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PMID: 2103444 Published · ppublish English Journal Article

Analysis of leaky viral translation termination codons in vivo by transient expression of improved beta-glucuronidase vectors.

Plant molecular biology ·Vol. 15 ·No. 1 ·1990-07-00 ·Pages 65-79

Skuzeski JM, Nichols LM, Gesteland RF

Abstract

Plant RNA viruses commonly exploit leaky translation termination signals in order to express internal protein coding regions. As a first step to elucidate the mechanism(s) by which ribosomes bypass leaky stop codons in vivo, we have devised a system in which readthrough is coupled to the transient expression of beta-glucuronidase (GUS) in tobacco protoplasts. GUS vectors that contain the stop codons and surrounding nucleotides from the readthrough regions of several different RNA viruses were constructed and the plasmids were tested for the ability to direct transient GUS expression. These studies indicated that ribosomes bypass the leaky termination sites at efficiencies ranging from essentially 0 to ca. 5% depending upon the viral sequence. The results suggest that the efficiency of readthrough is determined by the sequence surrounding the stop codon. We describe improved GUS expression vectors and optimized transfection conditions which made it possible to assay low-level translational events.

MeSH Terms
Base Sequence Codon/genetics DNA, Viral/genetics Genetic Vectors Glucuronidase/genetics Molecular Sequence Data Peptide Chain Termination, Translational Plant Viruses/enzymology,genetics Plants, Toxic Protein Biosynthesis Tobacco/enzymology,genetics
Chemicals
Codon DNA, Viral Glucuronidase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Skuzeski J M
Howard Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City 84132.
Nichols L M
Gesteland R F
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Article Info
Journal
Plant molecular biology
Abbr.
Plant Mol Biol
ISSN
0167-4412
DOI
10.1007/BF00017725
Published
1990-07-00
Pages
65-79
Language
English
Region
Netherlands
NLM ID
9106343
Subset
IM
External Links
PubMed source DOI: 10.1007/BF00017725 View journal details
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