Structural analysis of the genetic switch that regulates the expression of restriction-modification genes

Abstract

Controller (C) proteins regulate the timing of the expression of restriction and modification (R–M) genes through a combination of positive and negative feedback circuits. A single dimer bound to the operator switches on transcription of the C-gene and the endonuclease gene; at higher concentrations, a second dimer bound adjacently switches off these genes. Here we report the first structure of a C protein–DNA operator complex, consisting of two C protein dimers bound to the native 35 bp operator sequence of the R–M system Esp1396I. The structure reveals a role for both direct and indirect DNA sequence recognition. The structure of the DNA in the complex is highly distorted, with severe compression of the minor groove resulting in a 50° bend within each operator site, together with a large expansion of the major groove in the centre of the DNA sequence. Cooperative binding between dimers governs the concentration-dependent activation–repression switch and arises, in part, from the interaction of Glu25 and Arg35 side chains at the dimer–dimer interface. Competition between Arg35 and an equivalent residue of the σ70 subunit of RNA polymerase for the Glu25 site underpins the switch from activation to repression of the endonuclease gene.

Overexpression, purification and preliminary X-ray diffraction analysis of the controller protein C.Csp231I fromCitrobactersp. RFL231

“…This may be an indication of significant differences in the structure of C.Csp231I compared with the three available search models C.AhdI (McGeehan et al, 2005), C.Esp1396I (McGeehan et al, 2008) and C.BclI (Sawaya et al, 2005). Indeed, such differences in structure would not be surprising given the presence of the 33-aminoacid extension and 12-amino-acid deletion at the C-and N-termini, respectively.…”

“…To date, the structures of three C proteins have been reported (McGeehan et al, 2005(McGeehan et al, , 2008Sawaya et al, 2005); all are highly homologous proteins with similar folds and with similar DNA-recognition sites. Other groups, such as that exemplified by C.Csp231I and C.EcoO109I, have very different recognition sites and their structures are currently unknown.…”

RNA protects a nucleoprotein complex against radiation damage

“…Recently, for a well characterized bacterial protein-DNA complex (C.Esp1396I; PDB entry 3clc; resolution 2.8 Å ; McGeehan et al, 2008) it was concluded that over a wide dose range (2.1-44.6 MGy) the protein was far more susceptible to SRD than the DNA within the crystal (Bury et al, 2015). Only at doses above 20 MGy were precursors of phosphodiester-bond cleavage observed within AT-rich regions of the 35-mer DNA.…”

Structural analysis of the bacterial RM controller protein, C.Esp1396I, in complex with DNA

“…The affinity for the weakest operator site (O R ) is increased 100-fold when the directly adjacent O L site is occupied by a C-protein. X-ray crystallography revealed the structure of the adjacent O L and O R sites occupied by a pair of C.Esp1396I dimers forming the repression complex [1] as well as the C-protein-O M repression complex [2] and the transcriptional activation C-protein-O L complex [3]. These complexes revealed a combination of both direct and indirect readout between the C-protein and the DNA, as well as significant DNA distortion and protein-protein cooperativity.…”