eukaryotic initiation factor 4F and other mRNA-dependent initiation factors.