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Investigations into the NIpNerTMF family of DNA-binding proteins

Authors
Publisher
McGill University
Publication Date
Keywords
  • Biology
  • Microbiology.
Disciplines
  • Biology
  • Computer Science

Abstract

Our laboratory previously established that the E. coli Nlp protein (also known as SfsB, Sugar fermentation stimulation B) was a transcribed nonessential gene in E. coli. I have further established by Northern blot analysis that nlp is expressed as a monocistronic mRNA of approximately 320 nucleotides, and defined the precise nlp/sfsB transcriptional initiation site by primer extension. I also expressed and purified Nlp to homogeneity from a cell-free extract and showed, by electrophoretic mobility assay, that Nlp binds DNA with specificity for the transcriptional regulatory regions of two operons involved in sugar metabolism, mal and lac. E. coli Nlp shares high sequence similarity with the Ner proteins of coliphages Mu and D108 (~62%) and with an 85 amino acid region of the human TMF protein (~30%, named 'TMF'). The high sequence similarity of this human 'TMF' to these regulatory bacteriophage and bacterial proteins suggests the existence of an evolutionarily conserved functional motif. 'TMF' was cloned by PCR from a HeLa cDNA library, over-expressed in E. coli and purified by affinity chromatography. Electrophoretic mobility shift assays suggest specific, although weak, binding of this short 'TMF' protein to the binding site of the larger full length TMF. The Nlp protein acts as a positive activator of several sugar-metabolizing operons (such as mal) in E. coli, but only in strains with cya-crp *1 mutations. I have shown that Mu Ner and 'TMF' are also capable of acting as positive activators of maltose metabolism in the cya -crp*1 mutant, suggesting that the similarity is more than just sequence based. A malPQ::lacZ fusion strain was constructed and beta-galactosidase assays showed that Nlp has a positive effect on the expression of the malP promoter. RNA dot blot analyses further showed that Nlp increases the expression of the malT regulator gene as well as the malF and malK genes (involved in maltose transport). These results give ins

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