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 language | English (US) |
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Pages (from-to) | 9-22 |
Number of pages | 14 |
Journal | Journal of molecular biology |
Volume | 242 |
Issue number | 1 |
DOIs | |
State | Published - 1994 |
Externally published | Yes |
Keywords
- Domain 2.3/2.4
- Normal mobility
- Transcription factor σ
- Tryptophan mutation
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology