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Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

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Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

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Título Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense
Autor Passaglia, Luciane Maria Pereira
Van Soom, C.
Schrank, Augusto
Schrank, Irene Silveira
Abstract NifA protein activates transcription of nitrogen fixation operons by the alternative s54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGTN10- ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.
Contido em Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas. Ribeirão Preto, SP. Vol. 31, no. 11 (Nov. 1998), p. 1363-1374
Assunto Genética
[en] Azospirillum
[en] Gel shift assay
[en] NifA activity
[en] NifA antibodies
[en] NifA protein
[en] Upstream activator sequence
Origem Nacional
Tipo Artigo de periódico
URI http://hdl.handle.net/10183/21114
Arquivos Descrição Formato
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