CLARIFICATION OF THE DIMERIZATION DOMAIN AND ITS FUNCTIONAL-SIGNIFICANCE FOR THE ESCHERICHIA-COLI NUCLEOID PROTEIN H-NS

The Escherichia coli nucleoid protein, H-NS, functions as a global reg ulator for expression of a wide variety of genes. We recently analyzed the structure-function relationship of H-NS with special reference to the domains responsible for transcriptional repression and DNA-bindin g respectively. However, identification of the presumed dimerization d omain of H-NS and its functional significance was elusive. To address this particular issue, we first examined a set of N-terminally or C-te rminally truncated forms of H-NS, in terms of their so-called dominant -negative effect on the in vivo function of the wild-type H-NS. The re sults showed that certain truncated forms exhibit such a dominant-nega tive effect, but others did not. As judged by the results of the domin ant-negative effect, it was assumed that a relatively central portion of H-NS extending from residues 21 to 63 is involved in dimerization. This was confirmed by an in vitro chemical cross-linking analysis and a gel filtration analysis with these truncated forms of H-NS. Furtherm ore, the use of the dominant-negative phenotype, caused by a truncated form of H-NS (named N91), allowed us to isolate a missense mutant, wh ich was expected to be specifically defective in dimerization. This mu tant had an amino acid substitution at position 30 (Leu30 to Pro) in N 91 consisting of the N-terminal 91 amino acids of H-NS. This mutant wa s indeed defective in the in vitro ability to form a heterodimer with the wild-type H-NS. When this particular single amino acid substitutio n was introduced into the full-length H-NS, the resultant H-NS mutant had lost the ability to form dimers in vitro and to function as a tran scriptional repressor. These findings collectively provided us with ev idence that the ability of H-NS to form a dimer is crucial for H-NS to function as a transcriptional repressor. (C) 1997 Academic Press Limi ted.