4554 Nucleic Acids Research, 2009, Vol. 37, No. 14 Figure 7. Model of a hexadecameric array of two CrgA oligomers binding to the DNA target site. (A) Two CrgA octamers bound to the 63-bp CrgA DNA footprint (48). The ﬁrst octamer (blue) binds to CrgA site 1 and the second octamer (gray) to site 2. The LTTR motifs (boxed in green in the sequence) are bound between the two recognition helices of a DBD pair. One strand of the DNA is coloured to represent the sequence elements depicted in the schematic. The LTTR motifs are indicated by green colouring of the DNA. The three base pairs between CrgA site 1 and 2 are shown in yellow. The bases overexposed to DMS methylation (+) or hypersensitive to DNase I ( ) are indicated. The predicted transcription and trans- lation start points are represented with bent and broad arrows, respectively. The predicted –10 promoter element for crgA is underlined. The location of the –35 promoter (underlined) element of mdaB is indicated with a dashed orange line. (B) EMSA of puriﬁed CrgA binding to its DNA target site. The amount of DNA was kept constant at 2.5 pmol per reaction and the molarity of CrgA was varied as indicated. (C) Asymmetric unit of Crystal form B of CrgA. 25 S species, referred to above. The calculated sedimenta- tion coeﬃcients for an LTTR tetramer and octamer are 6.65 S and 11.03 S based on the atomic structures of CnBr [2] and CrgA, respectively. Therefore, we conclude that the major species observed for CrgA subjected to AUC in both the absence and presence of DNA is most likely to be an octamer consistent with the results of MS, described above. The slower sedimentation of CrgA, 9.5 S with or without DNA, is probably due to additional hydrody- namic drag resulting from the hollow nature of the octa- mer that is not allowed for in the atomic model-based calculation of the s value. Further drag is presumably introduced by the bound DNA since the added DNA does not increase the overall sedimentation coeﬃcient though it does signiﬁcantly improve the deﬁnition of the peak in the c(s,M) distribution. Most interestingly, we observed that the addition of DNA resulted in a proportion ( 25%) of CrgA molecules forming higher molecular weight/sedimentation coeﬃcient species. The fact that these oligomers were only observed in the presence of DNA strongly suggests that they were induced by DNA binding (Figure 5). They include species that presumably correspond to the hexadecamer observed by MS and also higher order assemblies up