Attenuation control of ilvBNC in Corynebacterium glutamicum: Evidence of leader peptide formation without the presence of a ribosome binding site

The ilvBNC operon of Corynebacterium glutamicum encodes acetohydroxy acid s ynthase and isomero-reductase, which are key enzymes of L-isoleucine, L-val ine and L-leucine syntheses. In this study we identified the transcript ini tiation site of ilvBNC operon 292 nucleotides in front of the first structu ral gene, and detected the formation of a short transcript from the leader region in addition to the full length transcript of the operon. This identi fies the control of ilvBNC transcription by an attenuation mechanism involv ing antitermination. Mutations in the leader region were made and their eff ect on the operon expression in ilvB'lacZ fusions was quantified, Although a presumed leader-peptide-coding region is only one nucleotide away from th e transcript initiation site determined, there is clear evidence to support the formation of this leader peptide: (i) the substitution of initiation c odon ATG of the peptide by AGG reduced lacZ expression of the appropriate f usion construct to 19%; (ii) the replacement of three subsequent Val codons by Ala codons resulted in the loss of Val-dependent expression; and (iii) a leader peptide LacZ fusion resulted in active beta -galactosidase. Based on these results, it is concluded that transcription of ilvBNC is controlle d by a translational-coupled attenuation mechanism. The absence of a riboso me binding site for leader peptide formation means that additional mechanis ms may contribute to the transcription control at the decoding initiation s tep in the leader peptide formation.