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

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


INTRODUCTION 26
Transcriptional regulation in cells is generally orchestrated by regulators, which, upon binding 27 to operator sites, either block the binding of RNA polymerase (RNAP) leading to repression (i.e., 28 repressors) or recruit RNAP leading to activation (i.e., activators) 1, 2 . One mechanistic paradigm for 29 these regulators is an on-off model in which they bind to their cognate operator sites tightly, while their 30 corresponding non-repressor/non-activator forms have insignificant affinity to DNA and stay 31 predominantly in the cytoplasm. Some exceptions recently emerged. For example, IscR, a member of 32 the MarA/SoxS/Rob family of transcription regulators in E. coli, is a repressor in its holo-form (i.e., 33 containing a Fe-S cluster); its apo-form, generally thought to not bind DNA, was shown to bind DNA 34 motifs different from its holo-repressor form 3, 4 . 35 Derepression or deactivation subsequently comes from the unbinding of the regulator from the 36 operator site. Here another mechanistic paradigm exists regarding the kinetics of regulator unbinding, 37 which is presumed to be a unimolecular reaction (i.e., spontaneous unbinding), whose first-order rate 38 constant is independent of surrounding regulator concentration. However, recent in vitro single-39 molecule and bulk measurements uncovered facilitated unbinding, in which the first-order unbinding 40 rate constant increases with increasing protein concentrations 5 . These proteins include nucleoid 41 associated proteins that bind double-stranded DNA nonspecifically 6 , replication protein A that binds 42 single-stranded DNA nonspecifically 7 , and DNA polymerases 8,9 . We also discovered that CueR and 43 ZntR, two MerR-family metal-sensing transcription regulators that bind to their cognate promoter 44 sequences specifically, also show facilitated unbinding 10 . Using single-molecule tracking (SMT) and 45 single cell quantification of protein concentration (SCQPC) that connect protein-DNA interaction 46 kinetics with cellular protein concentrations quantitatively, we further showed that the facilitated 47 unbinding of CueR and ZntR also operate in living E. coli cells 11 . A mechanistic consensus emerged, 48 involving multivalent contacts between the protein and DNA 5 , which enables the formation of ternary 49 complexes as intermediates that subsequently give rise to concentration-enhanced protein unbinding 50 kinetics. 51 Here we report a SMT and SCQPC study of Zur, a Fur-family homodimeric zinc-uptake 52 regulator, whose Zn 2+ -bound holo-form binds to its cognate operator site with nM affinity and represses 53 the transcription of zinc uptake genes under zinc stress 12-15 ; its apo-form is a non-repressor. We found 54 that in living E. coli cells, Zur's interactions with DNA challenge the above two paradigms. First, apo-55 Zur, long thought to not bind DNA, can bind to chromosome tightly, likely at non-consensus sites. 56 Second and more strikingly, the unbinding of both apo-and holo-Zur from chromosome not only show 57 facilitated unbinding with increasing cellular protein concentrations, but also exhibit repressed 58 unbinding at lower concentrations, giving a first-of-its-kind biphasic unbinding behavior. The repressed 59 unbinding of Zur likely stems from Zur oligomerization on DNA, where an inter-dimer salt bridge plays 60 a key role, and it likely facilitates transcription switching between repression and depression in cells. 61

SMT and SCQPC identify a tight DNA-binding state for both holo-and apo-Zur in cells 64
