RECOGNITION OF PROMOTER DNA BY SUBDOMAIN-4.2 OF ESCHERICHIA-COLI-SIGMA(70) - A KNOWLEDGE-BASED MODEL OF -35-HEXAMER INTERACTION WITH 4.2-HELIX-TURN-HELIX MOTIF

In Escherichia call, subdomains 2.4 and 4.2 of the primary transcripti on factor sigma(70) are the most highly conserved regions and are resp onsible for the recognition of -10 and -35 promoter elements respectiv ely Mutational studies provide evidence to this end and indicate that the side chains of subdomain 4.2 make specific contacts with the nucle otides at -35. Subdomain 4.2 is highly conserved among group-1 sigma f actors and is strongly homologous to the classical helix-turn-helix (H TH) motif shared by bacteriophage lambda cI, Cro, the CAP protein and other homeodomain proteins, suggesting that a factor also belongs to t he HTH class of proteins. In this study, a single point mutation of th e conserved hydrophobic residue valine at position 576, in the 4.2 sub domain results in a mutant that is transcriptionally inefficient altho ugh conformationally similar to wild-type sigma. The mutant sigma, lik e wild-type, migrates as a 87 kDa protein on SDS gels and has 50% heli city. However, transcription at 'extended -10 promoter' by RNA polymer ase containing mutant sigma(70)-V576G synthesized appreciable amount o f RNA product, when compared with that generated by sigma(70)-W434G, a mutation in -10 DNA binding domain. A model of HTH motif for the cons erved 20 residue region of 4.2 domain of E. coli sigma(70) as well as its mutant sigma(70)-V576G and sigma(70)-V576T were constructed based on five other homologous HTH motifs from DNA-protein complexes for whi ch X-ray or NMR structure is available. A B-DNA structure was designed for -35 region using sequence dependent base pair parameters. The mod eled HTH structure was docked into the major groove formed by the -35 hexamer DNA using the DNA-recognition rules and amino acid - nucleotid e base contact information of homologous DNA-protein complexes. Analys is of the residue contact information of the model was tested and foun d to have good agreement with the experimental reports.