The long term goal of our research has been to understand post-transcriptional regulation in Escherichia coli. The general objective is to address current and fundamental questions in translation and mRNA processing/decay. We have focused attention on a transcription unit encoding six genes from the filamentous phage f1 genome that are transcribed in huge amounts but regulated exclusively after transcription to yield proteins made in different amounts. Appropriate regulation of these genes is required to balance phage gene expression and permit the phage to maintain a persistent infection in its host. First, the proposed research will complete studies of controls in the last 2 of the 6 genes. These genes are an in-frame overlapping pair encoding essential DNA replication genes with opposing functions. Present evidence indicates that the genes are regulated at several points, including mRNA stability. We will strengthen evidence that RNA structure inhibits translation of the smaller gene and identify apparently multiple factors that limit internal initiation on the mRNA encoding both genes. Of broader interest, this will clarify how regulation of gene expression in in- frame overlapping gene pairs, numerous in bacterial, phage and plasmid genomes, is achieved. Second, the proposed research will focus heavily on the pathway by which these very abundant phage mRNAs undergo mRNA degradation. The specific rationales are that these mRNAs will reflect a major activity on the decay machinery in infected hosts, and more important, the phage mRNAs illustrate a 5' to 3' wave of decay that is the predominant pathway for decay of bacterial mRNAs. Messenger RNA decay has proven an important parameter that determines levels of gene expression and permits rapid responses to cellular signalling signals, but until recent accelerated progress, has been the slowest of the principal gene regulatory processes to be worked out. Currently, the field is poised to make major progress. As part of this effort, we will define the steps of the f1 mRNA decay pathway, the major aim being to test proposed molecular models for decay. We will identify the cleavages made by major enzymes with functions in endo- and exonucleolytic decay, and as part of planning for in vitro reconstitution studies, define the decay pathways for smaller f1 mRNA substrates. In a new direction, we will follow f1 mRNA decay and degradosome localization within the bacterial cell.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Rhoades, Marcus M
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Duke University
Schools of Medicine
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Yu, Jae-Sung; Kokoska, Robert J; Khemici, Vanessa et al. (2007) In-frame overlapping genes: the challenges for regulating gene expression. Mol Microbiol 63:1158-72
Yu, J S; Madison-Antenucci, S; Steege, D A (2001) Translation at higher than an optimal level interferes with coupling at an intercistronic junction. Mol Microbiol 42:821-34
Steege, D A (2000) Emerging features of mRNA decay in bacteria. RNA 6:1079-90
Goodrich, A F; Steege, D A (1999) Roles of polyadenylation and nucleolytic cleavage in the filamentous phage mRNA processing and decay pathways in Escherichia coli. RNA 5:972-85
Madison-Antenucci, S; Steege, D A (1998) Translation limits synthesis of an assembly-initiating coat protein of filamentous phage IKe. J Bacteriol 180:464-72
Kokoska, R J; Steege, D A (1998) Appropriate expression of filamentous phage f1 DNA replication genes II and X requires RNase E-dependent processing and separate mRNAs. J Bacteriol 180:3245-9
Stump, M D; Madison-Antenucci, S; Kokoska, R J et al. (1997) Filamentous phage IKe mRNAs conserve form and function despite divergence in regulatory elements. J Mol Biol 266:51-65
Stump, M D; Steege, D A (1996) Functional analysis of filamentous phage f1 mRNA processing sites. RNA 2:1286-94
Ivey-Hoyle, M; Steege, D A (1992) Mutational analysis of an inherently defective translation initiation site. J Mol Biol 224:1039-54
Pavco, P A; Steege, D A (1991) Characterization of elongating T7 and SP6 RNA polymerases and their response to a roadblock generated by a site-specific DNA binding protein. Nucleic Acids Res 19:4639-46

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