TableEdit

Sut, MV, Mihajlovic, S, Lang, S, Gruber, CJ and Zechner, EL (2009) Protein and DNA effectors control the TraI conjugative helicase of plasmid R1. J. Bacteriol. 191:6888-99

The mechanisms controlling progression of conjugative DNA processing from a preinitiation stage of specific plasmid strand cleavage at the transfer origin to a stage competent for unwinding the DNA strand destined for transfer remain obscure. Linear heteroduplex substrates containing double-stranded DNA binding sites for plasmid R1 relaxosome proteins and various regions of open duplex for TraI helicase loading were constructed to model putative intermediate structures in the initiation pathway. The activity of TraI was compared in steady-state multiple turnover experiments that measured the net production of unwound DNA as well as transesterase-catalyzed cleavage at nic. Helicase efficiency was enhanced by the relaxosome components TraM and integration host factor. The magnitude of stimulation depended on the proximity of the specific protein binding sites to the position of open DNA. The cytoplasmic domain of the R1 coupling protein, TraDDeltaN130, stimulated helicase efficiency on all substrates in a manner consistent with cooperative interaction and sequence-independent DNA binding. Variation in the position of duplex opening also revealed an unsuspected autoinhibition of the unwinding reaction catalyzed by full-length TraI. The activity reduction was sequence dependent and was not observed with a truncated helicase, TraIDeltaN308, lacking the site-specific DNA binding transesterase domain. Given that transesterase and helicase domains are physically tethered in the wild-type protein, this observation suggests that an intramolecular switch controls helicase activation. The data support a model where protein-protein and DNA ligand interactions at the coupling protein interface coordinate the transition initiating production and uptake of the nucleoprotein secretion substrate.

PubMed PMC2772500 Online version:10.1128/JB.00920-09

Bacterial Proteins/genetics; Bacterial Proteins/metabolism; DNA Helicases/genetics; DNA Helicases/metabolism; DNA, Bacterial/genetics; DNA, Bacterial/metabolism; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Integration Host Factors/genetics; Integration Host Factors/metabolism; Membrane Proteins/genetics; Membrane Proteins/metabolism; Models, Biological; Plasmids/genetics