PMID- 10064133
OWN - NLM
STAT- MEDLINE
DCOM- 19990423
LR  - 20240109
IS  - 1431-6730 (Print)
IS  - 1431-6730 (Linking)
VI  - 380
IP  - 1
DP  - 1999 Jan
TI  - Efficient control of raf gene expression by CAP and two Raf repressors that bend 
      DNA in opposite directions.
PG  - 19-29
AB  - The plasmid-borne raf operon of Escherichia coli encodes proteins involved in the 
      uptake and utilisation of the trisaccharide raffinose. The operon is subject to 
      dual regulation; to negative control by the binding of RafR repressor to twin 
      operators, O1 and O2, and to positive control by the cAMP-binding protein, CAP. 
      We have identified the CAP binding site (CBS) as a 22 bp palindromic sequence 
      with incomplete dyad symmetry by deletion analysis, DNasel footprinting and 
      electrophoretic mobility shift assays (EMSA) of CAP-DNA complexes. The CBS is 
      centred 60.5 bp upstream of the transcription start point and partially overlaps 
      O1. In vivo, CAP increases rafA (alpha-galactosidase) gene expression up to 
      50-fold. The 28 bp spacing between the centres of CBS and the - 35 box is 
      essential, since insertions of 4, 8, 12 or 16 bp completely eliminated rafA gene 
      expression. In vitro binding studies revealed that the CBS, O1 and O2 sites, can 
      be simultaneously occupied by their cognate proteins. However, no cooperativity 
      between binding of CAP and RafR was detected. EMSA with circularly permuted DNA 
      fragments demonstrated that CAP and RafR proteins bend raf promoter (rafP) DNA by 
      75 degrees +/- 5 degrees and 95 degrees +/- 5 degrees, respectively, in opposite 
      directions. Among sugar catabolic operons, the compact arrangement of three 
      protein-binding sites, a CBS and two operators bounding the - 35 promoter box, is 
      unique and provides a sensitive and highly efficient device for transcriptional 
      control.
FAU - Muiznieks, I
AU  - Muiznieks I
AD  - Faculty of Biology, University of Latvia, Riga, Republic of Latvia.
FAU - Rostoks, N
AU  - Rostoks N
FAU - Schmitt, R
AU  - Schmitt R
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Germany
TA  - Biol Chem
JT  - Biological chemistry
JID - 9700112
RN  - 0 (Bacterial Proteins)
RN  - 0 (Carrier Proteins)
RN  - 0 (Cyclic AMP Receptor Protein)
RN  - 0 (DNA, Bacterial)
RN  - E0399OZS9N (Cyclic AMP)
RN  - EC 2.7.11.1 (Proto-Oncogene Proteins c-raf)
SB  - IM
MH  - Bacterial Proteins/*genetics/metabolism
MH  - Base Sequence
MH  - Binding Sites
MH  - Carrier Proteins
MH  - Cyclic AMP/*metabolism
MH  - Cyclic AMP Receptor Protein/*physiology
MH  - DNA, Bacterial/*chemistry/metabolism
MH  - *Gene Expression Regulation, Bacterial
MH  - Genes, Bacterial
MH  - Molecular Sequence Data
MH  - Nucleic Acid Conformation
MH  - Proto-Oncogene Proteins c-raf/*genetics
EDAT- 1999/03/04 00:00
MHDA- 1999/03/04 00:01
CRDT- 1999/03/04 00:00
PHST- 1999/03/04 00:00 [pubmed]
PHST- 1999/03/04 00:01 [medline]
PHST- 1999/03/04 00:00 [entrez]
AID - 10.1515/BC.1999.003 [doi]
PST - ppublish
SO  - Biol Chem. 1999 Jan;380(1):19-29. doi: 10.1515/BC.1999.003.