A whole genome approach to in vivo DNA-protein interactions in E. coli

Abstract

The increasingly rapid pace at which genomic DNA sequences are being determined has created a need for more efficient techniques to determine which parts of these sequences are bound in vivo by the proteins controlling processes such as gene expression, DNA replication and chromosomal mechanics. Here we describe a whole-genome approach to identify and characterize such DNA sequences. The method uses endogenous or artificially introduced methylases to methylate all genomic targets except those protected in vivo by protein or non-protein factors interfering with methylase action. These protected targets remain unmethylated in purified genomic DNA and are identified using methylation-sensitive restriction endonucleases. When the method was applied to the Escherichia coli genome, 0.1% of the endogenous adenine methyl-transferase (Dam methylase) targets were found to be unmethylated. Five foreign methylases were examined by transfection. Database-matched DNA sequences flanking the in vivo-protected Dam sites all fell in the non-coding regions of seven E. coli operons (mtl, cdd, flh, gut, car, psp and fep). In the first four operons these DNA sequences closely matched the consensus sequence that binds to the cyclic AMP-receptor protein. The in vivo protection at the Dam site upstream of the car operon was correlated with a downregulation of car expression, as expected of a feedback repressor-binding model.

Modulation of Escherichia coli DNA Methyltransferase Activity by Biologically Derived GATC-flanking Sequences

“…DNA oligonucleotides were designed based on these GATC sites and the actual flanking DNA found in the E. coli genome (Table 1; [1][2][3][4][5][6][7][8]. GATC sites previously identified as undermethylated in vivo and located in the 5Ј-noncoding region of various genes within the E. coli genome were also used as templates for oligonucleotide design (18,19). These substrates were designed based on the actual GATC and flanking DNA sequences found in the E. coli genome and named according to the gene identified to be possibly impacted by the methylation state of that GATC ( Table 1, mtl-yihU/V).…”

“…Gene expression and proteomic studies of bacteria in which the EcoDam gene has been deleted show extreme and widespread changes in RNA and protein levels, in many cases involving well characterized virulence factors (14 -17). Within the E. coli genome, ϳ0.1-0.2% of the ϳ20,000 GATC sites are undermethylated in vivo (18,19). Most of these GATC sites are found in the 5Ј-noncoding region of various genes, indicating that they could be involved in gene regulation.…”

Epigenetic Gene Regulation in the Bacterial World

“…36 sites) in the genome of E. coli K-12, which lack pap DNA sequences, were stably nonmethylated (218,272). These sites were identified by digestion of chromosomal DNA with MboI, which cuts at nonmethylated GATC sites.…”

A comprehensive library of DNA-binding site matrices for 55 proteins applied to the complete Escherichia coli K-12 genome 1 1Edited by R. Ebright

“…The methylases Dam and Dcm methylate the sequences GATC and CCWGG, respectively (Palmer & Marinus, 1994). Some genomic sites with these sequences are unmethylated, presumably due to a bound DNA-binding protein blocking access by the cognate methylase (Hale et al, 1994;Ringquist & Smith, 1992;Wang & Church, 1992). Conversely, the methylation status of DNAbinding sites can affect the binding of proteins to these sites (Bolker & Kahmann, 1989;Braun & Wright, 1986;Charlier et al, 1995;van der Woude et al, 1992;Yin et al, 1988).…”