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Lon Protease Degrades Transfer-Messenger RNA-Tagged Proteins

Lon Protease Degrades Transfer-Messenger RNA-Tagged Proteins Lon Protease Degrades Transfer-Messenger RNA-Tagged Proteins ▿ † Jennifer S. Choy 1 , 2 , Latt Latt Aung 1 , and A. Wali Karzai 1 , 2 , * 1 Department of Biochemistry and Cell Biology 2 Center for Infectious Diseases, Stony Brook University, Stony Brook, New York 11794 ABSTRACT Bacterial trans translation is activated when translating ribosomes are unable to elongate or terminate properly. Small protein B (SmpB) and transfer-messenger RNA (tmRNA) are the two known factors required for and dedicated to trans translation. tmRNA, encoded by the ssrA gene, is a bifunctional molecule that acts both as a tRNA and as an mRNA during trans translation. The functions of tmRNA ensure that stalled ribosomes are rescued, the causative defective mRNAs are degraded, and the incomplete polypeptides are marked for targeted proteolysis. We present in vivo and in vitro evidence that demonstrates a direct role for the Lon ATP-dependent protease in the degradation of tmRNA-tagged proteins. In an endogenous protein tagging assay, lon mutants accumulated excessive levels of tmRNA-tagged proteins. In a reporter protein tagging assay with λ-CI-N, the protein product of a nonstop mRNA construct designed to activate trans translation, lon mutant cells efficiently tagged the reporter protein, but the tagged protein exhibited increased stability. Similarly, a green fluorescent protein (GFP) construct containing a hard-coded C-terminal tmRNA tag (GFP-SsrA) exhibited increased stability in lon mutant cells. Most significantly, highly purified Lon preferentially degraded the tmRNA-tagged forms of proteins compared to the untagged forms. Based on these results, we conclude that Lon protease participates directly in the degradation of tmRNA-tagged proteins. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Bacteriology American Society For Microbiology

Lon Protease Degrades Transfer-Messenger RNA-Tagged Proteins

Journal of Bacteriology , Volume 189 (18): 6564 – Sep 15, 2007

Lon Protease Degrades Transfer-Messenger RNA-Tagged Proteins

Journal of Bacteriology , Volume 189 (18): 6564 – Sep 15, 2007

Abstract

Lon Protease Degrades Transfer-Messenger RNA-Tagged Proteins ▿ † Jennifer S. Choy 1 , 2 , Latt Latt Aung 1 , and A. Wali Karzai 1 , 2 , * 1 Department of Biochemistry and Cell Biology 2 Center for Infectious Diseases, Stony Brook University, Stony Brook, New York 11794 ABSTRACT Bacterial trans translation is activated when translating ribosomes are unable to elongate or terminate properly. Small protein B (SmpB) and transfer-messenger RNA (tmRNA) are the two known factors required for and dedicated to trans translation. tmRNA, encoded by the ssrA gene, is a bifunctional molecule that acts both as a tRNA and as an mRNA during trans translation. The functions of tmRNA ensure that stalled ribosomes are rescued, the causative defective mRNAs are degraded, and the incomplete polypeptides are marked for targeted proteolysis. We present in vivo and in vitro evidence that demonstrates a direct role for the Lon ATP-dependent protease in the degradation of tmRNA-tagged proteins. In an endogenous protein tagging assay, lon mutants accumulated excessive levels of tmRNA-tagged proteins. In a reporter protein tagging assay with λ-CI-N, the protein product of a nonstop mRNA construct designed to activate trans translation, lon mutant cells efficiently tagged the reporter protein, but the tagged protein exhibited increased stability. Similarly, a green fluorescent protein (GFP) construct containing a hard-coded C-terminal tmRNA tag (GFP-SsrA) exhibited increased stability in lon mutant cells. Most significantly, highly purified Lon preferentially degraded the tmRNA-tagged forms of proteins compared to the untagged forms. Based on these results, we conclude that Lon protease participates directly in the degradation of tmRNA-tagged proteins.

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Publisher
American Society For Microbiology
Copyright
Copyright © 2007 by the American society for Microbiology.
ISSN
0021-9193
eISSN
1098-5530
DOI
10.1128/JB.00860-07
pmid
17616591
Publisher site
See Article on Publisher Site

Abstract

Lon Protease Degrades Transfer-Messenger RNA-Tagged Proteins ▿ † Jennifer S. Choy 1 , 2 , Latt Latt Aung 1 , and A. Wali Karzai 1 , 2 , * 1 Department of Biochemistry and Cell Biology 2 Center for Infectious Diseases, Stony Brook University, Stony Brook, New York 11794 ABSTRACT Bacterial trans translation is activated when translating ribosomes are unable to elongate or terminate properly. Small protein B (SmpB) and transfer-messenger RNA (tmRNA) are the two known factors required for and dedicated to trans translation. tmRNA, encoded by the ssrA gene, is a bifunctional molecule that acts both as a tRNA and as an mRNA during trans translation. The functions of tmRNA ensure that stalled ribosomes are rescued, the causative defective mRNAs are degraded, and the incomplete polypeptides are marked for targeted proteolysis. We present in vivo and in vitro evidence that demonstrates a direct role for the Lon ATP-dependent protease in the degradation of tmRNA-tagged proteins. In an endogenous protein tagging assay, lon mutants accumulated excessive levels of tmRNA-tagged proteins. In a reporter protein tagging assay with λ-CI-N, the protein product of a nonstop mRNA construct designed to activate trans translation, lon mutant cells efficiently tagged the reporter protein, but the tagged protein exhibited increased stability. Similarly, a green fluorescent protein (GFP) construct containing a hard-coded C-terminal tmRNA tag (GFP-SsrA) exhibited increased stability in lon mutant cells. Most significantly, highly purified Lon preferentially degraded the tmRNA-tagged forms of proteins compared to the untagged forms. Based on these results, we conclude that Lon protease participates directly in the degradation of tmRNA-tagged proteins.

Journal

Journal of BacteriologyAmerican Society For Microbiology

Published: Sep 15, 2007

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