Binding site specificity of the transcriptional regulator Ss-LrpB from Sulfolobus solfataricus and identification of Ss-LrpB regulated genes and operons

Ss-LrpB from Sulfolobus solfataricus is a dimeric transcriptional regulator that binds cooperatively to three targets, with as consensus the 15 bp palindrome TTGYAWWWWWTRCAA, and condenses about 100 bp of the own operator DNA into a globular nucleoprotein complex (1, 2). To determine the sequence requirements and specificity of Ss-LrpB binding we have analysed complex formation with a very large set of binding sites derived from the idealized consensus box, including all possible single bp substitutions in one half-site, several double bp substitutions, single and double bp insertions or deletions, targets containing the unusual bases inosine or deoxyuridine, or single abasic sites, altogether nearly sixty variants. Binding profiles were studied systematically with the EMSA technique (Electrophoretic Mobility Shift Assay) and extended, refined by SPR (Surface Plasmon resonance) for the wild type and a few mutant operators. SPR also allowed us to determine the kinetic on and off rate constants (kon and koff). This analysis has allowed us to establish the hierarchy in the binding preference of Ss-LrpB at each and every position of the binding site, to identify the most discriminating positions and the most contributing bases and base-specific groups, and to determine the importance of bp composition of the central minor groove segment in bending of the target site. Such an extended analysis has not yet been performed for an archaeal regulatory system. Thus we found for instance that the C-G bp at position five of the Ss-LrpB Box is the most discriminating one. These data constitute an extremely valuable tool in our search for physiologically relevant binding sites for Ss-LrpB that have to be extracted from the bunch of potential targets that was obtained by in silico analysis of the genome sequence. It is indeed crucial to discriminate between Ss-LrpB targets directly involved in regulation of promoter activity and less specific binding sites that nevertheless may play an important role in vivo, both thermodynamically, as a buffer that will set the concentration of free ss-LrpB protein, and kinetically, by regulating the rate of location of specific targets. Moreover, even very weak binding sites may play a crucial role in the regulatory process if they occur in combination with higher affinity sites and are bound in a cooperative fashion, as it happens in the own control region. The interaction of Ss-LrpB with five particularly promising control regions showing variable numbers of Ss-LrpB binding sites and organizations have been analyzed for the interaction with Ss-LrpB by EMSA, DNase I footprinting and in gel copper-phenantroline footprinting and further dissected by subcloning of the various isolated 15 bp boxes. The genes and operons associated with these control regions are the porDAB operon, encoding pyruvate ferredoxine oxidorectase, two potential transport proteins, a potential methylthioadenosine phosphorylase and a conserved gene of unknown function.

References

1. Peeters, E., Thia-Toong, T.-L., Gigot, D., Maes, D. and Charlier, D. (2004). Mol. Microbiol. 54: 321-336.
2. Peeters, E., Willaert, R., Maes, D. and Charlier, D. (2006). J. Biol. Chem. 281: 11721-11728.