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Papers In Press, published online ahead of print March 9, 2005
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI 53706
Corresponding Author: rburgess{at}wisc.edu
J. Biol. Chem, 10.1074/jbc.M500393200
Submitted on January 12, 2005
Revised on March 9, 2005
Accepted on March 8, 2005
The global transcriptional response of Escherichia coli to induced sigma protein involves sigma regulon activation followed by inactivation and degradation of sigma in vivo
32 is the first alternative sigma factor discovered in E. coli and can direct transcription of many genes in response to heat shock stress. To define the physiological role of 32, we have used transcription profiling experiments to identify, on a genome-wide basis, genes under the control of 32 in E. coli by moderate induction of a plasmid-borne rpoH gene under defined, steady-state growth conditions. Together with a bioinformatics approach, we successfully confirmed genes previously known to be directly under the control of 32 and also assigned many additional genes to the 32 regulon. In addition, to better understand the functional relevance of the increased amount of 32 to changes in the transcriptional level of 32-dependent genes, we measured the protein level of 32 both before and after induction by a newly developed quantitative Western blot method. We found that, at a normal constant growth temperature (37C), the 32 protein level rapidly increased, plateaued, and then gradually decreased after induction, indicating 32 can be regulated by genes in its regulon and that the mechanisms of 32 synthesis, inactivation, and degradation are not strictly temperature dependent. The decrease in the transcriptional level of 32-dependent genes occurs earlier than the decrease in full-length 32 in wild-type strain, and the decrease in the transcriptional level of 32-dependent genes is greatly diminished in a DnaK deletion strain, suggesting that DnaK can act as an anti-sigma factor to functionally inactivate 32 and thus reduce 32-dependent transcription in vivo.
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