Protein and DNA Effectors Control the TraI Conjugative Helicase of Plasmid R1

Abstract

Controlled duplex DNA unwinding is a crucial prerequisite for the expression and maintenance of genomes. Genomemanipulating and -regulating proteins are central to that biological function in recognizing appropriate DNA targets at initiation sequences and unwinding the complementary strands to provide single-stranded DNA (ssDNA) templates for nucleic acid synthesis and other processing reactions. The protein machineries involved include nucleic acid helicases. DNA helicases are powerful enzymes that convert the energy of nucleoside triphosphate hydrolysis to directional DNA strand translocation and separation of the double helix into its constituent single strands (for reviews, see references 13, 14, 16, 38, 55, and 64). By necessity, these enzymes interact with DNA strands via mechanisms independent of sequence recognition. At replication initiation helicases gain controlled access to the doublestranded genome at positions determined by the DNA binding properties of initiator proteins that comprise an origin recognition complex (1,9,17,31,45,66). The mechanisms supporting localized unwinding within the complex include initiatorinduced DNA looping, wrapping, and bending and feature regions of low thermodynamic stability. The exposed ssDNA mediates helicase binding followed by directional translocation along that strand until the enzyme engages the duplex for unwinding.In the MOB F family of conjugation systems, the plasmid DNA strand destined for transfer (T strand) is unwound from its complement by a dedicated conjugative helicase, TraI of F-like plasmids or TrwC of the IncW paradigm. These enzymes are remarkable in that the same polypeptides additionally harbor in a distinct domain a DNA transesterase activity. That function is required to recognize and cleave the precise phosphodiester bond, nic, in the T strand where unwinding of the secretion substrate begins. In current models the conjugative helicases are thus targeted to the transfer origin (oriT) of their cognate plasmid by the high-affinity DNA sequence interactions of their N-terminal DNA transesterase domains. In the bacterial cell, recruitment and activation of the conjugative helicase occur not on naked DNA but within an initiator complex called the relaxosome (67). For the F-like plasmid R1, sequence-specific DNA binding properties of the plasmid proteins TraI, TraY, TraM, and the host integration factor (IHF) direct assembly of the relaxosome at oriT (10,12,29,33,51,52). Integration of protein TraM confers recognition features to the relaxosome, which permit its selective docking to TraD, the coupling protein associated with the conjugative type IV secretion system (T4CP) (2,15,49). In current models, the T4CP forms a hexameric translocation pore at the cytoplasmic membrane that not only governs substrate entry to the envelope spanning type IV secretion machinery but also provides energy for macromolecular transport via ATP hydrolysis (36,50). These models propose that T4CPs provide not only a physical bridge between the plasmid and the type IV ...

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

“…The number of alternative components in the R1 relaxosome that enhance the cleaving reaction in vitro and in vivo (34,39) hints at a complex series of (yet-undefined) interactions that coordinate the production of T strand for uptake to the secretion pathway. As demonstrated in the accompanying report, TraD, TraM, and IHF indeed enhance the efficiency of TraI helicase activity at oriT, presumably by facilitating an early step in the multistep process of duplex unwinding (72).…”

Single-Stranded DNA Binding by F TraI Relaxase and Helicase Domains Is Coordinately Regulated

“…R1 TraI⌬N308 binds ssDNA with an affinity reduced approximately 10-fold relative to the level for R1 TraI ( Table 2), indicating that amino acids Nterminal to residue 309 affect TraI ssDNA binding. The R1 TraI⌬N308 fragment, however, displays wild-type helicase activity (41).…”

Functional Organization of MobB, a Small Protein Required for Efficient Conjugal Transfer of Plasmid R1162

“…Thus, TraM could directly promote the entry of plasmid DNA into the type IV secretory pathway. At the same time, TraM also stimulates both relaxosome-induced nicking and unwinding by the relaxosome-linked helicase (17,21,33).…”