Characterizing a Functional Helicase Domain within the E. coli F plasmid TraI Protein (2008)

Undergraduates: Jiayin Xue, Dr. Scott Lujan Dr. Laura Guogas

Faculty Advisor: Matthew Redinbo
Department: Chemistry

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TraI is a 1,756 amino acid plasmid-encoded protein central to conjugative DNA transfer. TraI functions both as a transesterase and a helicase; it nicks one strand of DNA on the E. coli F plasmid origin of transfer site, and then unwinds the plasmid in the 5’ to 3’ direction. While the structure and active site mechanism of the transesterase domain have been elucidated, details about the functional helicase domain remain largely unresolved. When isolated, the TraI960-1504 amino acid region, which contains all the necessary helicase primary sequence motifs, exhibits minimal helicase activity. Thus, a range of TraI constructs encompassing the helicase domain and surrounding regions were expressed and purified to examine their DNA binding and ATP hydrolysis activities. I found that there are at least two DNA binding sites adjacent to the helicase domain, and that the conserved helicase region does not bind DNA well. In addition, I identified a 650 amino acid region N-terminal to the conserved helicase domain that is essential to both DNA binding and efficient ATP hydrolysis activity. Taken together, my results show that the funcitonal TraI helicase domain encompasses ~1,200 residues of this 1,745 amino acid enzyme.