Characterization of consensus operator site for Streptococcus pneumoniae copper repressor, CopY

Copper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems ( operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repression. In (the pneumococcus), CopY is the copper repressor for the operon. Previously, these homologs have been characterized to bind a 10-base consensus sequence T/GACAnnTGTA. Here, we bioinformatically and empirically characterize these operator sites across species using . CopY as a guide for binding. By examining the 21-base repeat operators for the pneumococcal operon and comparing binding of recombinant CopY to this, and the operator sites found in , we show using biolayer interferometry that the T/GACAnnTGTA sequence is essential to binding, but it is not sufficient. We determine a more comprehensive . CopY operator sequence to be RnYKACAAATGTARnY (where “R” is purine, “Y” is pyrimidine, and “K” is either G or T) binding with an affinity of 28 nM. We further propose that the operon operator consensus site of pneumococcal homologs be RnYKACAnnYGTARnY. This study illustrates the necessity to explore bacterial operator sites further to better understand bacterial gene regulation.