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Nitrogen fixation genes are shown to undergo a complex positive and negative regulation in Rhizobium meliloti. Activation of fixN by fixLJ is shown to require a third regulatory gene, fixK. As fixK is activated by fixLJ, we propose a cascade model for fixN regulation such that fixLJ activates fixN via fixK. In addition fixK negatively regulates expression of the nif‐specific activator nifA as well as its own expression by autoregulation. Thus nifA and fixK are subject to a mixed regulation, positive (by fixLJ) and negative (by fixK). The sequence of fixK shows homology with the Escherichia coli regulators fnr and crp, which makes fixK the third characterized member of this family of prokaryotic regulators.

The cauliflower mosaic virus (CaMV) 35S RNA promoter has been dissected and examined in a transient expression system using the firefly luciferase gene as a reporter of promoter activity. Deletion analysis has shown that the 35S RNA promoter is composed of at least three regions-distal, medial, and proximal-which are essential for activity. The distal region contains three smaller elements homologous to the simian virus 40 "core" enhancer element, the medial region possesses a CCAAT-like box, and the proximal region contains a TATA box. A DNA segment encompassing the distal region is capable of activating the CaMV 35S core promoter in an orientation-independent, but not position-independent, fashion. The distal region can also activate a heterologous weak promoter, the CaMV 19S RNA promoter, albeit not to the high levels of the 35S RNA promoter. Multimers of the distal region are able to activate the 35S RNA promoter core to even greater levels of expression than the native 35S promoter. These experiments demonstrate that elements outside the boundaries of the core promoter (composed of proximal and medial elements) are recognized in a plant cell transient expression system. Eukaryotic promoters that have been examined in detail are composed of multiple cis-acting elements, which are required for promoter function. These elements have been recognized in DNA manipulation experiments (deletions, mutagenesis, linker scanning analysis, element relocation, etc.) and bind trans-acting factors, which activate transcription (reviewed in refs. 1 and 2). Many, but not all, plant and animal promoters are composed of a TATA box, 25-30 bases upstream from the start of transcription, often a CCAAT box at about -80, and other elements that may confer greater or regulated activity on a promoter. The TATA box establishes polarity to the eukaryotic promoter; binds protein factors (3, 4); and alterations in sequence, orientation, or position dramatically reduce transcription altogether or from the normal start site (5). The CCAAT box, in promoters, in which it i,s found, is affected by alterations in sequence (5), appear,s in one instance (HV thymidine kinase promoter) in an inverted orientation with respect to the start of transcription (6), and binds distinct protein factors (7). Other upstream elements may differ from one promoter to another and presumably bind different trans-acting protein factors that may confer specialized functions, such as high level constitutive expression, tissue-specific expression, or response to environmental cues (reviewed in ref.2). The elements that bind the transcriptional factor SP1 are well characterized and are found in a number of animal promoters, such as those in the 21-base-pair (bp) repeats of the simian virus 40 (SV40) early promoter (8, 9). Other upstream elements can act as enhancers, such as those in the 72-bp repeats of the SV40 promoter-that is, they can function in a position-and orientation-independent fashion and are able to empower or regulate heterologous core promoters...

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