To visualize individual Zur proteins in E. coli cells, we fused the photoconvertible fluorescent 65 protein mEos3.2 16, 17 to its C-terminus creating Zur mE , either at its chromosomal locus to have 66 physiological expression or in an inducible plasmid in a zur deletion strain to have a wider range of 67 cellular protein concentrations (Methods). This Zur mE fusion-protein is intact and as functional a 68 repressor as the wild-type (WT) in the cell under Zn stress growth conditions ( Supplementary Fig. 1a-69 b). 70 Using sparse photoconversion and time-lapse stroboscopic imaging, we tracked the motions 71 of photoconverted Zur mE proteins individually in single E. coli cells at tens of nanometer precision until 72 their mEos3.2 tags photobleached (Fig. 1a). This SMT allows for measuring Zur mE 's mobility, which 73 reports on whether the molecule is freely diffusing in the cell or bound to DNA. We repeated this 74 3 photoconversion and SMT cycle 500 times for each cell, during which we counted the number of 75 tracked protein molecules. We then used the SCQPC protocol to quantify the remaining number of 76 Zur mE protein molecules in the same cell 11 , eventually determining the Zur mE concentration in each cell 77 (i.e., [Zur mE ]cell). This single-cell protein quantitation allowed for sorting the cells into groups of similar 78 protein concentrations and subsequently examining protein-concentrationdependent processes, 79 without being limited by the large cell-to-cell heterogeneity in protein expression. 80 We first examined Zur apo mE whose regulatory Zn-binding site was mutated (i.e., C88S) to make 81 it permanent apo and a non-repressor 15 ( Supplementary Fig. 1b). To quantify its mobility in cells, we 82 determined the distribution of its displacement length r between successive images and the 83 corresponding cumulative distribution function (CDF) of r for each cell group having similar cellular 84 Zur apo mE concentrations (Fig. 1b- The resolution of CDFs of r also gave the fractional populations of the three states across the 96 range of cellular protein concentrations (Fig. 1d) whereas that of the TB state decreases (Fig. 1d). states are included based on the resolved distributions of r (Fig. 1b), to select these small displacements 122 and obtain estimates of the individual residence time  of a single Zur protein at a chromosomal tight 123 binding site (Fig. 2a). Each  starts when r drops below r0 and ends when r jumps above r0 (e.g., 's in 124 Fig. 2a), which are expected to reflect dominantly protein unbinding from DNA, or when the mEos3.2-125 tag photobleaches/blinks. 126 We analyzed trajectories from many cells of similar cellular Zur concentrations to obtain their 127 corresponding distribution of  (Fig. 2b). We used a quantitative three-state model (i.e., FD, NB, and two MerR-family metalloregulators that we discovered in vitro and in living cells 10, 11 ; the repressed 141 unbinding of Zur apo mE is a first-of-its-kind discovery, however. 142 In contrast, kd for Zur Zn mE only shows the facilitated unbinding within the accessible cellular 143 protein concentration range (~30 to ~900 nM) -it increases consistently with increasing cellular 144 protein concentrations (Fig. 2d, left, red points). The different behaviors of Zur Zn mE from that of Zur apo mE 145 indicate that we could indeed observe the behaviors of the holo-repressor. 146

Mechanism of biphasic unbinding of Zur from DNA 147
Amid the biphasic unbinding of Zur from DNA (Fig. 2d, left), the concentration-facilitated 148 unbinding at higher protein concentrations is analogous to those of CueR and ZntR 11 . There it stems 149 from an assisted dissociation pathway, in which an incoming protein from solution helps an incumbent 150 protein on DNA to unbind, or a direct substitution pathway, in which the incoming protein directly 151 replaces the incumbent one ( Fig. 2e, lower) 10, 11 . The rates of both pathways depend linearly on the free 152 protein concentration, and both likely occur through a common ternary protein2DNA complex, in 153 which the two homodimeric proteins each use one DNA-binding domain to bind to half of the dyad 154 recognition sequence 5, 24 . As Zur is also a homodimer, Zur also could form this ternary complex and 155 undergo assisted dissociation or direct substitution, leading to its concentration-facilitated unbinding 156 from DNA. 157 Regarding the repressed unbinding of apo-Zur in the lower concentration regime, we propose 158 that it likely results from protein oligomerization around the DNA binding site, in which the number of 159 proteins in the oligomer increases with increasing protein concentration and the resulting protein-160 protein interactions contribute to additional stabilization, thereby repressing protein unbinding rate (Fig.  161 2e, upper sites should lead to more populations at shorter pair-wise distances. This PWD for Zur apo mE indeed shows 171 a higher population at distances shorter than ~500 nm relative to the simulated random sites (Fig. 3a). 