Reid C. Johnson
Research in the Johnson lab revolves around three areas. The first concerns the mechanisms responsible for the control and catalysis of a site-specific DNA inversion reaction from Salmonella. This reaction regulates the alternative expression of flagellar proteins by switching the orientation of 995 bp DNA fragment containing a promoter. Since flagellin is one of the major surface proteins on the bacterium, this specialized DNA recombination reaction enables the organism to evade a host immune response. One of the most unique aspects of this reaction is the requirement for a 65 bp cis-acting recombinational enhancer sequence together with the Fis protein. We have shown that the two recombination sites bound by the Hin recombinase assemble into a synaptic complex at the Fis-bound enhancer segment. Current evidence indicates that upon assembly of the invertasome, Hin undergoes a conformational change to activate DNA catalysis. Hin catalyzes site-specific double strand cleavages within the center of the recombination sites, rearranges the DNA strands, and ligates the DNA such that the intervening DNA segment is in the inverted orientation. A variety of genetic, biochemical, and physical approaches are being pursued to elucidate the molecular structure of reaction complexes, the specific role of the enhancer in activating Hin, and the enzymology of DNA strand exchange.
A second project is the identification and characterization of nucleoprotein assembly factors from eukaryotes. The primary assay to identify such non-specific DNA binding proteins is the Hin-mediated DNA inversion reaction. We have shown the HU from E. coli or HMG1/2 from mammals promotes assembly of the invertasome when the enhancer is located close to a recombination site by facilitating the looping of intervening DNA. Current emphasis is on the biochemical and genetic characterizaiton of 2 HMG1/2-related proteins from S. cerevisiae called NHP6A and NHP6B. In particular, we are interested in how the "HMG" DNA binding motif is binding to and deforming the DNA molecule as well as how these proteins are functioning in particular transcription reactions and in chromosome segregation in yeast. We have shown that the yeast HMG proteins are required for activated transcription of certain promoters but not of others and are determining what features of the promoter specify this requirement.
Fis is one of several general nucleoid-associated proteins found in enteric bacteria. In addition to this role in regulating DNA inversion, Fis has recently been found to participate in many other diverse reactions including transcription, DNA replication, and phage lambda excision. We have found that the expression of the gene encoding Fis varies enormously with respect to the growth phase and nutrient availability in Salmonella and E. coli. For example, when stationary phase cells are subcultured into fresh rich media, Fis protein levels increase from less than 100 dimers/cell to 40,000 dimers/cell within the first cell division. The mechanism responsible for this regulation remains to be determined. We have recently identified a large number of genes that are either positively or negatively controlled by Fis by screening transposon-induced lac fusions to chromosomal genes. Many of these are only expressed under certain physiological conditions. An example is the negatively controlled aldehyde dehydrogenase gene, aldB, which is expressed only in stationary phase. The high Fis levels in exponential phase repress aldB expression, but this repression is relieved in stationary phase due to reduced Fis levels. In the four Fis-repressed promoters studied so far, multiple binding sites for Fis are clustered withing the promoter region, with at least one site dierectly overlapping the RNA polymerse binding site. Fis also activates transcription of a variety of genes. We are intensively studying the regulation of the proline permease gene, proP, in which transcription from its P2 promoter is dependent upon Fis binding at -41 and is further synergistically stimulated by CRP-cAMP binding at -121. Current genetic and biochemical studies indicated that the mechanism by which Fis is activating transcription is very different from the mechanism of activation of DNA inversion.