Use of molecular beacons to probe for messenger RNA release from ribosomes during 5'-translational blockage by consecutive low-usage codons in Escherichia coli

Wenwu Gao; Sanjay Tyagi; Fred Russell Kramer; Emanuel Goldman

doi:10.1117/12.380508

22 March 2000 Use of molecular beacons to probe for messenger RNA release from ribosomes during 5'-translational blockage by consecutive low-usage codons in Escherichia coli

Wenwu Gao, Sanjay Tyagi, Fred Russell Kramer, Emanuel Goldman

Author Affiliations +

Proceedings Volume 3926, Advances in Nucleic Acid and Protein Analyses, Manipulation, and Sequencing; (2000) https://doi.org/10.1117/12.380508
Event: BiOS 2000 The International Symposium on Biomedical Optics, 2000, San Jose, CA, United States

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.

Citation Download Citation

Wenwu Gao, Sanjay Tyagi, Fred Russell Kramer, and Emanuel Goldman "Use of molecular beacons to probe for messenger RNA release from ribosomes during 5'-translational blockage by consecutive low-usage codons in Escherichia coli", Proc. SPIE 3926, Advances in Nucleic Acid and Protein Analyses, Manipulation, and Sequencing, (22 March 2000); https://doi.org/10.1117/12.380508

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KEYWORDS

Proteins

In vitro testing

Polymers

Luminescence

Sodium

Computer programming

Ultraviolet radiation

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