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Proceedings Paper

Use of molecular beacons to probe for messenger RNA release from ribosomes during 5'-translational blockage by consecutive low-usage codons in Escherichia coli
Author(s): Wenwu Gao; Sanjay Tyagi; Fred Russell Kramer; Emanuel Goldman
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Paper Abstract

In `5'-translational blockage,' significantly reduced yields of proteins are synthesized in Escherichia coli when consecutive low-usage codons are inserted near translation starts of messages (with reduced or no effect when these same codons are inserted downstream). We tested the hypothesis that ribosomes encountering these low-usage codons prematurely release the mRNA. RNA from polysome gradients was fractionated into pools of polysomes, monosomes and ribosomes-free. New hybridization probes, called `molecular beacons,' and standard slot-blots, were used to detect test messages containing either consecutive low-usage AGG (arginine) or synonymous high-usage CGU insertions near the 5' end. The results show an approximately twofold increase in the ratio of free to bound mRNA when the low-usage codons were present compared to high-usage codons. In contrast, there was no difference in the ratio of free to bound mRNA when consecutive low-usage CUA or high-usage CUG (leucine) codons were inserted, or when the arginine codons were inserted near the 3' end. These data indicate that at least some mRNA is released from ribosomes during 5'-translational blockage by arginine but not leucine codons, and they support proposals that premature termination of translation can occur in some conditions in vivo in the absence of a stop codon.

Paper Details

Date Published: 22 March 2000
PDF: 12 pages
Proc. SPIE 3926, Advances in Nucleic Acid and Protein Analyses, Manipulation, and Sequencing, (22 March 2000); doi: 10.1117/12.380508
Show Author Affiliations
Wenwu Gao, New Jersey Medical School (United States)
Sanjay Tyagi, Public Health Research Institute (United States)
Fred Russell Kramer, Public Health Research Institute (United States)
Emanuel Goldman, New Jersey Medical School (United States)


Published in SPIE Proceedings Vol. 3926:
Advances in Nucleic Acid and Protein Analyses, Manipulation, and Sequencing
Patrick A. Limbach; John C. Owicki; Ramesh Raghavachari; Weihong Tan, Editor(s)

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