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Correlations of substitutions predict specific protein-DNA contacts in the MerR family of transcriptional factors Ilya Zharov,
Kharkevich Institute for Information Transmission Problems, Russian Academy of Sciences, Moscow, 127994 , peshwalk@gmail.com

Yuriy Korostelev
Kharkevich Institute for Information Transmission Problems, Russian Academy of Sciences, Moscow, 127994 , Department of Bioengineering and Bioinformatics, Moscow State University, Moscow, Russia, 119991 , lan787@yko.name

Correlations of substitutions between the sequences of transcriptional factors (TFs) and their binding sites may be used to predict specific contacts between the protein and DNA residues. Here we apply this method to the group of heavy metal resistance TFs of the bacterial MerR family using the Prot-DNA-Korr program. MerR family TFs regulate response to different stresses (antimicrobial agents, heavy metals, heat shock, oxidative stress, nitrosative stress) as well as a number of metabolic processes (nitrogen metabolism, carotenoid biosynthesis, degradation of isoprenoids and branchedchain amino acids, curli and biofilm formation) in bacteria. A separate group of this family is responsible for transcriptional activation of heavy metal resistance (HMR) systems that detoxify and export mercury, copper, zinc, cadmium, lead, silver, and gold ions. Experimentally studied TFs from this group include MerR, CueR, HmrR, ZntR, CadR, PbrR, and GolS [1-4]. Most of the MerR family transcriptional activators including the studied TFs bind their palindromic sites between the -35 and -10 promoter boxes. Spacers of the regulated promoters are characterized by the increased length (19-20 bp comparing with average 16-17 bp). Upon binding of the TF the distance between the promoter boxes decreases and they realign for optimal DNA polymerase binding [5]. Unsurprisingly TFs that adopt this mode of regulation share very similar spatial conformation of DNA binding and dimerization domains [5-8, 10]. Their ligand-binding domains differ in sequence, size and structure. 3D-structures of DNA-bound TFs are known for BmrR, MtaN, SoxR, TipAL, GlnR and TnrA [5-11] as


well as DNA-free structures for BmrR, MtaN, CueR, ZntR, NmlR, LMOf2365_2715 and SCO5550 [11-14]. Among them only CueR and ZntR belong to the studied group. GlnR, TnrA and SCO5550 have a different DNA binding and dimer formation modes. Nevertheless these structures show that the conformations of the DNA-binding winged helix-turn-helix (WHTH) domains of the MerR family TFs and therefore their contacts with the half-sites are highly similar. For the current work we selected proteins from GenBank RefSeq database that contain HTH_CueR, HTH_MerR1, HTH_CadR-PbrR, HTH_CadR-PbrR-like and HTH_HMRTR conserved domains of GenBank CDD (1516 TFs in total). In this study they are referred to CueR, MerR, CadR-PbrR, CadR-PbrR-like and HMRTR subfamilies, respectively Structurebased sequence alignments and phylogenetic trees were built for each subfamily. After filtering out the TFs that impair the alignments as well as nearly identical TFs encoded in the genomes of close strains, 906 TFs remained for further study. Selective positional weighted matrices (PWMs) were built for searching the genomes for the binding sites. The genomes were searched in regions from -400 to +50 bp using the GenomeExplorer package. Only sites located in the long (19-20 bp) spacers of the putative 70 promoters were retained for the correlation study. 884 sites for 763 TFs were identified using this procedure.
Subfamily Number of TFs in completely assembled genomes 511 205 253 189 358 1516 Number of TFs after filtering 260 123 193 147 183 906 Number of TFs with identified TFBSs 238 105 172 100 148 763 Number of TFBSs 324 106 174 110 170 884

CueR MerR CadR-PbrR CadR-PbrR-like HMRTR Total

Table 1. TF and TFBS statistics The aligned sequences of the DNA-binding domains and their binding sites were sent to ProtDNA-Korr program. 32 correlates pairs of positions of these two alignments were identified. Then we searched the literature and the NPIDB database for the experimentally identified contacts of the MerR family TFs with DNA. A pair of positions was marked as interacting if the contact was reported at least once. In total, 36 pairs of positions with side chain to base


interactions were found. 9 pairs appear both contacting and correlated. Given 74 protein and 20 DNA alignment positions (1480 possible pairs), Fisher's exact test gives p-value of 1.96в10­8 for this overlap. We mapped several correlated pairs of positions with large (over 50) counts for overrepresented pairs of residues on the general phylogenetic tree of the studied TFs. The same overrepresented pairs `nucleotide ­ amino acid' in fixed alignment positions appeared several times independently during the evolution. We reconstructed ancestral sequences of the studied TFs and their binding sites using PAML package to test whether mutations in TFs lead to changes in binding sites. Unfortunately we could not find evidence neither for this order of changes nor for the opposite. Our results show that correlations of substitutions in the sequences of TFs and their binding sites can be used as predictors of specific contacts This is joint work with Mikhail Gelfand. 1. N.L. Brown, J.V. Stoyanov, S.P. Kidd, and J.L. Hobman (2003) The MerR family of transcriptional regulators. FEMS Microbiol. Rev., 27: 145-163. 2. J.L. Hobman, J. Wilkie and N.L. Brown (2005) A design for life: prokaryotic metalbinding MerR family regulators. Biometals, 18: 429-436. 3. P.R. Chen and C. He (2008) Selective recognition of metal ions by metalloregulatory proteins. Curr. Opin. Chem. Biol., 12: 214-221. 4. A.O. Summers (2009) Damage control: regulating defenses against toxic metals and metalloids. Curr. Opin. Microbiol., 12: 138-144. 5. E.E. Heldwein and R.G. Brennan (2001) Crystal structure of the transcription activator BmrR bound to DNA and a drug. Nature, 409: 378-382. 6. K.J. Newberry and R.G. Brennan (2004) The structural mechanism for transcription activation by MerR family member multidrug transporter activation, N terminus. J. Biol. Chem., 279: 20356-20362. 7. Newberry,K.J. et al. (2008) Structures of BmrR-drug complexes reveal a rigid multidrug binding pocket and transcription activation through tyrosine expulsion. J. Biol. Chem., 283: 26795-26804.


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