Tuberculosis has reemerged as a serious threat to human health because of the increasing prevalence of drug-resistant strains and synergetic infection with HIV, prompting an urgent need for new and more efficient treatments. The PhoP-PhoR two-component system of Mycobacterium tuberculosis plays an important role in the virulence of the pathogen and thus represents a potential drug target. To study the mechanism of gene transcription regulation by response regulator PhoP, we identified a high-affinity DNA sequence for PhoP binding using systematic evolution of ligands by exponential enrichment. The sequence contains a direct repeat of two 7 bp motifs separated by a 4 bp spacer, TCACAGC(N4)TCACAGC. The specificity of the direct-repeat sequence for PhoP binding was confirmed by isothermal titration calorimetry and electrophoretic mobility shift assays. PhoP binds to the direct repeat as a dimer in a highly cooperative manner. We found many genes previously identified to be regulated by PhoP that contain the direct-repeat motif in their promoter sequences. Synthetic DNA fragments at the putative promoter-binding sites bind PhoP with variable affinity, which is related to the number of mismatches in the 7 bp motifs, the positions of the mismatches, and the spacer and flanking sequences. Phosphorylation of PhoP increases the affinity but does not change the specificity of DNA binding. Overall, our results confirm the direct-repeat sequence as the consensus motif for PhoP binding and thus pave the way for identification of PhoP directly regulated genes in different mycobacterial genomes.

Proteins with a conserved Cys- and His-rich SQUAMOSA promoter binding protein (SBP) domain are transcription factors restricted to photosynthetic organisms that possess a novel two Zn-finger structure DNA-binding domain. Despite the fact that altered expression of some SBP-encoding genes has profound effects on organism growth and development, little is known about SBP domain protein target genes. Misexpression of the Arabidopsis thaliana AtSPL14 SBP domain gene confers resistance to programmed cell death and modifies plant architecture. A consensus DNA-binding motif for AtSPL14 was identified by systematic evolution of ligands by exponential enrichment (SELEX) or random binding site selection (RBSS). DNA recognized by AtSPL14 contained the core binding motif (GTAC) found for other SBP domain proteins, but mutational analyses indicated that at least one additional flanking nucleotide is necessary for effective AtSPL14-DNA interaction. Comparison of several SBP domain amino acid sequences allows us to hypothesize which specific amino acids might participate in this sequence-specific DNA recognition. Electrophoretic mobility shift assays (EMSA) with mutant AtSPL14 DNA-binding domain proteins indicated that not all of the Zn (2+) ion coordinating ligands in the second Zn structure are strictly required for DNA binding. Surface plasmon resonance (SPR) was used to evaluate AtSPL14 in vitro binding kinetics for comparison of equilibrium binding constants with other SBP domain proteins. These data provide a strong basis for further experiments aimed at defining and distinguishing the sets of genes regulated by the closely related SBP domain family members.

The TraM protein encoded by plasmid R1 has three functions in conjugative transfer: (i) it positively controls transfer gene expression, (ii) it stimulates efficient site-specific ssDNA cleavage at the oriT in vivo and (iii) it couples the relaxosome to the envelope-bound transport complex. Plasmid R1 contains two binding regions for TraM, sbmA and sbmB, either of which comprises several minimal TraM targets. SELEX of a randomized minimal (18 bp) TraM binding sequence was used to search for sequences that bind TraM most efficiently. This approach resulted in a sequence with a modular structure with two 7 bp consensus motifs 5\' T1G2A3N4T5C6R7 and 5\' T1G2A3N4T5Y6R7 spaced by a 4 bp linker with the consensus sequence 5\' Y1A2/C2T3/G3A4. EMSAs revealed that evolution in vivo was for maximal binding affinity. R1 derivatives with mutated sbmA and sbmB sites based on one in vitro selected sequence were found to be either 5-fold impaired (mut sbmA) in conjugation or could not be transferred by conjugation anymore (mut sbmB). Transcriptional lacZ fusions demonstrated that the introduced sbmB mutations affected promoter activity severely and abrogated autoregulation by TraM, thereby explaining this drastic effect on conjugal transfer. The implications of these findings are discussed in the context of the role of TraM.