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

Abstract

Restriction-modification controller proteins play an essential role in regulating the temporal expression of restriction-modification genes. The controller protein C.Csp231I represents a new class of controller proteins. The gene was sublconed to allow overexpression in Escherichia coli. The protein was purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.0 Å resolution and belonged to space group P2 1. An electrophoretic mobility-shift assay provided evidence of strong binding of C.Csp231I to a sequence located upstream of the csp231IC start codon. crystallization communications Acta Cryst. (2009). F65, 898-901 Streeter et al. C.Csp231i 901 Figure 5 Self-rotation function showing NCS. The self-rotation function is shown using a angle of 180 and was calculated using data in the resolution range 20.0-4.0 Å. The radius of integration was 35 Å .

Structural Analysis of a Novel Class of R–M Controller Proteins: C.Csp231I from Citrobacter sp. RFL231

“…Previous sequence alignments indicated that C.Csp231I has a large C-terminal extension compared with other C-proteins 28 . The predicted α-helical nature of this extension is confirmed by the crystal structures presented here, where the overall topology comprises a compact five-helix bundle with two extended C-terminal helices (Fig.…”

“… 14 We were particularly interested in the class exemplified by the R–M systems EcoO1091I and Csp231I, since their recognition sequences (classified by Sorokin et al as motif 8) have unusual features. 25–28 The DNA binding site of C.Csp231I consists of two sets of palindromic sequences (operators); however, unusually, there is a large (~ 18 bp) separation between them. 28 C.Csp231I has the recognition sequence CTAAGN 5 CTTAG, where the inverted repeat sequences are separated by A-rich pentanucleotides (GAAAA and AAAAT, respectively, for the distal and proximal operators).…”

“… 25–28 The DNA binding site of C.Csp231I consists of two sets of palindromic sequences (operators); however, unusually, there is a large (~ 18 bp) separation between them. 28 C.Csp231I has the recognition sequence CTAAGN 5 CTTAG, where the inverted repeat sequences are separated by A-rich pentanucleotides (GAAAA and AAAAT, respectively, for the distal and proximal operators). The sequence between the two operator sites is also notably rich in polyA and polyT tracts, the significance of which is unknown but suggests a structural constraint on the DNA ( Fig.…”

Structural analysis of DNA binding by C.Csp231I, a member of a novel class of R-M controller proteins regulating gene expression

“…However, bioinformatic analysis revealed additional classes of C-proteins based on a variety of distinct DNA-recognition sites (Sorokin et al, 2009), and 517 putative C-proteins have now been reported in ReBase (http://rebase.neb.com/cgi-bin/ azlist?cp). The control regions of the R-M systems classified by Sorokin and coworkers as motif 8 [typified by EcoO1091I (Imasaki et al, 2004;Kita et al, 2002) and Csp231I (Streeter et al, 2009;McGeehan et al, 2011)] have very different sequence motifs to those previously studied. In C.Csp231I, this region consists of two sets of 5 bp palindromic sequences with a 5 bp spacer (Fig.…”

“…For simplicity, only one strand of the DNA is shown for all duplexes. (Streeter et al, 2009). For crystallization trials, the protein was dialysed against buffer consisting of 50 mM Tris-HCl pH 8.0, 0.1 M NaCl, 1 mM Na 2 EDTA.…”