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A virB-lacZ translational fusion was constructed to monitor expression of the Agrobacterium tumefaciens virB operon. Expression of the fusion gene was dependent on the presence of pTiA6 virA, virG, and a plant factor acetosyringone. Analysis of deletion mutants, constructed by exonuclease Bal31 digestion, showed that 68 residues upstream of the virB transcription initiation site was necessary for its expression. A TT----CC substitution at positions -62 and -61 led to a 7 fold reduction in virB expression. The virB upstream region contains a tetradecameric sequence, dPuT/ATDCAATGHAAPy (D = A, G or T; H = A, C or T), that is conserved in the non-transcribed regions of all vir genes. Alteration of the position of this sequence relative to the promoter region sequences had a drastic negative effect on virB expression.