Regulation der Transkription des hspA-Gens in Stigmatella aurantiaca und Analyse der Funktion des HspA-Proteins

HspA (formerly SP21) of S. aurantiaca is synthesised during development or under stress. Two transcripts of the hspA gene were detected after heat shock and only one after indole treatment. A unique transcription initiation site of the monocistronic hspA gene was detected by primer extension. Analysis of the promoter proved various upstream regions to be required for maximum expression of hspA under stress conditions. To delimit the regulatory elements involved in hspA transcription that depends on heat shock, deletion / insertion mutagenesis as well as gel shift assays were performed. Three regulatory promoter regions involved in heat shock response were defined. The first region bears the sigma factor and core RNA polymerase binding sites. Deletion of this region completely blocks hspA transcription. The second domain carries putative regulator-binding sites. Heat shock and phosphorylation enhance the binding of the regulator(s) to the hspA promoter. Deletion of this region reduces hspA transcription by more than half. Deleting a sequence ranging from bp 86 to bp 140 upstream of the hspA ORF abolishes hspA transcription suggesting a cis-acting element to exist just upstream of the -35, -10 regions of the hspA promoter. These results suggest the transcription of hspA to be mainly positively regulated under heat shock conditions. hspA was expressed in E. coli resulted in a His-tagged fusion protein (HspAHis). This HspAHis tends to assemble into a large complex that consists of 26 subunits with a molecular mass of 560 kDa as judged by SEC. This oligomer of HspAHis was able to interact with unfolded CS to prevent its precipitation from solution, but not with the unfolded B-chain of insulin. A stable complex may thus formed between HspAHis and unfolded CS. Although thermotolerance and differentiation of S. aurantiaca cells is not affected when this protein is absent, it is speculated that HspA plays a protective function in vivo mainly due to its chaperone properties

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Heidelberg

Year

1999

Author dc:creator

• Shen, Hui

Contributors dc:contributor

• Dr. H. U. Schairer

Repository record source_url

http://www.ub.uni-heidelberg.de/archiv/610

OAI identifier oai:identifier

oai:archiv.ub.uni-heidelberg.de:610