The objective of this research proposal is to understand in detail the structure and functional role of tmRNA as part of a newly discovered mechanism which regulates the translation and turnover of proteins in all bacteria. The investigators will be using a combination of site- directed mutagenesis, site-directed cross-linking, and phylogenetic comparisons with the cloned components of the tmRNA system. The major topics to be investigated in this proposal are: (1) The molecular mechanisms by which tmRNA recognizes the ribosome. As a prerequisite, the three-dimensional folding of the tmRNA will be studied in its isolated form. Subsequently, the investigators will identify in detail the contacts between tmRNA and several sites of the Escherichia coli ribosome at various stages of translation. (2) The identification of those proteins that are tagged by a tmRNA-dependent process in vivo. This goal will be accomplished by altering the sequence of the tmRNA- encoded tag peptide such that the associated protein can be identified directly. (3) The biochemical identification and characterization of tmRNA-protein contacts. The investigators will determine the nucleotides and the amino acids in covalent cross-links between tmRNA and ribosomal protein S1. They will monitor the association of protein S1 and other RNA-protein contacts on the ribosome during the various stages of tmRNA function. Additional proteins that may be in proximity to tmRNA will be found by the use of established photoaffinity labeling approaches. These studies will focus on the specific features of several critical interactions between tmRNA, its associated proteins, and the ribosome. As such the investigators will define the relationship of tmRNA to the overall process of protein translation.
| Fu, Jie; Hashem, Yaser; Wower, Jacek et al. (2011) tmRNA on its way through the ribosome: two steps of resume, and what next? RNA Biol 8:586-90 |
| Fu, Jie; Hashem, Yaser; Wower, Iwona et al. (2010) Visualizing the transfer-messenger RNA as the ribosome resumes translation. EMBO J 29:3819-25 |
| Wower, Iwona K; Zwieb, Christian; Wower, Jacek (2009) Escherichia coli tmRNA lacking pseudoknot 1 tags truncated proteins in vivo and in vitro. RNA 15:128-37 |
| Wower, Jacek; Wower, Iwona K; Zwieb, Christian (2008) Making the jump: new insights into the mechanism of trans-translation. J Biol 7:17 |
| Andersen, Ebbe Sloth; Rosenblad, Magnus Alm; Larsen, Niels et al. (2006) The tmRDB and SRPDB resources. Nucleic Acids Res 34:D163-8 |
| Burks, Jody; Zwieb, Christian; Muller, Florian et al. (2005) Comparative 3-D modeling of tmRNA. BMC Mol Biol 6:14 |
| Wower, Iwona K; Zwieb, Christian; Wower, Jacek (2004) Contributions of pseudoknots and protein SmpB to the structure and function of tmRNA in trans-translation. J Biol Chem 279:54202-9 |
| Okada, Takahiro; Wower, Iwona K; Wower, Jacek et al. (2004) Contribution of the second OB fold of ribosomal protein S1 from Escherichia coli to the recognition of TmRNA. Biosci Biotechnol Biochem 68:2319-25 |
| Zwieb, Christian; Gorodkin, Jan; Knudsen, Bjarne et al. (2003) tmRDB (tmRNA database). Nucleic Acids Res 31:446-7 |
| Wower, Jacek; Zwieb, Christian W; Hoffman, David W et al. (2002) SmpB: a protein that binds to double-stranded segments in tmRNA and tRNA. Biochemistry 41:8826-36 |
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