Gene F of plasmid RSF1010 codes for a low-molecular-weight repressor protein that autoregulates expression of the repAC operon

The repAC operon of plasmid RSF1010 consists of the genes for proteins E, F, RepA (DNA helicase), and RepC (origin-binding initiator protein) and is transcriptionally initiated by a promoter called P4. We have studied the expression of the repAC operon in vivo by using fusions to the lacZ reporter g...

Full description

Saved in:
Bibliographic Details
Published in:Nucleic acids research 1990-11, Vol.18 (21), p.6215-6222
Main Authors: MAESER, S, SCHOLZ, P, OTTO, S, SCHERZINGER, E
Format: Article
Language:English
Subjects:
Base Sequence
Binding Sites
Biological and medical sciences
DNA-Directed RNA Polymerases - metabolism
Escherichia coli - genetics
F protein
Fundamental and applied biological sciences. Psychology
Gene expression
Gene Expression Regulation, Bacterial
Genes, Bacterial
Molecular and cellular biology
Molecular genetics
Molecular Sequence Data
Nucleotide Mapping
Operon
Plasmids
Repressor Proteins - genetics
Repressor Proteins - isolation & purification
Repressor Proteins - metabolism
Restriction Mapping
Transcription, Genetic
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:The repAC operon of plasmid RSF1010 consists of the genes for proteins E, F, RepA (DNA helicase), and RepC (origin-binding initiator protein) and is transcriptionally initiated by a promoter called P4. We have studied the expression of the repAC operon in vivo by using fusions to the lacZ reporter gene. The results show that the product of the second gene, F, autoregulates the operon by inhibiting transcription from P4. To verify its properties postulated from the in vivo studies and to initiate its biochemical characterization, we have purified the F protein from an overproducing E.coli strain constructed in vitro. Purification was based on a gel retardation assay for detection of P4-specific DNA binding. Subsequent DNase footprinting of the F binding sites showed clear protection around two partially symmetric P4 sequences of 16 bp, each of which matches the symmetric consensus sequence, GCGTGAGTACTCACGC, in at least 13 positions. The native repressor, as judged from gel filtration, velocity sedimentation and crosslinking studies, exists as a dimer in dilute solution; its monomeric subunit, as predicted from DNA sequence and N-terminal protein sequence data, consists of 68 amino acids and has a calculated M tau = 7,673.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/18.21.6215