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Mechanism for nitrogenase mrna stabilization by the nifa transcriptional activator

Objective



Research objectives and content
The nitrogen fixation gene control is one of the tightest regulated systems in bacteria. The transcription of the structural genes (nifHDKTY) is activated through the RpoN-dependent transcriptional activator, NifA. In addition, there seems to be a posttranscriptional control involving the stabilization of the nitrogenase mRNAs depending on the NifA activity whose mechanism has not been determined. We would try to clarify how this process is accomplished by NifA. To determine which are the requirements for mRNA stabilization, we will determine the half-lives of nifH mRNA compared to that of the glnA mRNA which is induced by the RpoN-dependent regulator NtrC. RNase E mutants will be used to know whether that RNase is involved in the mRNA decay. Using hybrid promoters with heterolog binding sites, we will analyze if the stabilization is dependent specifically on NifA or on any RpoN-transcriptional activator. The conservation of the
postranscriptional mechanism will be tested by using either distinct nifA genes or nitrogenase genes from different species. Structural and functional comparison of nifH-lacZ half-life will be also assessed by in vitro translation. A possible interaction of nifH mRNA with NifA or any other elements will be analyzed by biochemical studies of RNA binding. Training content (objective, benefit and expected impact)
The planned project may find a novel mechanism of gene expression which may be attributed to a prokaryotic transcriptional activator with common features to eukaryotic ones. Moreover, postranscriptional controls of gene expression are not yet as well known as the transcriptional ones, but can be the bottleneck for the production of several proteins. Working in posttranscriptional steps of gene expression would also improve my experience in the field of gene expression.
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Coordinator

Universidad de Sevilla
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41080 Sevilla
Spain