THE MINIMAL SEQUENCE NEEDED TO DEFINE A FUNCTIONAL DNA TERMINATOR IN BACILLUS-SUBTILIS

The 47 bp DNA replication terminator (IRI) of Bacillus subtilis, conta ins two binding sites, A and B, for the replication terminator protein (RTP). Each site binds a dimer of RTP. Removal of the first two base- pairs (bp 1-2) from IRI completely destroyed in vivo terminator (fork arrest) function and was accompanied by loss of RTP binding to the A s ite, which is distal to the approaching fork that is arrested. Removal of base-pairs 34 to 47 from the other end, proximal to the approachin g fork, lowered in vivo function to approximately 50% of the complete IRI. RTP binding appeared to be largely unaffected. Terminator functio n remained at the approximately 50 % level with further deletions that proceeded as far as to include base-pair 28; and RTP binding remained largely unaffected. Removal of more of the sequence beyond base-pair 27 and into the region that makes extensive contact with RTP resulted in a further impairment to in vivo function, and caused altered RTP bi nding. The base-pairs 1 to 24 segment retained only 16% fork arrest ac tivity and the effect on RTP binding was largely evidenced by an elimi nation of the ability of this extensively truncated sequence to fill t he B site alone. The behaviour of the various terminator deletions emp hasize the importance of the previously defined RTP-DNA contacts which allow the binding of RTP to the two overlapping sites, A and B, of IR I for terminator function. A comparison of the affinities of selected truncated terminators for RTP raises the possibility that the overall affinity of RTP for its DNA terminator is not the sole determinant of terminator function.