TrfA dimers play a role in copy-number control of RK2 replication.

摘要: Copy-number regulation of the broad-host-range plasmid RK2 is dependent on plasmid-encoded initiator protein, TrfA, and origin replication. The handcuffing model for copy-number control proposes that TrfA-bound oris reversibly couple to prevent further initiation replication when copy number in vivo at or above replicon-specific number. TrfA mutants have been isolated which allow oriV elevated numbers. To better understand mechanism 'handcuffing', copy-up TrfA(G254D/S267L) mutant was characterized further. In present study we show by size exclusion chromatography native gel electrophoresis unlike wt largely dimeric, purified His6-TrfA(G254D/S267L) primarily monomeric. vivo, TrfA33(G254D/S267L) supports an ori trans a greatly number, while cis exhibits runaway However, expression either two previously DNA-binding defective mutants, TrfA33(P151S) TrfA33(S257F), cell transformed with mini-RK2 replicon encoding results suppression phenotype. His6-TrfA(P151S) His6-TrfA(S257F) purify as dimers, expressed are incapable supporting contrast, combination trfA(P151S) trfA(S257F) mutation trfA(G254D/S267L) mutations proteins mainly monomers can no longer restore directed vivo. On basis these findings proposed, whereby oriV-bound coupled dimeric molecules.