Messenger RNA release from ribosomes during 5′-translational blockage by consecutive low-usage arginine but not leucine codons in Escherichia coli

Authors

  • Wenwu Gao,

    1. Department of Microbiology and Molecular Genetics, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA.,
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  • Sanjay Tyagi,

    1. Department of Molecular Genetics, Public Health Research Institute, New York, NY 10016, USA.,
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  • Fred Russell Kramer,

    1. Department of Molecular Genetics, Public Health Research Institute, New York, NY 10016, USA.,
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  • Emanuel Goldman

    1. Department of Microbiology and Molecular Genetics, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA.,
    2. New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA.
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Emanuel Goldman Department of Microbiology and Molecular Genetics, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA. E-mail egoldman@umdnj.edu; Tel. (973) 972 4367; Fax (973) 972 3644.

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 near the translation start prematurely release the mRNA. RNA from polysome gradients was fractionated into pools of polysomes and monosomes and a ribosome-free pool. 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 in the message compared with when high-usage codons were present. 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.

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