Recognition of Promoter DNA by Subdomain 4.2 of Escherichia Coli σ⁷⁰: A Knowledge Based Model of -35 Hexamer Interaction With 4.2 Helix-Turn-Helix Motif

Abstract

In Escherichia coli, subdomains 2.4 and 4.2 of the primary transcription factor σ⁷⁰ are the most highly conserved regions and are responsible for the recognition of −10 and −35 promoter elements respectively. Mutational studies provide evidence to this end and indicate that the side chains of subdomain 4.2 make specific contacts with the nucleotides at −35. Subdomain 4.2 is highly conserved among group-1 sigma factors and is strongly homologous to the classical helix-turn-helix (HTH) motif shared by bacteriophage λcI, Cro, the CAP protein and other homeodomain proteins, suggesting that ? factor also belongs to the HTH class of proteins. In this study, a single point mutation of the conserved hydrophobic residue valine at position 576, in the 4.2 subdomain results in a mutant that is transcriptionally inefficient although conformationally similar to wild-type sigma. The mutant sigma, like wild-type, migrates as a 87 kDa protein on SDS gels and has 50% helicity. However, transcription at ‘extended −10 promoter’ by RNA polymerase containing mutant δ⁷⁰-V576G, synthesized appreciable amount of RNA product, when compared with that generated by δ⁷⁰-W434G, a mutation in −10 DNA binding domain. A model of HTH motif for the conserved 20 residue region of 4.2 domain of E. coli δ⁷⁰ as well as its mutant δ⁷⁰-V576G and δ⁷⁰-V576T were constructed based on five other homologous HTH motifs from DNA-protein complexes for which X-ray or NMR structure is available. A B-DNA structure was designed for −35 region using sequence dependent base pair parameters. The modeled HTH structure was docked into the major groove formed by the −35 hexamer DNA using the DNA-recognition rules and amino acid—;nucleotide base contact information of homologous DNA-protein complexes. Analysis of the residue contact information of the model was tested and found to have good agreement with the experimental reports.