A CONSENSUS SEQUENCE FOR BINDING OF LRP TO DNA

Lrp (leucine-responsive regulatory protein) is a major regulatory prot ein involved in the expression of numerous operons in Escherichia coli . For ilvIH, one of the operons positively regulated by Lrp, Lrp binds to multiple sites upstream of the transcriptional start site and acti vates transcription. An alignment of 12 Lrp binding sites within ilvIH DNA from two different organisms revealed a tentative consensus seque nce AGAAT TTTATTCT (Q. Wang, M. Sacco, E. Ricca, C. T. Lago, M. DeFeli ce, and J. M. Calvo, Mol. Microbiol. 7:883-891, 1993). To further char acterize the binding specificity of Lrp, we used a variation of the Se lex procedure of C. Tuerk and L. Gold (Science 249:505-510, 1990) to i dentify sequences that bound Lrp out of a pool of 10(12) different DNA molecules. We identified 63 related DNA sequences that bound Lrp and estimated their relative binding affinities for Lrp. A consensus seque nce derived from analysis of these sequences, YAGHAWATTWT DCTR, where Y = C or T, H = not G, W = A or T, D = not C, and R = A or G, contains clear dyad symmetry and is very similar to the one defined earlier. T o test the idea that Lrp in the presence of leucine might bind to a di fferent subset of DNA sequences, we carried out a second selection exp eriment,vith leucine present during the binding reactions. DNA sequenc es selected in the presence or absence of leucine were similar, and le ucine did not stimulate binding to any of the sequences that were sele cted in the presence of leucine. Therefore, it is unlikely that leucin e changes the specificity of Lrp binding.