Figure 3. Genes encoding TktD and TktE are promoter-distal in a five-gene operon. A. The pagR gene is divergently transcribed from the operon containing the genes encoding the two subunits of TktC (tktD and tktE ). The first three genes of the operon are annotated as putative phosphotransferase system (PTS)-type transporters.B. Operon PCR of the putative polycistronic operon containing the transketolase genes tktD and tktE encoding the subunits of TktC. RNA, cDNA and gDNA were PCR amplified using primers specific to overlapping regions between genes of the putative operon designated 1-4 in panel A. Positive controls are amplification of gDNA; negative controls are no amplification of RNA samples; experimental samples were the cDNA amplifications. Ladder on left shows corresponding amplification size (2-Log DNA Ladder, NEB).
PagR binds directly to the promoter region for its own gene and the polycistronic mRNA encoding tktD and tktE. To determine whether PagR directly bound to the promoter driving the expression of the tktD and tktE genes or was acting indirectly to repress the expression of the operon, PagR protein was purified to >95% homogeneity (Fig. S1) and tested for its ability to bind to the putative promoter region between stm2344 andpagR (Fig. 3A). The putative promoter fragment of pagR and the polycistronic operon comprised of the stm2340 -stm2344genes was incubated with increasing concentrations of PagR protein then separated by native polyacrylamide electrophoresis (Fig. 4). The mobility of the DNA fragment (2,457,048 to 2,457,454 nt of the chromosome; 406 nt) that included the putative promoter region decreased as a function of increasing concentrations of PagR protein as compared to the no-PagR-protein-added control. This suggested that the oligomerization state of PagR changed as its concentration increased or that multiple PagR-binding sites were present in the DNA fragment.