Mode of action of the mobile genetic element Tn5: A study of the roles of the RecA protease in Tn5 excision and transposition in Escherichia coli
Abstract
Tn5 is a bacterial composite transposon that is composed of two insertion sequences. It ordinarily excises and transposes in Escherichia coli by recA-independent mechanisms. However, I have found that RecA proteins that are constitutively activated to the protease state (Prt$\sp{\rm c})$ greatly enhance both excision and transposition of Tn5. Contrary to current models, the two events occur concomitantly and without destruction of the donor DNA. By studying the excision of Tn5 from the umuC gene in the presence of the recA(Prt$\sp{\rm c})$ gene, I found that the cells were heterogeneous in the extent of restored UmuC$\sp+$ function, indicating that excision of the Tn5 left the gene in a variety of states from nearly nonfunctional to fully functional. This result suggests that excision can occur with a variable degree of precision. The RecA encodes both a protease and a recombinase activity. With a split-phenotype recA(Prt$\sp{\rm c}$ Rec$\sp-)$ mutant I found that the phenomenon of highly stimulated excision and transposition does not require the recombinase function of the RecA protein. Several lines of evidence indicate that the frequency of Tn5 transposition under constitutive SOS conditions is site-dependent. I have shown that a lexA(Def) mutation derepresses expression of the Tn5 transposase (tnp) gene in vivo and the LexA protein binds to an SOS box within the tnp promoter site in vitro. These features show that the IS50R tnp gene is a member of the SOS regulon. By measuring the LexA binding affinities for recA and tnp operators, I learned that ionic strength plays an important role in determining the specificity of binding to the SOS box. Genetic evidence indicates that the RecA(Prt$\sp{\rm c})$ proteins play at least two roles in the phenomenon of Tn5 excision and transposition. One is to turn on SOS genes, including the Tn5 transposase gene, by mediating the proteolytic cleavage of LexA. A second, and even more important, role also seems to involve the proteolytic function. To investigate this additional role of RecA(Prt$\sp{\rm c})$ in stimulating Tn5 transposition, anti-Tnp antibody was prepared and used in analyzing the IS50R-encoded proteins in recA(Prt$\sp{\rm c})$ cells. The results suggest that two transposase species may exist, one a precursor and the other the functional product. The RecA(Prt$\sp{\rm c})$ protease activity may somehow mediate the cleavage of Tnp, thus shifting the ratio of these two species. Therefore, the combination of RecA(Prt$\sp{\rm c})$ protein and cleaved LexA protein would result in a higher ratio of functional Tnp protein to Inh protein, which would produce the observed increase in frequency.
Degree
Ph.D.
Advisors
Tessman, Purdue University.
Subject Area
Molecular biology|Microbiology|Genetics
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