Figure 1.

MvaT forms oligomers in vivo. (A) Schematic representation of the bacterial genetic assay used to study MvaT oligomerization in vivo. In this system MvaT functionally replaces the dimerization domain of the bacteriophage λ CI protein (λCI). Occupancy of the λ operator located between the promoter −35 and −10 elements) by λCI prevents RNAP from binding the promoter and represses transcription. (B) Transcription repression by λCI and the λCI_NTD-MvaT fusion protein. FW123 cells containing a plasmid directing the IPTG-dependent synthesis of either the N-terminal domain and linker of λCI (λCI_NTD), λCI, or the λCI_NTD-MvaT fusion protein were grown in the presence of 20 µM IPTG and assayed for β-galactosidase activity. (C) Schematic representation of the bacterial two-hybrid assay used to detect MvaT higher-order oligomer formation. Contact between the λCI_NTD-MvaT fusion protein and MvaT (residues 2–124) fused to the N-terminal domain and linker of the α subunit of E. coli RNAP (α-MvaT), activates transcription from the test promoter which contains the λ operator O_R2 centered 42 bp upstream of the transcription start site of promoter P_RM. Note that when the λCI_NTD-MvaT fusion protein is bound to O_R2 at this position, the CI moiety of the fusion protein contacts region 4 of σ⁷⁰ bound to the −35 element of the test promoter (interaction not depicted). In E. coli strain BN30, the P_RM test promoter is linked to lacZ on an F′ episome. (D) Transcription activation by the λCI_NTD-MvaT fusion protein in the presence of the α-MvaT fusion protein. BN30 cells harboring compatible plasmids synthesizing the indicated proteins were grown and assayed for β-galactosidase activity.