A point mutation at the junction of domain 2.3/2.4 of transcription factor σ70 abrogates productive transcription and restores its expected mobility on a denaturing gel

Vijaya Gopal, Hui Wen Ma, Kumaran Kuppusamy Mande, Dipankar Chatterji*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    25 Scopus citations

    Abstract

    Region 2 of eubacterial σ 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 σ 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 σ70 of Escherichia coli RNA polymerase. These are W433F, W433G and W434G. σ70-W433F is found to be indistinguishable from the native σ factor by both structural and functional analysis. σ70-W433G shows anomalous mobility on SDS-PAGK like the native σ factor, is α-helical in conformation (50% helicity) although found to be less active in total transcription when reconstituted with core RNA polymerase. Free σ70-W434G, unlike the native a factor, shows the expected mobility of a 70 kDa protein on SDS-PAGE and has 20% helicity. Time-resolved fluorescence analysis indicates that free σ70-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 σ70 giving rise to the stability of the protein under denaturing conditions.

    Original languageEnglish (US)
    Pages (from-to)9-22
    Number of pages14
    JournalJournal of Molecular Biology
    Volume242
    Issue number1
    DOIs
    StatePublished - Jan 1 1994

    Keywords

    • Domain 2.3/2.4
    • Normal mobility
    • Transcription factor σ
    • Tryptophan mutation

    ASJC Scopus subject areas

    • Structural Biology
    • Molecular Biology

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