[Research Interests] [Representative Publications] [Lab Members] RESEARCH INTERESTSResearch Overview My lab studies the structural biology of bacterial conjugation. Conjugation is a method of DNA transfer that can occur between even distantly related bacterial species, facilitating the dissemination of drug resistance and virulence factors throughout bacterial populations. By exploiting a variety of biophysical and biochemical techniques including X-ray crystallography, calorimetry, fluorescence spectroscopy, and mutagenesis, we intend to describe the various steps in this complex biological process in structural terms, attaining atomic resolution wherever possible. The information we obtain will enable us to better understand the function, organization, regulation and structures of the macromolecular assemblies that carry out this process. During bacterial conjugation, plasmid-encoded Tra proteins direct transfer of DNA, in single-stranded form, from a donor to a recipient cell. For F plasmid of E. coli, the first identified of the conjugative plasmids, the process initially involves expression of pili by the donor bacterium. Recipient bacteria can adhere to the pili, and through retraction of a pilus, the donor and recipient come into contact, eventually forming a stable “mating pair”. Plasmid DNA in the donor is nicked, unwound, and separated into single strands. One strand is transferred to the site of contact between the cells and transported across the membranes into the recipient. The DNA in the recipient is circularized, and complementary DNA strands are synthesized in both donor and recipient. While the F plasmid tra genes have all been sequenced and the system thoroughly studied on the genetic level, comparatively little is known about the biochemical or structural basis of the activity of the Tra proteins. Our current focus is the DNA nicking and initiation of unwinding of F plasmid. The TraI protein is central to both of these steps, as TraI possesses both DNA nucleolytic and DNA helicase activities. TraI does not act alone, however, with its optimal nicking activity requiring two accessory proteins. These are the F plasmid-encoded TraY, and the host-encoded integration host factor (IHF), both of which bind to DNA sequences proximal to the TraI nicking site. Following DNA nicking, unwinding of the plasmid by TraI is delayed until a signal indicating formation of a stable mating complex is received. Responding to this signal, the TraI molecule is converted from an apparently quiescent state to a functioning helicase. We are attempting to answer two questions. First, how do TraY and IHF enhance the TraI nicking activity? TraI recognizes and cuts a single-stranded DNA conformation. IHF and TraY may induce this single-stranded conformation by binding to nearby DNA sequences. Alternatively, IHF and TraY may directly interact with TraI, properly orienting TraI relative to the DNA. Second, what is the nature of the signal that indicates mating pair formation, and what is the nature of the TraI conversion? To successfully answer these questions, we must employ a multidisciplinary approach. We are examining the in vitro functions of the proteins, characterizing their interactions, and attempting to gather both low and high resolution structural data. The data on protein function will be analyzed with reference to the structural data, and these data will be used in turn to plan experiments to further characterize the proteins and to test and refine proposed models of function. By careful in vitro examination of these (and eventually additional) Tra proteins, we will piece together a detailed model of how this complex biological process proceeds in vivo.
TraI is among the largest soluble proteins in E. coli. It initiates F plasmid transfer by creating a site specific break in the plasmid backbone at the origin of transfer. In order to facilitate the study of the single stranded DNA binding and nicking functions of TraI a 36 kDa fragment of the full length protein has been created (Street, 2003). This fragment (known as TraI36) retains the specific nicking and binding of the full length protein but lacks the helicase activity. This fragment has been characterized via mutagenesis where we targeted conserved residues in an attempt to elucidate the important residues for DNA binding and cleavage. These mutagenesis studies have revealed residues important for DNA cleavage, binding affinity and binding specificity. Additionally the structure of TraI36 has recently been solved by x-ray crystallography (Datta, 2003). The structure will facilitate new directions in the study of TraI36 as well as the full length TraI. Further mutagenic and crystallographic studies will be perused with TraI36 in order to fully elucidate its DNA cleavage and binding functions.
Overall structure of the TraI relaxase domain (TraI36) Catalytically important residues shown as sticks.
