Transcript analysis reveals an extended regulon and the importance of protein-protein co-operativity for the Escherichia coli methionine repressor.
We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterized previously in vitro. Repression is largely restricted to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5' promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent electrophoretic mobility-shift assay analysis showed that for two such loci their repressor affinity is higher than or comparable with the known metB operator, suggesting that they are directly regulated. This can be rationalized for one of the loci, folE, by the metabolic role of its encoded enzyme; however, the links to the other regulated loci are unclear, suggesting both an extension to the known met regulon and additional complexity to the role of the repressor. The plasmid gene replacement system has been used to examine the importance of protein-protein co-operativity in operator saturation using the structurally characterized mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein-protein and protein-DNA affinity.
Marincs
Ferenc
F
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Manfield
Iain W
IW
Stead
Jonathan A
JA
McDowall
Kenneth J
KJ
Stockley
Peter G
PG
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D009875:Operator Regions, Genetic; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D018085:Regulon; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D012097:Repressor Proteins; C489220:methionine repressor protein, E coli; D014364:Tryptophan