172 However, at the distance scale of a few hundred nanometers, the compaction of chromosome also 173 contributes to the PWD of residence sites 11 . To decouple the contribution of protein oligomerization 174 from chromosome compaction, we examined the fraction of residence sites within a radius threshold R. 175 At small R (e.g., <100 nm), the contribution of Zur oligomerization to this fraction should dominate 176 over chromosome compaction, as oligomerization is at molecular scale whereas the most compact 177 chromosome in a E. coli cell is still around hundreds of nanometer in dimension 11, 26 . At any specified 178 R (e.g., 200 nm), the fraction of Zur apo mE residence sites within the radius R increases expectedly with 179 increasing cellular protein concentrations (Fig. 3b, red points), because higher protein concentrations 180 gave higher sampling frequency of residence sites. More important, at lower R (e.g., 100 nm), the 181 fraction of Zur apo mE residence sites is larger than that of simulated random sites (Fig 3b, red vs. blue 182 points), and their ratio is larger at lower protein concentrations (Fig. 3b, ko is a first-order intrinsic unbinding rate constant. The kr n term accounts for the repressed unbinding 192 from protein oligomerization, where a first-order rate constant kr is attenuated by the factor  (0 <  < as reported for CueR/ ZntR 11 . In the limit of weak oligomerization and low free protein concentrations, 197 the apparent unbinding rate constant kd from any TB site is: 198 ; it has the units of protein concentration, reflecting the effective dissociation constant of the 199 protein oligomer on the chromosome. k o off = ko + kr; it is a first-order spontaneous unbinding rate 200 constant at the limit of zero cellular protein concentration. Equation (2) satisfactorily fits the biphasic 201 unbinding kinetics of Zur apo mE (Fig. 2d, left), giving the associated kinetic parameters (Table 1 and  202  Supplementary Table 6). In particular, Km of Zur apo mE is ~5 nM, indicating that apo-Zur can oligomerize 203 on chromosome at its physiological concentrations in the cells (Fig. 4a). 204 The same model also allowed for analyzing the relative populations of FD, NB, and TB states 205 of Zur across all cellular protein concentrations, giving additional thermodynamic and kinetic 206 parameters ( behavior, however the minimum of the apparent unbinding rate constant kd shifted to a higher cellular 220 protein concentration (Fig. 2d, right). Its Km is 16.2  7.5 nM, three times larger than that of Zur apo mE 221 (Table 1), indicating a weakened oligomerization affinity and thus a significant role of these salt bridges. 222 More strikingly, for Zur Zn mE , which only showed facilitated unbinding (Fig. 2d, left), the 223 resulting mutant Zur Zn, D49A mE clearly shows biphasic unbinding with Km = 3.2  1.9 nM (Fig. 2d,  and holo-Zur not only exhibit facilitated unbinding, a newly discovered phenomenon for a few DNA-237 binding proteins 6, 7, 9, 28 , but also show repressed unbinding, a first-of-its-kind phenomenon that likely 238 results from Zur oligomerization on chromosome, facilitated by inter-dimer salt bridges. Overall, Zur 239 has biphasic unbinding kinetics from chromosome with increasing cellular protein concentrations, 240 which challenges the paradigm of protein unbinding being typically unimolecular processes whose first-241 order rate constants do not depend on the protein concentration. 242 To probe whether the biphasic unbinding of Zur occurs within the physiological cellular protein 243 concentrations, we quantified cellular Zur mE concentration when it is encoded only at the chromosomal 244 locus (Fig. 4a). In minimal medium without Zn stress, the cellular Zur mE , which is mostly in the apo-245 form, ranges from ~24 to 108 nM (mean = 50  14 nM), within which apo-Zur unbinding from TB sites 246 is in the repressed unbinding regime and slows down by ~42% from the lowest to the highest protein 247 concentration (Fig. 4b). When stressed by 20 M Zn 2+ , the cellular Zur mE , now mostly in the holo-form, 248 ranges from ~26 to 124 nM (mean = 63  20 nM), reflecting an average of ~28% protein concentration 249 increase induced by Zn stress. In this protein concentration range, holo-Zur is already in the facilitated 250 unbinding regime, and its unbinding rate from a recognition site can increase by ~36% (Fig. 4b). 