COMMUNICATION Suppression Analysis of Positive Control Mutants of NifA Reveals Two Overlapping Promoters for Klebsiella pneumoniae rpoN Ricardo A. Grande 1 , Brenda Valderrama 1 and Enrique Morett 1,2 * 1 Instituto de Biotecnologõ Âa Universidad Nacional Auto Ânoma de Me Âxico AP 510- 3, Cuernavaca, Morelos Me Âxico 2 European Molecular Biology Laboratory, Meyerhofstrasse 1 69117, Heidelberg, Germany Activation of gene expression relies on direct molecular interactions between the RNA polymerase and transcription factors. Eubacterial enhancer-binding proteins (EBPs) activate transcription by binding to dis- tant sites and, simultaneously, contacting the s 54 -holoenzyme form of the RNA polymerase (Es 54 ). The interaction between the EBP and Es 54 is transient, such that it has been dif®cult to be studied biochemically. Therefore, the details of this molecular recognition event are not known. Genetic and physical evidences suggest that the highly conserved C3 region in the activation domain of the EBP has major determinants for positive control and for the interaction with Es 54 . To further investigate the target of this region we searched for extragenic suppressors of some C3 region mutant derivatives of NifA. As a ®rst step we mutagenized Klebsiella pneumoniae rpoN, the gene that codes for s 54 . A mutant allele, rpoN1320, that suppressed two different NifA derivatives was obtained. Immunodetection of s 54 and transcriptional initiation studies demon- strated that the cause of the suppression was an enhanced expression of rpoN. A single point mutation was responsible for the phenotype. It mapped at the 10 region of an unidenti®ed promoter, here denomi- nated rpoNp1, and increased its similarity to the consensus. A second upstream promoter, denominated rpoNp2, was also identi®ed. Its 10 region partially overlaps with the 35 region of rpoNp1. Interestingly, the promoter-up 10 mutation in rpoNp1 caused a reduction in the expression from rpoNp2, likely re¯ecting a stronger occupancy of the for- mer promoter by the RNA polymerase at the expense of the latter. The presence of two overlapping promoters competing for the RNA polymer- ase implies a complex regulatory pattern that needs elucidation. The fact that increasing the concentration of s 54 in the cell can suppress positive control mutants of NifA adds further evidence for their direct interaction in the activation process. # 1999 Academic Press Keywords: second-site mutations; transcriptional activation; sigma factor; nitrogen ®xation; enhancer-binding protein *Corresponding author Introduction Activation of transcription in eubacteria results from the recruitment of the RNA polymerase holoenzyme to a promoter and/or by increasing the rate of the subsequent steps until the polymer- ase is engaged in elongation (reviewed by Ptashne & Gann, 1997). The function of transcription factors is to stabilize, by a direct and productive inter- action with the RNA polymerase holoenzyme, one or more of the intermediate states (Roy et al., 1998). RNA polymerases are complex enzymes composed of at least ®ve subunits, with a stoichiometry of a2bb's (von Hippel et al., 1984). The well-charac- terized Escherichia coli housekeeping RNA poly- merase holoenzyme (Es 70 ) provides a paradigm for understanding how transcription factors inter- act with different subunits to activate transcription. E-mail address of the corresponding author: emorett@ibt.unam.mx Abbreviations used: EBP, enhancer-binding protein. Article No. jmbi.1999.3232 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 294, 291±298 0022-2836/99/470291±8 $30.00/0 # 1999 Academic Press