A POINT MUTATION AT THE JUNCTION OF DOMAIN 2-CENTER-DOT-3 2-CENTER-DOT-4 OF TRANSCRIPTION FACTOR SIGMA(70) ABROGATES PRODUCTIVE TRANSCRIPTION AND RESTORES ITS EXPECTED MOBILITY ON A DENATURING GEL/

Region 2 of eubacterial sigma factors is highly conserved and the subd omain 2.4 is involved in -10 promoter recognition. An evolutionary con served ''RpoD box'' has been identified at the junction of subdomain 2 .3/2.4 in class I and class II sigma factors and there are two tryptop han residues at position 433 and 434 which can be used as intrinsic fl uorescent markers to study their structure-function relationship. Site -directed mutagenesis of these two tryptophan residues has been carrie d out to generate three variants of sigma(70) of Escherichia coli RNA polymerase. These are W433F, W433G and W434G. sigma(70)-W433F is found to be indistinguishable from the native sigma factor by both structur al and functional analysis. sigma(70)-W433G shows anomalous mobility o n SDS-PAGE like the native sigma factor, is alpha-helical in conformat ion (50% helicity) although found to be less active in total transcrip tion when reconstituted with core RNA polymerase. Free alpha(70)-W434G , unlike the native sigma factor, shows the expected mobility of a 70 kDa protein on SDS-PAGE and has 20% helicity. Time-resolved fluorescen ce analysis indicates that free sigma(70)-W434G has DNA binding abilit y, and displays a normal abortive initiation reaction but a decreased level of productive transcription after reconstitution with core RNA p olymerase. A model is proposed in which tryptophan at position 434 int eracts with the hydrophobic 1.1 domain of sigma(70) giving rise to the stability of the protein under denaturing conditions.