251 Within the physiological protein concentration range, the opposite dependences of unbinding 252 kinetics on the cellular protein concentration between apo-and holo-Zur could provide functional 253 advantages for an E. coli cell to repress or de-repress Zn uptake genes. When cell encounters 254 environmental Zn stress that demands strong repression of Zn uptake, the cellular concentration of Zur 255 swings upward and it becomes dominantly in the holo-repressor form. The unbinding of holo-repressor 256 from recognition sites could be facilitated by its increasing concentration (Fig. 5a), but the facilitated 257 unbinding via direct substitution by another holo-repressor has no functional consequence while 258 7 facilitated unbinding via assisted dissociation will be immediately compensated by a rebinding of a 259 holo-repressor (the rebinding would occur within ~0.014 s; Supplementary Note 7). For those cellular 260 Zur in the apo non-repressor form, its unbinding from DNA slows down, keeping them longer (i.e., 261 stored) at non-consensus chromosomal sites (Fig. 5b). On the other hand, when cell transitions to a Zn-262 deficient environment that demands derepression of Zn uptake, the cellular Zur protein concentration 263 goes down. Here unbinding of the holo-repressor would be slower (Fig. 5c), which is undesirable for 264 derepression, while the unbinding of the apo-form would become faster, releasing them from the non-265 consensus "storage" sites on the chromosome into the cytosol (Fig. 5d). If the cytosolic apo-Zur could 266 possibly facilitate the unbinding of holo-Zur from promoter recognition sites (e.g., through assisted 267 dissociation), it would give a more facile transition to derepression. To support this possibility, we 268 measured the apparent unbinding rate constant kd for chromosomally encoded Zur Zn mE in cells that 269 contains a plasmid encoding an untagged Zurapo mutant (i.e., C88S). When the expression of this Zurapo 270 mutant is induced, kd of Zur Zn mE increases by ~28% at any cellular Zur Zn mE concentration (Fig. 4b, green  271 vs. red points), indicating that apo-Zur can indeed facilitate the unbinding of holo-Zur from recognition 272 sites (Fig. 5e) were generated via site-directed mutagenesis in pBAD24, which was introduced into the Δzur strain. of these effects are most significant on the FD state, less on the NB state, and negligible on the TB state, 317 and were evaluated quantitatively in a previous study of metal-responsive transcription regulators of a 318 different family 11 . 319

Determination and analysis of kd 320
A three-state (FD, NB, and TB state) kinetic model, including the interconversion between 321 states and photobleaching/blinking rates (Fig. 2c), was used to analyze the distribution of residence 322 times (upper thresholded by r0; Fig. 2a Here k eff FD = k bl and Ai is the fractional population of i th -state. 329 The dependence of kd on the cellular free diffusing protein concentration [P]FD was analyzed 330 with Eq. (2), containing three terms representing spontaneous, repressed, and facilitated unbinding with 331 the corresponding rate constants k o off , kr, and k f , respectively (derivation in Supplementary Note 6). 332

Analysis of relative populations 333
The same three-state kinetic model (Fig. 2c)   in Fig. 1b)  while that of apo-Zur from storage site is repressed (b) due to increase in cellular protein concentration. 524 Upon zinc deficiency, the facilitated unbinding of holo-Zur is attenuated (c) while the unbinding of apo-525 Zur is less repressed (d) due to decrease in cellular protein concentration. Released apo-Zur into cytosol 526 could facilitate holo-Zur to unbind (e), helping transition to de-repression of zinc uptake. 527 528