Toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mRNAs
Graduate School of Biomedical Sciences; Department of Biochemistry and Molecular Biology; Department of Molecular Genetics and Microbiology
Medical Subject Headings
Base Sequence; Chromatography; Codon, Terminator; Genes, Fungal; Molecular Sequence Data; Neurospora crassa; Nitrogen; Open Reading Frames; *Protein Biosynthesis; RNA, Messenger; Saccharomyces cerevisiae; Temperature
Life Sciences | Medicine and Health Sciences
The ability to map the position of ribosomes and their associated factors on mRNAs is critical for an understanding of translation mechanisms. Earlier approaches to monitoring these important cellular events characterized nucleotide sequences rendered nuclease-resistant by ribosome binding. While these approaches furthered our understanding of translation initiation and ribosome pausing, the pertinent techniques were technically challenging and not widely applied. Here we describe an alternative assay for determining the mRNA sites at which ribosomes or other factors are bound. This approach uses primer extension inhibition, or "toeprinting," to map the 3' boundaries of mRNA-associated complexes. This methodology, previously used to characterize initiation mechanisms in prokaryotic and eukaryotic systems, is used here to gain an understanding of two interesting translational regulatory phenomena in the fungi Neurospora crassa and Saccharomyces cerevisiae: (a) regulation of translation in response to arginine concentration by an evolutionarily conserved upstream open reading frame, and (b) atypical termination events that occur as a consequence of the presence of premature stop codons.
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Citation: Methods. 2002 Feb;26(2):105-14. Link to article on publisher's site
DOI of Published Version
Methods (San Diego, Calif.)
Sachs, Matthew S.; Wang, Zhong; Gaba, Anthony; Fang, Peng; Belk, Jonathan Philip; Ganesan, Robin; Amrani, Nadia; and Jacobson, Allan, "Toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mRNAs" (2002). GSBS Student Publications. 1061.