DNA binding activity of Anabaena sensory rhodopsin transducer probed by fluorescence correlation spectroscopy

Abstract

Anabaena sensory rhodopsin transducer (ASRT) is believed to be a major player in the photo-signal transduction cascade, which is triggered by Anabaena sensory rhodopsin. Here, we characterized DNA binding activity of ASRT probed by using fluorescence correlation spectroscopy. We observed clear decrease of diffusion coefficient of DNA upon binding of ASRT. The dissociation constant, K(D), of ASRT to 20 bp-long DNA fragments lied in micro-molar range and varied moderately with DNA sequence. Our results suggest that ASRT may interact with several different regions of DNA with different binding affinity for global regulation of several genes that need to be activated depending on the light illumination.

Kinetics of DNA looping by Anabaena sensory rhodopsin transducer (ASRT) by using DNA cyclization assay

“…To observe ASRT-induced DNA bending, excess amount of ASRT (100 μM) was put into the sample chamber and the changes in the FRET value of individual DNA was monitored. We used the salt condition of 50 mM NaCl which was used in the previous FCS and NMR studies for ASRT-DNA binding 28 , 30 . Note that 50 mM NaCl was too weak to induce spontaneous DNA bending (Figure S2 , upper left panel).…”

“…By using EMSA, it has been shown that multiple regions in the promoter region and transcription start sites of the phycoerythrocyanin gene have higher binding affinity with ASRT 29 . The FCS study 30 found a K D of ~ 10 μM between ASRT and 20 bp fragment of phycoerythrocyanin promotor region and reported a weak sequence dependent changes of the K D . Together, a hypothesis emerged: ASRT might induce changes in the structure of DNA, such as in the cases of CAP and AraC proteins, for gene regulation purpose.…”

“…Previously, the binding between ASRT and DNA was investigated using various experimental methods such as NMR 28 , Electrophoretic Mobility Shift Assay (EMSA) 29 , and Fluorescence correlation spectroscopy (FCS) 30 . The NMR study found that three amino acid residues (R109, N113, Q110) in the C-terminal of ASRT are the main contact points with the DNA 28 .…”

“…The detailed structure of ASRT was reported by X-ray crystallography and solution NMR studies 27 , 28 . It has been reported from EMSA and fluorescence correlation spectroscopy (FCS) that ASRT binds to the promotor region of the phycoerythrocyanin gene 29 , 30 . In particular, it showed high binding affinity in the region containing transcriptional and translation start sites.…”

Structure of an Inward Proton-Transporting Anabaena Sensory Rhodopsin Mutant: Mechanistic Insights

“…However, rather than pumping ions, it has been shown to regulate the expression of phycocyanin and phycoerythrocyanin, part of the phycobilisome light-harvesting complex, via a soluble 14-kDa cytoplasmic transducer (ASRT) (29)(30)(31)(32)(33)(34), found on a single operon (27). Unlike the haloarchaeal sensory rhodopsins, which interact with their cognate transmembrane transducers to mediate light-stimulated phototaxis (35,36), it has been proposed that ASR's photosignal is relayed to ASRT, which then translocates to the nucleus where it can bind specifically to the promoter regions of multiple genes (33,34). Other studies have shown evidence that ASR alone in E. coli can bind DNA and regulate gene activity upon photostimulation (31).…”