In vivo and in vitro investigation of transcriptional regulation by DntR.
DntR is a bacterial transcription factor that has been isolated from Burkholderia species that are able to degrade the nitro-aromatic compound 2,4-dinitrotoluene. We recently solved the X-ray crystal structure of DntR, which suggested a putative location of an inducer-binding cavity (IBC). In this study, we constructed mutants of DntR in which residues lining the proposed IBC were modified in order to identify the structural elements involved in inducer binding, to modulate the inducer binding specificity, and to investigate the mechanism of transcriptional regulation by DntR. The transcriptional activation of the reporter gene gfp induced by the wild-type and mutant DntRs was monitored by analysing whole-cell fluorescence using flow-cytometry after addition of a number of potential inducer compounds. Three of the mutant proteins (F111L; F111V/H169V and Y110S/F111V) were purified and the binding constants for several of the potential inducers to these mutants were estimated. Furthermore, crystal structures of the F111L and Y110S/F111V mutant proteins were solved and used to explain changes in the inducer binding specificity at an atomic level. A comparison of the inducing capability in the whole-cell system and binding constants for a number of potential inducers suggests a mechanism where binding of an inducer molecule is not the sole requirement for transcriptional activation. In addition, specific interactions between DntR and the inducer molecule resulting in a conformational change of the protein are needed.
Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Rosa
R
Lönneborg
Irina
I
Smirnova
Cyril
C
Dian
Gordon A
GA
Leonard
Peter
P
Brzezinski
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D019117:Burkholderia / Q000235:genetics*; D018360:Crystallography, X-Ray; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007700:Kinetics; D050505:Mutant Proteins / Q000378:metabolism; D009574:Nitro Compounds / Q000494:pharmacology; D010957:Plasmids; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000378:metabolism; D020156:Salicylic Acid / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
D001426:Bacterial Proteins; D050505:Mutant Proteins; D009574:Nitro Compounds; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins; D020156:Salicylic Acid