Left: The metal binding cluster of TraI36. A bound Mg++ was identified in the structure Right: The proposed DNA binding cleft of TraI36, residues identified as contributing to DNA binding are highlighted Here is another page of TraI structure pictures. TraY - Regulation and Relaxosome Formation TraY is an important part of the relaxosome. It specifically binds to double stranded DNA and is believed to be responsible for creating the ssDNA conformation necessary for TraI to nick and initiate transfer. TraY also specifically binds to the TraYI promoter to up regulate many tra genes. As the lab is interested in the structure of the relaxosome we are focusing on how TraY contacts its DNA recognition site and how it may influence relaxosome formation. Both structural and biochemical studies need to be completed with TraY to better understand the nature of the relaxosome. REPRESENTATIVE PUBLICATIONSLarkin C, Haft RJ, Harley MJ, Traxler B, Schildbach JF. (2007) Roles of active site residues and the HUH motif of the F plasmid TraI relaxase. J. Biol Chem. 282, 33707-13. Williams SL, Schildbach JF. (2007) TraY and integration host factor oriT binding sites and F conjugal transfer: sequence variations, but not altered spacing, are tolerated. J Bacteriol. 189, 3813-23. Williams SL, Schildbach JF. (2006) Examination of an inverted repeat within the F factor origin of transfer: context dependence of F TraI relaxase DNA specificity. Nucleic Acids Res. 34, 426-35. Larkin C, Datta S, Harley MJ, Anderson BJ, Ebie A, Hargreaves V, Schildbach JF. (2005) Inter- and intramolecular determinants of the specificity of single-stranded DNA binding and cleavage by the F factor relaxase. Structure (Camb). 13, 1533-44. Stern JC, Anderson BJ, Owens TJ, Schildbach JF. Energetics of the sequence-specific binding of single-stranded DNA by the F factor relaxase domain. J. Biol. Chem. 2004 279, 29155-9. Datta S, Larkin C, Schildbach JF. Structural Insights into Single-Stranded DNA Binding and Cleavage by F Factor TraI. Structure (Camb). 11, 1369-79. Harley MJ, Schildbach JF. (2003) Swapping single-stranded DNA sequence specificities of relaxases from conjugative plasmids F and R100. Proc. Natl. Acad. Sci. U.S.A. 100, 11243-8. Larkin C, Datta S, Nezami A, Dohm JA, Schildbach JF. (2003) Crystallization and preliminary X-ray characterization of the relaxase domain of F factor TraI. Acta Crystallogr D Biol Crystallogr. 59:1514-6. Street LM, Harley MJ, Stern JC, Larkin C,
Williams SL, Miller DL, Dohm JA, Rodgers
ME, Schildbach JF. (2003) Subdomain
organization and catalytic residues of the
F factor TraI relaxase domain. Biochim
Biophys Acta. 1646, 86-99. Miller DL, Schildbach JF. (2003) Evidence for a monomeric intermediate in the reversible unfolding of F factor TraM J. Mol. Biol. 321, 563-78 Lum PL, Rodgers ME, Schildbach JF. (2002) TraY DNA recognition of its two F factor binding sites. J. Mol. Biol. 4, 563-578. Harley MJ, Toptygin D, Troxler T, Schildbach JF. (2002) R150A mutant of F TraI relaxase domain: reduced affinity and specificity for single-stranded DNA and altered fluorescence anisotropy of a bound labeled oligonucleotide. Biochemistry. 40, 6460-6468 Stern, J.C., and Schildbach, J. F. (2001) DNA Recognition by F Factor TraI36; Highly Sequence-specific Binding ofÝ Single-Stranded DNA. Biochemistry. 40, 11586-11595. Lum, P. L. & Schildbach, J.F. (1999) Specific DNA Recognition by F Factor TraY Involves Beta Sheet Residues. J. Biol. Chem. 274, 19644-19658. Schildbach, J.F., Karzai, A. W., Raumann, B.E., & Sauer, R.T. (1999) Origins of DNA-binding Specificity: Role of protein contacts with the DNA backbone. Proc. Natl. Acad. Sci. U.S.A. 96, 811-817. Schildbach, J.F., Robinson, C.R., & Sauer, R.T. (1998) Biophysical Characterization of the TraY Protein of Escherichia coli F Factor. J. Biol. Chem. 273, 1329-1333.
Lab MembersGraduate Students: Casey Hemmis Sarah Williams
Laura Tsang Jia Wang
Former Graduate
Students: Matt Harley, currently a teacher at the Harker School Jenn Stern, currently a Clinical Research Scientist with BioCure Medical Dana Miller, currently a post-doc at Fred Hutchinson Cancer Institute Chris Larkin, currently a post-doc at NIH Kip Guja (M.S.), currently technician in lab Please visit our lab webpage as well!!! All molecular images were created with pymol. Can't locate your heavy atoms with a Patterson ? Try SnB. Please do the world a favor and buy a Mac, see the light! Better yet, try linux. The Mighty Beavers of Oregon State University: Back-to-Back NCAA Baseball National Champs
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n that TraI
preferentially cuts, or
stabilizing TraI-DNA recognition through
interactions with TraI. The
TraI-nicked DNA complex remains intact
until a signal indicating formation of a stable mating pair is
received. TraI then converts
into an active helicase and proceeds to
unwind and separate the plasmid DNA strands.
