MUTATIONAL ANALYSIS OF THE REGULATORY REGION OF THE MYCOBACTERIUM PLASMID PAL5000

The regulatory region of the Mycobacterium fortultum plasmid pAL5000 w as studied in vivo and in vitro by mutational analysis. This region co mprises the origin of replication for the plasmid and the start point of transcription for the repA/B genes, which encode the two replicatio n proteins RepA and RepB, In this region there are two binding sites f or RepB: a low-affinity site which is probably the origin of replicati on and a high-affinity-site which overlaps the promoter and implies an autoregulated expression of RepB. The high-affinity site contains two 8 bp pallndromes, as well as an inverted repeat structure, By introdu cing point mutations into each of these motifs and monitoring changes to RepB binding in a gel-retardation assay, it was shown that the cent ral, GC-rich palindrome (the CC-box) is the most important motif for p rotein binding. Mutations in the second, AT-rich palindrome (the AT-bo x) had no effect on protein binding and the inverted repeat structure per se was not needed, though some single-base changes affected bindin g to one or other of the DNA strands, These mutations were subsequentl y tested in vivo for their effects on plasmid replication in Mycobacte rium smegmatis., Any change to the CC-box abolished replication, but c hanges to the other motifs were dependent on the position of the chang ed base, again indicating that the inverted repeats are not essential and that the AT-box is part of the promoter and not primarily recognis ed by RepB, The mutated plasmids did not show any changes in copy numb er to that of the wild-type, The expression of RepB was boosted by int roducing a stronger promoter upstream of the repA/B genes, The resulti ng plasmid was capable of increasing to a degree in trans the copy num ber of other plasmids carrying the ori region, but was unstable when p resent on its own in M.smegmatis.