A single aromatic residue in transcriptional repressor protein KorA is critical for cooperativity with its co-regulator KorB.
A central feature of broad host range IncP-1 plasmids is the set of regulatory circuits that tightly control plasmid core functions under steady-state conditions. Cooperativity between KorB and either KorA or TrbA repressor proteins is a key element of these circuits and deletion analysis has implicated the conserved C-terminal domain of KorA and TrbA in this interaction. By NMR we show that KorA and KorB interact directly and identify KorA amino acids that are affected on KorB binding. Studies on mutants showed that tyrosine 84 (or phenylalanine, in some alleles) is dispensable for repressor activity but critical for the specific interaction with KorB in both in vivo reporter gene assays and in vitro electrophoretic mobility shift and co-purification assays. This confirms that direct and specific protein-protein interactions are responsible for the cooperativity observed between KorB and its corepressors and lays the basis for determining the biological importance of this cooperativity.
Bingle
Lewis E H
LE
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Rajasekar
Karthik V
KV
Muntaha
Sidra tul
St
Nadella
Vinod
V
Hyde
Eva I
EI
Thomas
Christopher M
CM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; C090051:KorA protein, bacteria; C053863:KorB protein, Plasmid RK2; D012097:Repressor Proteins; D014443:Tyrosine; D010649:Phenylalanine; D004247:DNA