PMID:15465046
| Citation |
Geszvain, K, Gruber, TM, Mooney, RA, Gross, CA and Landick, R (2004) A hydrophobic patch on the flap-tip helix of E.coli RNA polymerase mediates sigma(70) region 4 function. J. Mol. Biol. 343:569-87 |
|---|---|
| Abstract |
The Escherichia coli RNA polymerase beta subunit contains a flexible flap domain that interacts with region 4 of sigma(70) to position it for recognition of the -35 element of promoters. We report that this function depends on a hydrophobic patch on one face of the short stretch of alpha helix located at the tip of the flap domain, called the flap-tip helix. Disruption of the hydrophobic patch by the substitution of hydrophilic or charged amino acids resulted in a loss of the interaction between the flap and sigma region 4, as determined by protease sensitivity assays, and impaired transcription from -35-dependent promoters. We suggest that contact of the flap-tip helix hydrophobic patch to the sigma region 4 hydrophobic core is essential for stable interaction of the flap-tip helix with region 4. This contact allowed region 4.2 recognition of the -35 promoter element and appeared to stabilize region 4 interaction with the beta' Zn(2+) binding domain. Our studies failed to detect any role for sigma region 1.1 in establishing or maintaining the flap-sigma region 4 interaction, consistent with recent reports placing sigma region 1.1 in the downstream DNA channel. |
| Links |
PubMed Online version:10.1016/j.jmb.2004.08.063 |
| Keywords |
Amino Acid Sequence; DNA-Directed RNA Polymerases/chemistry; DNA-Directed RNA Polymerases/genetics; DNA-Directed RNA Polymerases/metabolism; Escherichia coli; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Molecular Sequence Data; Promoter Regions, Genetic; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Sequence Alignment; Sigma Factor/chemistry; Sigma Factor/genetics; Sigma Factor/metabolism; Transcription, Genetic |
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