Genome-wide identification of targets for the archaeal heat shock regulator phr by cell-free transcription of genomic DNA.
The hyperthermophilic archaeon Pyrococcus furiosus grows optimally near 100 degrees C and undergoes a heat shock response at 105 degrees C, mediated at least in part by the heat shock regulator Phr. Genes encoding a small heat shock protein (HSP20) and a member of the AAA(+) ATPase are the only known targets of the regulator, but a genetic mutant of Phr has yet to be characterized. We describe here an alternative approach for the identification of the regulon of Phr based on cell-free transcription of fragmented chromosomal DNA in the presence or absence of the regulator and hybridization of in vitro RNA to P. furiosus whole-genome microarrays. Our results confirmed the phr, the hsp20, and the aaa(+) ATPase genes as targets of Phr and also identified six additional open reading frames, PF0624, PF1042, PF1291, PF1292, PF1488, and PF1616, as Phr-responsive genes, which include that encoding di-myo-inositol phosphate synthase. Transcription of the identified novel genes was inhibited by Phr in standard transcription assays, and the novel consensus sequence 5'-TTTAnnnACnnnnnGTnAnnAAAA-3' (uppercase letters denote a high conservation of the bases) was inferred from our data as the Phr recognition motif. Mutational evidence for the significance of this sequence as Phr recognition was provided in DNA-binding experiments.
Keese
Annette M
AM
Department of Microbiology, University of Regensburg, Universitaetsstr. 31, D-93053 Regensburg, Germany.
Schut
Gerrit J
GJ
Ouhammouch
Mohamed
M
Adams
Michael W W
MW
Thomm
Michael
M
D019843:Archaeal Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D019641:DNA, Archaeal / Q000378:metabolism*; D019848:Gene Expression Regulation, Archaeal; D006358:Hot Temperature; D046228:Microarray Analysis / Q000379:methods; D008969:Molecular Sequence Data; D011485:Protein Binding; D020027:Pyrococcus furiosus / Q000235:genetics / Q000502:physiology*; D018085:Regulon; D012097:Repressor Proteins / Q000378:metabolism*; D013312:Stress, Physiological; D014158:Transcription, Genetic
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D012097:Repressor Proteins