Toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mRNAs.
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.
Sachs
Matthew S
MS
Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering, Oregon Health and Science University, 20000 NW Walker Road, Beaverton 97006-8921, USA.
Wang
Zhong
Z
Gaba
Anthony
A
Fang
Peng
P
Belk
Jonathan
J
Ganesan
Robin
R
Amrani
Nadia
N
Jacobson
Allan
A
D001483:Base Sequence; D002845:Chromatography / Q000379:methods; D018388:Codon, Terminator; D005800:Genes, Fungal / Q000235:genetics*; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013696:Temperature
D018388:Codon, Terminator; D012333:RNA, Messenger; D009584:Nitrogen