A point mutation at the junction of domain 2.3/2.4 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 subdomain 2.4 is involved in -10 promoter recognition. An evolutionary conserved "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 tryptophan residues at position 433 and 434 which can be used as intrinsic fluorescent markers to study their structure-function relationship. Site-directed mutagenesis of these two tryptophan residues has been carried 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 structural and functional analysis. sigma 70-W433G shows anomalous mobility on SDS-PAGE like the native sigma factor, is alpha-helical in conformation (50% helicity) although found to be less active in total transcription when reconstituted with core RNA polymerase. Free sigma 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 fluorescence analysis indicates that free sigma 70-W434G has DNA binding ability, and displays a normal abortive initiation reaction but a decreased level of productive transcription after reconstitution with core RNA polymerase. A model is proposed in which tryptophan at position 434 interacts with the hydrophobic 1.1 domain of sigma 70 giving rise to the stability of the protein under denaturing conditions.
Gopal
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Centre for Cellular and Molecular Biology, Hyderabad, India.
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Kumaran
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Chatterji
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D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014364:Tryptophan / Q000737:chemistry
D004268:DNA-Binding Proteins; D012808:Sigma Factor; D014364:Tryptophan