Subdomain organization and catalytic residues of the F factor TraI relaxase domain.
TraI from conjugative plasmid F factor is both a "relaxase" that sequence-specifically binds and cleaves single-stranded DNA (ssDNA) and a helicase that unwinds the plasmid during transfer. Using limited proteolysis of a TraI fragment, we generated a 36-kDa fragment (TraI36) retaining TraI ssDNA binding specificity and relaxase activity but lacking the ssDNA-dependent ATPase activity of the helicase. Further proteolytic digestion of TraI36 generates stable N-terminal 26-kDa (TraI26) and C-terminal 7-kDa fragments. Both TraI36 and TraI26 are stably folded and unfold in a highly cooperative manner, but TraI26 lacks affinity for ssDNA. Mutational analysis of TraI36 indicates that N-terminal residues Tyr(16) and Tyr(17) are required for efficient ssDNA cleavage but not for high-affinity ssDNA binding. Although the TraI36 N-terminus provides the relaxase catalytic residues, both N- and C-terminal structural domains participate in binding, suggesting that both domains combine to form the TraI relaxase active site.
Street
Lara M
LM
Department of Biology, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
Harley
Matthew J
MJ
Stern
Jennifer C
JC
Larkin
Chris
C
Williams
Sarah L
SL
Miller
Dana L
DL
Dohm
Julie A
JA
Rodgers
Michael E
ME
Schildbach
Joel F
JF
D001426:Bacterial Proteins; D001665:Binding Sites; D002942:Circular Dichroism; D004265:DNA Helicases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry; D004706:Endodeoxyribonucleases / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005144:F Factor / Q000737:chemistry*; D005822:Genetic Vectors; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry; D011489:Protein Denaturation; D014357:Trypsin; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D004706:Endodeoxyribonucleases; C120220:MobM protein, bacteria; D014357:Trypsin; C027093:TraI protein, E coli; D004265:DNA Helicases