Kinetic analysis of the interaction between the QutA and QutR transcription-regulating proteins.
The QutR protein is a multidomain repressor protein that interacts with the QutA activator protein. Both proteins are active in the signal transduction pathway that regulates transcription of the quinic acid utilization (qut) gene cluster of the microbial eukaryote Aspergillus nidulans. In the presence of quinate, production of mRNA from the eight genes of the qut pathway is stimulated by the QutA activator protein. The QutR protein plays a key role in signal recognition and transduction, and a deletion analysis has shown that the N-terminal 88 amino acids are sufficient to inactivate QutA function in vivo. Using surface plasmon resonance we show here that the N-terminal 88 amino acids of QutR are able to bind in vitro to a region of QutA that genetic analysis has previously implicated in transcription activation. We further show that increasing the concentration of a full-length (missense) mutant QutR protein in the original mutant strain can restore its repressing function. This is interpreted to mean that the qutR mutation in this strain increases the equilibrium dissociation constant for the interaction between QutA and QutR. We propose a model in which the QutA and QutR proteins are in dynamic equilibrium between bound (transcriptionally inactive) and unbound (transcriptionally active) states.
Department of Biochemistry and Genetics, Catherine Cookson Building, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom.
Carys
C
Watts
Shi Ming
SM
Si-Hoe
Heather K
HK
Lamb
Lisa J
LJ
Levett
John R
JR
Coggins
Alastair R
AR
Hawkins
D001233:Aspergillus nidulans / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D007700:Kinetics; D008958:Models, Molecular; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C098152:QutA protein, Aspergillus nidulans; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors