THE BACILLUS-SUBTILIS DNA-REPLICATION TERMINATOR

The recent discovery of the Bacillus subtilis plasmid terminator TerLS 20 with bidirectional fork arrest activity has provided the opportunit y to probe further the structural and functional features of B. subtil is replication terminators in general. The minimal TerI and TerLS20 te rminators each comprise two 13 nt segments flanking a central trinucle otide, which is almost completely conserved in all terminators. It cor responds to the region of overlap of the two RTP binding sites (A and B) on the DNA. It has been shown that, despite this conservation, cons iderable variation in this trinucleotide region still allows fork arre st activity. Thus, the productive interaction of the RTP dimers, which presumably occurs in the vicinity of this trinucleotide region,is not dependent upon stringently defined contacts with the bases in this re gion. A completely synthetic and highly symmetrical terminator was con structed by replacing the 13 nt segment of the A site of TerI with an opposed segment identical to that in the B site. The efficient bidirec tional activity of this new terminator, TerSymB, established more firm ly the need for two opposed RTP binding sites in a functional terminat or. TerSymB was used to investigate the effect of sequence deviation i n one of the 13 nt segments, from that in the B site, on bidirectional ity of the terminator. It was found that the deviations introduced con verted the terminator significantly towards polarity of action. The pa rtial symmetry within each of the 13 nt segments of TerSymB, and the p resumed recognition of this symmetry in the binding of a symmetrical d imer of RTP to each overlapping site, suggest that the bound dimers ar e centred over positions in the DNA sequence separated by 15 nt. This separation distance has been used in conjunction with the mode of bind ing of RTP to DNA proposed by Bussiere rt nl., based on their crystal structure fur Rm, to model the interaction of the two dimers of RTP wi th unbent B-form DNA. Increased separation of the two binding sites of TerSymB was performed by inserting an extra three, seven or ten nucle otides centrally within the TerSymB sequence. The effects of these ins ertions on RTP binding and fork arrest activity were consistent with t he proposed positioning of the RTP dimers within the terminator sequen ce, and interaction between the dimers bound to TerSymB. A model to ac count for the generation of RTP-terminator complexes with bidirectiona l or polar fork arrest activity utilising TerSymB or TerI-VI is presen ted. (C) 1996 Academic Press Limited