期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1993
卷号：90
期号：6
页码：2266-2270
DOI：10.1073/pnas.90.6.2266
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The NIFA protein activates transcription of nitrogen fixation (nif) operons by the sigma 54-holoenzyme form of RNA polymerase. We purified active NIFA from Klebsiella pneumoniae in the form of a maltose-binding protein (MBP)-NIFA fusion; proteolytic release of MBP yielded inactive and insoluble NIFA. MBP-NIFA activated transcription from the nifHDK promoter in a purified transcription system. Like the related transcriptional activator NTRC, MBP-NIFA catalyzed the ATP-dependent isomerization of closed complexes between sigma 54-holoenzyme and a promoter to open complexes. MBP-NIFA had a broader nucleotide specificity than NTRC, being able to utilize pyrimidine in addition to purine nucleoside triphosphates. Both MBP-NIFA and a purified C-terminal fragment of NIFA bound to the upstream activation sequence for the nifHDK promoter, as assessed by DNAse I footprinting. When assays were performed at 37 degrees C instead of the usual 30 degrees C, transcriptional activation, open complex formation, and DNA binding by MBP-NIFA were all abolished, consistent with the known heat lability of NIFA. However, the purified C-terminal fragment of NIFA still bound the upstream activation sequence at 37 degrees C, indicating that the function of the helix-turn-helix DNA-binding motif is not inherently heat-labile.
