Bacterial ArtA protein specifically binds to the internal region of IS1 in vitro
Author(s)
Sachiko Matsutani*
ABSTRACT
The internal region of bacterial translocatable IS1 acts as a cis-element to stimulate transcription from the various promoters located upstream. The product of the artA gene is genetically shown to stimulate transcription with the cis-element. Here, a codon-optimized artA gene was synthesized and cloned to express the ArtA protein. ArtA was purified as the Histagged protein. Nitrocellulose filter binding assay showed that ArtA specifically binds to the IS1 internal region. Electrophoretic mobility shift assay also showed specific binding of ArtA to the IS1 internal region. These results imply that ArtA directly binds to the IS1 internal region and stimulates transcription.
The internal region of bacterial translocatable IS1 acts as a cis-element to stimulate transcription from the various promoters located upstream. The product of the artA gene is genetically shown to stimulate transcription with the cis-element. Here, a codon-optimized artA gene was synthesized and cloned to express the ArtA protein. ArtA was purified as the Histagged protein. Nitrocellulose filter binding assay showed that ArtA specifically binds to the IS1 internal region. Electrophoretic mobility shift assay also showed specific binding of ArtA to the IS1 internal region. These results imply that ArtA directly binds to the IS1 internal region and stimulates transcription.
Cite this paper
Matsutani, S. (2012) Bacterial ArtA protein specifically binds to the internal region of IS1 in vitro. Advances in Bioscience and Biotechnology, 3, 869-875. doi: 10.4236/abb.2012.37108.
Matsutani, S. (2012) Bacterial ArtA protein specifically binds to the internal region of IS1 in vitro. Advances in Bioscience and Biotechnology, 3, 869-875. doi: 10.4236/abb.2012.37108.
References
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[1] Mahillon, J. and Chandler, M. (1998) Insertion sequences. Microbiology and Molecular Biology Reviews, 2, 725-774. [2] Machida, C., Machida, Y. and Ohtsubo, E. (1984) Both inverted repeat sequences located at the ends of IS1 provide promoter functions. Journal of Molecular Biology, 177, 247-267. doi:10.1016/0022-2836(84)90455-8 [3] Matsutani, S. (1994) Genetic evidence for IS1 transposition regulated by InsA and the InsA-B’-InsB species, which is generated by translation from two alternative internal initiation sites and frameshifting. Journal of Molecular Biology, 240, 52-65. doi:10.1006/jmbi.1994.1417 [4] Zerbib, D., Jakowec, M., Prentki, P., Galas, D.J. and Chandler, M. (1987) Expression of proteins essential for IS1 transposition: Specific binding of InsA to the ends of IS1. EMBO Journal, 6, 3163-3169. [5] Matsutani, S. (2005) The internal sequence of IS1 stimulates RNA synthesis from IS1 own and exogenous promoters. Journal of Biological Systems, 13, 313-329. doi:10.1142/S0218339005001513 [6] Mendoza-Vargas, A., Olvera, L., Olvera, M., Grande, R., Vega-Alvarado, L., Taboada, B., Jimenez-Jacinto, V., Salgado, H., Juárez, K., Contreras-Moreira, B., Huerta, A.M., Collado-Vides, J. and Morett, E. (2009) Genome-wide identification of transcription start sites, promoters and transcription factor binding sites in E. coli. PLoS One, 4, e7526. doi:10.1371/journal.pone.0007526 [7] Wu, J.H. and Ippen-Ihler, K. (1989) Nucleotide sequence of traQ and adjacent loci in the Escherichia coli K-12 F-plasmid transfer operon. Journal of Bacteriology, 171, 213-221. [8] Gustafsson, C., Govindarajan, S. and Minshull, J. (2004) Codon bias and heterologous protein expression. Trends in Biotechnology, 22, 346-353. doi:10.1016/j.tibtech.2004.04.006 [9] Etchegaray, J.P. and Inouye, M. (1999) Translational enhancement by an element downstream of the initiation codon in Escherichia coli. Journal of Biological Chemistry, 274, 10079-10085. doi:10.1074/jbc.274.15.10079 [10] Gottesman, S., Halpern, E. and Trisler, P. (1981) Role of sulA and sulB in filamentation by lon mutants of Escherichia coli K-12. Journal of Bacteriology, 148, 265-273. [11] Hussain, M., Ichihara, S. and Mizushima, S. (1980) Accumulation of glyceride-containing precursor of the outer membrane lipoprotein in the cytoplasmic membrane of Escherichia coli treated with globomycin. Journal of Biological Chemistry, 255, 3707-3712. [12] Matsutani, S. (2007) Possible presence and role of the promoter sequence for eukaryotic RNA polymerase III in bacteria. Genetica, 131, 127-134. doi:10.1007/s10709-006-9122-5 [13] Geiduschek, E.P. and Kassavetis, G.A. (2001) The RNA polymerase III transcription apparatus. Journal of Molecular Biology, 310, 1-26. doi:10.1006/jmbi.2001.4732 [14] Galli, G., Hofstetter, H. and Birnstiel, M.L. (1981) Two conserved sequence blocks within eukaryotic tRNA genes are major promoter elements. Nature, 294, 626-631. [15] Folk, W.R., Hofstetter, H. and Birnstiel, M.L. (1982) Some bacterial tRNA genes are transcribed by eukaryotic RNA polymerase III. Nucleic Acids Research, 10, 7153-7162. doi:10.1093/nar/10.22.7153