Activator-independent formation of a closed complex between ?54-holoenzyme and nifH and nifU promoters of Klebsiella pneumoniae

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Abstract
The alternative sigma factor sigma 54 is required for transcription of nitrogen fixation genes in Klebsiella pneumoniae and other diazotrophs. The nif genes, and other E sigma 54-dependent genes whose products are necessary for a wide range of processes, are postively regulated. A unifying model that is well supported by studies on nif and other nitrogen-regulated (ntr) genes includes the central tenet that sigma 54 confers upon core RNA polymerase the ability to recognize and bind specific promoter sequences, but not the ability to isomerize to the open complex without assistance from the appropriate activator protein. Direct physical evidence for formation of an activator-independent complex between E sigma 54 and the NifA-dependent K. pneumoniae nifH and nifU promoters has, to date, been lacking. Using purified components we have now demonstrated formation of the closed complex at these promoters, indicating that it is an intermediate along the pathway to open complex formation. The closed complex was not detected when conserved features of the promoter were altered by mutation, nor was its stability increased when integration host factor protein was bound adjacent to the E sigma 54 recognition sequence.