Affiliation:
1. Department of Biochemistry and Cell Biology
2. Center for Infectious Diseases, Stony Brook University, Stony Brook, New York 11794
Abstract
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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Reference56 articles.
1. Amerik, A., V. K. Antonov, A. E. Gorbalenya, S. A. Kotova, T. V. Rotanova, and E. V. Shimbarevich. 1991. Site-directed mutagenesis of La protease. A catalytically active serine residue. FEBS Lett. 287 : 211-214.
2. New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria
3. Current protocols in molecular biology. 1995
4. Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. A. Datsenko, M. Tomita, B. L. Wanner, and H. Mori. 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2 : 2006-2008.
5. Barends, S., J. Wower, and B. Kraal. 2000. Kinetic parameters for tmRNA binding to alanyl-tRNA synthetase and elongation factor Tu from Escherichia coli. Biochemistry 39 : 2652-2658.
Cited by
91 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献