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PutA DNA-binding transcriptional repressor

Synonyms: PutA
Summary:
PutA is a flavoprotein with mutually exclusive functions as a transcriptional repressor and membrane-associated enzyme. The switch between the two activities is due to conformational changes triggered by the redox state of FAD. In the presence of proline, PutA is associated with the cytoplasmic membrane and acts a bifunctional enzyme catalyzing both reactions of the |FRAME: PROUT-PWY-I| pathway: the oxidation of proline by proline dehydrogenase and subsequent oxidation to glutamate by pyrroline-5-carboxylate (P5C) dehydrogenase. The kinetics of the coupled reaction is best described by substrate channeling. In the absence of proline, PutA is cytoplasmic and functions as a transcriptional repressor of the put regulon. The N-terminal 47 residues with a ribbon-helix-helix fold contain the dimerization domain and the specific DNA-binding activity of PutA [3, 4]. The Lys9 residue is essential for recognition of put promoter DNA []. Crystal structures of this domain have been solved [3]. In the absence of proline, PutA binds to operator sequences in the putA-putP intergenic region and represses transcription, most likely by keeping RNA polymerase from binding to the putA promoter [3].
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Transcription factor      
TF conformation(s):
Name Conformation Type TF-Effector Interaction Type Apo/Holo Conformation Evidence (Confirmed, Strong, Weak) References
PutA Functional   nd [1]
Evolutionary Family: PutA
Sensing class: External sensing using transported metabolites
Connectivity class: Local Regulator
Gene name: putA
  Genome position: 1074920-1078882
  Length: 3963 bp / 1320 aa
Operon name: putA
TU(s) encoding the TF:
Transcription unit        Promoter
putA
putAp


Regulon       
Regulated gene(s) putA, putP
Multifun term(s) of regulated gene(s) MultiFun Term (List of genes associated to the multifun term)
amino acids (1)
electron donors (1)
Transcription related (1)
repressor (1)
Porters (Uni-, Sym- and Antiporters) (1)
Regulated operon(s) putA, putP
First gene in the operon(s) putA, putP
Simple and complex regulons BasR,MarA,PutA
PutA
Simple and complex regulatory phrases Regulatory phrase (List of promoters regulated by the phrase)
[PutA,-](2)


Transcription factor regulation    


Transcription factor binding sites (TFBSs) arrangements
      

  Functional conformation Function Promoter Sigma factor Central Rel-Pos Distance to first Gene Genes Sequence
LeftPos RightPos Evidence (Confirmed, Strong, Weak) References
  PutA repressor putAp Sigma70 -31.5 -74.5 putA
tgcaactttgTGCAACcatgttaaat
1078954 1078959 [BPP], [GEA], [SM] [1], [2], [3]
  PutA repressor putAp Sigma70 -21.5 -64.5 putA
tccatgatgtTGCAACtttgtgcaac
1078944 1078949 [BPP], [GEA], [SM] [3]
  PutA repressor putAp Sigma70 14.5 -29.5 putA
ttcagaaaggTGCAACttaacgttat
1078909 1078914 [BPP], [GEA], [SM] [3]
  PutA repressor putPp1 Sigma70 -81.5 -218.5 putP
atttttgaaaGGTGCAaccgcaaaaa
1079084 1079089 [BPP], [GEA], [SM] [3]
  PutA repressor putPp1 Sigma70 -54.5 -191.5 putP
tgtgagagagTGCAACctgatgaaaa
1079111 1079116 [BPP], [GEA], [SM] [3]


Alignment and PSSM for PutA TFBSs    

Aligned TFBS of PutA   
  Sequence
  GGTGCAACT
  GTTGCAACT
  TGTGCAACC
  GGTGCAACC
  AGTGCAACC

Position weight matrix (PWM).   
A	1	0	0	0	0	5	5	0	0
C	0	0	0	0	5	0	0	5	3
G	3	4	0	5	0	0	0	0	0
T	1	1	5	0	0	0	0	0	2

PWM logo   


 


Evolutionary conservation of regulatory elements    
     Note: Evolutionary conservation of regulatory interactions and promoters is limited to gammaproteobacteria.
TF-target gene evolutionary conservation
Promoter-target gene evolutionary conservation


Evidence    

 [BPP] Binding of purified proteins

 [GEA] Gene expression analysis

 [SM] Site mutation



Reference(s)    

 [1] Becker DF., Thomas EA., 2001, Redox properties of the PutA protein from Escherichia coli and the influence of the flavin redox state on PutA-DNA interactions., Biochemistry 40(15):4714-21

 [2] Brown ED., Wood JM., 1992, Redesigned purification yields a fully functional PutA protein dimer from Escherichia coli., J Biol Chem 267(18):13086-92

 [3] Zhou Y., Larson JD., Bottoms CA., Arturo EC., Henzl MT., Jenkins JL., Nix JC., Becker DF., Tanner JJ., 2008, Structural basis of the transcriptional regulation of the proline utilization regulon by multifunctional PutA., J Mol Biol 381(1):174-88

 [4] Gu D., Zhou Y., Kallhoff V., Baban B., Tanner JJ., Becker DF., 2004, Identification and characterization of the DNA-binding domain of the multifunctional PutA flavoenzyme., J Biol Chem 279(30):31171-6

 [5] Vinod MP., Bellur P., Becker DF., 2002, Electrochemical and functional characterization of the proline dehydrogenase domain of the PutA flavoprotein from Escherichia coli., Biochemistry 41(20):6525-32

 [6] Moxley MA., Tanner JJ., Becker DF., 2011, Steady-state kinetic mechanism of the proline:ubiquinone oxidoreductase activity of proline utilization A (PutA) from Escherichia coli., Arch Biochem Biophys 516(2):113-20

 [7] Lee YH., Nadaraia S., Gu D., Becker DF., Tanner JJ., 2003, Structure of the proline dehydrogenase domain of the multifunctional PutA flavoprotein., Nat Struct Biol 10(2):109-14

 [8] Ostrander EL., Larson JD., Schuermann JP., Tanner JJ., 2009, A conserved active site tyrosine residue of proline dehydrogenase helps enforce the preference for proline over hydroxyproline as the substrate., Biochemistry 48(5):951-9

 [9] Ling M., Allen SW., Wood JM., 1994, Sequence analysis identifies the proline dehydrogenase and delta 1-pyrroline-5-carboxylate dehydrogenase domains of the multifunctional Escherichia coli PutA protein., J Mol Biol 243(5):950-6

 [10] Zhang L., Alfano JR., Becker DF., 2015, Proline metabolism increases katG expression and oxidative stress resistance in Escherichia coli., J Bacteriol 197(3):431-40

 [11] Wood JM., Zadworny D., 1980, Amplification of the put genes and identification of the put gene products in Escherichia coli K12., Can J Biochem 58(10):787-96



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