Specificity of Mnt 'master residue' obtained from in vivo and in vitro selections

Abstract

Mnt is a repressor from phage P22 that belongs to the ribbon-helix-helix family of DNA binding factors. Four amino acids from the N-terminus of the protein, Arg2, His6, Asn8 and Arg10, interact with the base pairs of the DNA to provide the sequence specificity. Raumann et al. (Nature Struct. Biol., 2, 1115-1122) identified position 6 as a 'master residue' that controls the specificity of the protein. Models for the interaction have residue 6 of Mnt interacting directly with position 5 of the operator. In vivo selections demonstrated that protein variants at residue 6 bound specifically to operator mutations at that position. Operators in which the wild-type G at position 5 was replaced by T specifically bound to several different protein variants, primarily hydrophobic residues. The obtained protein variants, plus some others, were used in in vitro selections to determine their preferred binding sites. The results showed that the residue at position 6 influenced the preference for binding site bases predominantly at position 5, but that the effects of altering it can extend over longer distances, consistent with its designation as a 'master residue'. The similarities of binding sites for different residues do not correlate strongly with common measures of amino acid similarities.

Figure 1

Model for the Mnt–operator interaction from Knight and Sauer (3) and Raumann et al. (8). Positions 3–19 are thought to include all of the direct interactions with the protein. The amino acids modeled to interact directly with the DNA are Arg2, His6, Asn8 and Arg10. The figure shows the two dimers, one underlined the other not, that each bind to one half-site. Each dimer contains an anti-parallel β-ribbon of the N-terminal amino acids. Amino acids R2, H6 and N8 from one monomer of each dimer (in blue) and amino acids N8 and R10 of the other monomer (in green) are thought to interact with the base pairs indicated.

Figure 2

Plasmid designs for interference selections. Plasmid pSR192 contains the Mnt protein that is randomized at codons 2 and 6. Plasmid pSR200 contains one of the selection operators where Mnt binding will repress transcription from P_strong and allow the cells to be Sp resistant.

Figure 3

Selected binding sites for each Mnt variant obtained in the SELEX experiments. The binding sites, which contain the conserved core operator sequence CCACSGTGG (shaded), are aligned between the spaces in the sequences. Upper case letters are from the randomized regions and lower case letters are from the fixed regions. Sometimes a fixed region obtained a mutation, and those have been capitalized (e.g. H6A:1). The number in parentheses following some of the sequences are the number of times that sequence appeared in the selected collection.