Dept. of Microbiology & Immunology

University of Tennessee, Memphis

Martha M. Howe, Ph.D.

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Office:  (901) 448-8215

Lab:  (901) 448-8216

mhowe@utmem.edu 

Office: 701A M.S.B.

Lab: 711 M.S.B.

Laboratory web page

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Research Program

Mechanisms of genetic regulation during lytic and lysogenic development of bacteriophage Mu.    Our research program is focused on elucidating the mechanisms of genetic regulation as they are exemplified in the processes occurring during lysogenic and lytic development of bacteriophage Mu, a temperate phage of Escherichia coli K-12 and several other enteric bacteria. Mu has a number of unusual features that make it an interesting model system for analysis. Furthermore, its relative simplicity (37 kb and -50 genes) and the extensive existing body of research on both Mu and E. coli make it particularly amenable to sophisticated genetic, molecular, and biochemical analysis.

    Early work on Mu from our lab and others focused on the novel characteristics of Mu DNA, especially its integration and replication. Mu integrates randomly into host DNA during both lytic and lysogenic phases of growth and exhibits characteristics similar to those of transposable elements. Mu replication occurs within the host chromosome by a process called replicative transposition in which the Mu DNA is duplicated as it transposes from one site to another in the host DNA. Mu DNA is then packaged from these integrated forms, leading to the incorporation of adjacent host sequences into the mature DNA molecule in the phage particle. Another unusual feature of Mu DNA is the presence of an invertible DNA segment whose inversion is catalyzed by a site-specific invertase called Gin and whose orientation determines the Mu host range. To protect its DNA from restriction upon infection of its different hosts, Mu also encodes its owns unique DNA modification system (mom).

    More recent work, including that from our laboratory, has focused on the novel regulatory mechanisms used to control Mu lysogenic and lytic development. Transcription during lytic development occurs in three phases. Early transcription utilized the host RNA polymerase and is stimulated by the host IHF protein. Middle and late transcription are regulated by a cascade of Mu-encoded activator proteins which promote transcription initiation by the host RNA polymerase at the middle and late promoters. The early transcript encodes Mor, the activator of middle transcription, and the middle transcript encodes C, the activator of late transcription. Despite their considerable amino acid sequence similarity, Mor and C exhibit mutually exclusive promoter specificity. In addition to Mor, middle transcription also requires Mu DNA replication, perhaps to generate a DNA template that is competent for transcription initiation. Mu also makes two repressor proteins: c which maintains the prophage in a dormant state in a lysogen, and Ner which acts to reduce early transcription during the middle and late phases of growth. Constitutive low-level expression of the Mu lig/gem gene in a lysogen alters the level of host DNA supercoiling, with consequent modulation of expression of at least several host genes. Mu utilizes a number of host functions to stimulate or reduce expression of its genes. Examples include positive regulation of early transcription by IHF, enhancement of repressor binding by H-NS, and negative regulation of mom expression by the OxyR protein. Mom syntheses is also positively regulated at the level of transcription by dam-methylation of the mom promoter and post-transcriptionally by binding the Mu-encoded com protein to the mom transcript. Lastly, the late transcripts encoding Mu morphogenetic functions are processed at one or more sites by RNase E, and the resulting RNAs are found at greatly different levels, suggesting possible post-transcriptional regulation of specific late gene expression.

    Current and prospective research in our laboratory is directed toward the following goals: 1) determination of the middle and late promoter sequences which are important for promoter activity; 2) analysis of the mechanism by which C and Mor promote initiation of transcription by host RNA polymerase, for example, by promoting RNA polymerase binding or by stimulating open complex formation; 3) definition of the DNA binding domains of C and Mor through the isolation and characterization of C and Mor mutations that suppress the "down" phenotype of specific promoter mutations; 4) identification of the activation domain of C and Mor through the isolation of mutant prteins which still bind to the appropriate promoter but cannot activate transcription; 5) determination of the RNA polymerase subunit which interacts with each activator protein; 6) determination as to whether the middle and late promoters have an unusual DNA conformation such as a bend or kink in the absence of C and Mor binding, and whether the binding of the activator protein and/or RNA polymerase alters the DNA conformation; 7) isolation and use of monoclonal antibodies directed against C and Mor to facilitate protein purification, identify structural changes in the mutant proteins, and quantitate protein levels at different stages during the lytic cycle; 8) determination of the role that DNA replication plays in activation of the middle promoter; 9) determination as to whether transcription initiation at the late promoters also requires DNA replication and, if so, why; 10) analysis of the sits and enzymes used for porcessing of the late transcript initiated at P1; 11) determination of the role RNA processing plays in regulation of specific late gene expression; and 12) definition of the mechanisms by which certain of the RNA processing products are stabilized relative to others.

    We expect that these studies will continue to reveal novel regulatory mechanisms and insights relevant to a wide variety of organisms.

Selected Publications