Abstract

<p>(A) Purified YhaJ was tested for its ability to bind the <i>yhaO</i> promoter region (<i>pyhaO;</i> ~300 bp region upstream of the <i>yhaO</i> coding sequence) by EMSA. DIG-labeled <i>pyhaO</i> was incubated with increasing concentrations of YhaJ that corresponded to a shift in free-DNA indicating a YhaJ-DNA complex. Specificity of the binding reaction was tested by the addition of a 100-fold excess (+) of unlabeled <i>pyhaO</i> probe to the binding reaction to outcompete binding of the DIG-labeled probe to YhaJ. These reaction conditions were carried out using a fragment of the <i>kan</i> gene as a negative control. Additionally, the unlabeled <i>kan</i> probe in 100-fold excess was used as a non-specific competitor for YhaJ binding to the DIG-labeled <i>yhaO</i> probe (p<i>yhaO</i> vs <i>kan</i>). (B) Activity of the <i>yhaO</i> promoter in the <i>ΔyhaJ</i> mutant background. A plasmid containing a GFP-<i>yhaO</i> promoter fusion was transformed into TUV93-0 and <i>ΔyhaJ</i> to monitor transcription of <i>yhaO</i> in RFU during growth in MEM-HEPES. Data was calculated from three biological replicates and plotted at increasing OD<sup>600</sup> values. * denotes P ≤ 0.05.</p

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oai:figshare.com:article/1632511Last time updated on 2/12/2018

This paper was published in FigShare.

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