Biphasic unbinding of Zur from DNA for transcription (de)repression in Live Bacteria

Abstract

11Transcription regulator on-off binding to DNA constitutes a mechanistic paradigm in gene 12 regulation, in which the repressors/activators bind to operator sites tightly while the corresponding non-13repressors/non-activators do not. Another paradigm regards regulator unbinding from DNA to be a 14 unimolecular process whose kinetics is independent of regulator concentration. Using single-molecule 15 single-cell measurements, we find that the behaviors of the zinc-responsive uptake regulator Zur 16 challenges these paradigms. Apo-Zur, a non-repressor and presumed non-DNA binder, can bind to 17 chromosome tightly in live E. coli cells, likely at non-consensus sequence sites. Moreover, the 18 unbinding from DNA of its apo-non-repressor and holo-repressor forms both show a biphasic, 19repressed-followed-by-facilitated kinetics with increasing cellular protein concentrations. The 20 facilitated unbinding likely occurs via a ternary complex formation mechanism; the repressed unbinding 21is first-of-its-kind and likely results from protein oligomerization on chromosome, in which an inter-22 protein salt-bridge plays a key role. This biphasic unbinding could provide functional advantages in 23Zur's facile switching between repression and derepression. 24 25

Unraveling the Impact of Cysteine-to-Serine Mutations on the Structural and Functional Properties of Cu(I)-Binding Proteins

“…Cysteines are characterized by a distinctive affinity for Cu(I) ions, making them a uniquely appropriate amino acid for maintaining Cu(I) homeostasis [56,57]. Cys-to-Ser mutation is often employed in Cu(I) binding proteins to inspect selected mechanistic aspects [17,34,36], based on the similarity between these two amino acids. This substitution relies on the hypothesis that, in spite of the similarity between cysteines and serines, the SXXS motif will not be able to bind to the Cu(I) ion, and therefore will form non-functional proteins.…”

“…This substitution relies on the hypothesis that, in spite of the similarity between cysteines and serines, the SXXS motif will not be able to bind to the Cu(I) ion, and therefore will form non-functional proteins. This proposal is mainly based on functional measurements, which showed that double mutation of CXXC to SXXS aborts all functions [34,36,37]. However, in vitro studies to validate this hypothesis and to clarify the specific variations occurring at both the structural and functional levels have not hitherto been performed.…”

“…In order to compare a functional protein with a non-functional/inhibited/repressed protein, cysteine (Cys)-to-serine (Ser) mutations have often been applied in in vitro studies [17,34,35,36,37], relying on the hypothesis that the SXXS site is unable to bind Cu(I) ions. This hypothesis is mainly based on functional measurements, showing that double mutation of CXXC to SXXS aborts all functions of the hosting protein.…”

“…Nevertheless, EXAFS studies suggested that double Cys-to-Ser mutations of the human copper chaperone CCS can still bind Cu(I) ions, although in a distinct manner with respect to the wild-type (WT) protein, possibly leading to its oligomerization or precipitation (aggregation) [37]. An oligomerization of various ZuR monomers in the presence of Cys-to-Ser mutation was also observed in-cell [34]. Such oligomerization can be detected in vitro as aggregation or precipitation of the protein.…”

How do DNA-bound proteins leave their binding sites? The role of facilitated dissociation

“…A relation between dissociation rate and chromosome condensation levels was also reported: residence times for passive (i.e., apo) forms of the metalloregulators were reported to be longer than those of active (i.e., metalbound) forms in condensed chromosomes, with the opposite trend in cells with more loosely compacted chromosomes [5 ]. Similar effects for the zinc-responsive uptake regulator Zur in E. coli cells were also reported [22].…”