In the domain Archaea, the mechanism of transcription appears to resemble that in eucaryotes. However, almost nothing is known about the regulation of transcription. Nitrogen fixation is a highly regulated function that can be used as a model system for studying regulatory mechanisms. The regulation of nif (nitrogen fixation) gene expression in the archaeon Methanococcus maripaludis will be investigated. Using an established genetic system, mutants with derepressed nif gene transcription will be isolated, and the genes and gene products controlling repression will be investigated. Binding of factors to a previously-characterized operator will be studied. Activation of nifH expression will be studied by 5' deletion of the upstream region and analysis of activator proteins. A gene, nifX, appears to be involved I the control of nifH mRNA levels. This observation will be confirmed by making an in-frame deletion mutation in nifX. Because of their relative simplicity and the advent of facile genetic methods in certain species such as M. maripaludis, the Archaea could provide an effective system for studying eucaryotic-like regulatory mechanisms that are important in human development and disease. The nif genes (six genes) have been cloned from M. maripaludis and good evidence obtained for a single transcript initiated upstream of nifH. Regulation appears to be at the level of transcription in response to fixed nitrogen levels. The hypothesis that these genes are regulated both by repression and activation is to be tested. Evidence has been obtained for a trans-acting repressor that binds to a palindromic sequence (operator) downstream of the transcript start site. The conditions under which the repressor binds to the operator and the involvement of ligands will be investigated. Genes and their products necessary for repression will be identified through a mutational analysis. Proteins will be purified and antibodies raised. The presence or absence of the proteins in repressed vs. derepressed cells will be assessed. Experiments to establish activation will explore both cis features of the nif genes and possible trans factors. Although preliminary evidence for activation has not yet been generated, the possibility is great that activation is at work here. Two lines of reasoning suggest that it may play a role. First, the Archaea transcriptional apparatus resembles that from Eucarya where activation is required for full expression of almost all transcripts. Second, the regulation of nif genes in Bacteria offers a striking example of a positive control via nifA gene product. These studies will proceed by deletion analyses of the upstream region of nifH and by searches for unlinked mutations lacking expression of nif genes. Finally, the effects of nifX mutations and the gene product levels on the expression of nif genes will be pursued. Mutations apparently affecting nifX expression lead to derepressed levels of nif mRNA, although gel shifts of the operator region of nifH are unaffected in extracts of these mutants. NifX may influence mRNA stability or act at an alternate site.
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