Identification of activating region (AR) of Escherichia coli LysR-type transcription factor CysB and CysB contact site on RNA polymerase alpha subunit at the cysP promoter.
CysB is a LysR-type transcriptional regulator (LTTR) controlling the expression of numerous genes involved in bacterial sulphur assimilation via cysteine biosynthesis. Our previous mutational analysis of CysB identified several residues within the N-terminal domain crucial for DNA-binding function. Here, we focus on the functional significance of CysB residues localized in the turn between the alpha2 and alpha3 helices forming an N-terminal helix-turn-helix motif. On the basis of the characteristics of alanine-substituted mutants, we propose that CysB residues Y27, T28 and S29, lying in this turn region, comprise an 'activating region' (AR) that is crucial for positive control of the cysP promoter, but not for DNA binding and inducer response activities of CysB. Using a library of alanine substitutions in the C-terminal domain of the RNAP alpha subunit (alpha-CTD), we identify several residues in alpha-CTD that are important for CysB-dependent transcription from the cysP promoter. After probing potential protein-protein contacts in vivo with a LexA-based two-hybrid system, we propose that the '273 determinant' on alpha-CTD, including residues K271 and E273, represents a target for interaction with CysB at the cysP promoter.
Lochowska
Anna
A
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland.
Iwanicka-Nowicka
Roksana
R
Zaim
Jolanta
J
Witkowska-Zimny
Malgorzata
M
Bolewska
Krystyna
K
Hryniewicz
Monika M
MM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019077:Helix-Turn-Helix Motifs; D008958:Models, Molecular; D008969:Molecular Sequence Data; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics
D001426:Bacterial Proteins; C062886:CysB protein, Bacteria; C094586:CysP protein, E coli; D029968:Escherichia coli Proteins; D033902:Periplasmic Binding Proteins; D021122:Protein Subunits; D014157:Transcription Factors; C073470:LysR protein, Bacteria; D003850:Deoxyribonuclease I