Bacterial Transcription Factors Research Articles

A contact-based analysis of local energetic frustration dynamics identifies key residues enabling RfaH fold-switch.

Fold-switching enables metamorphic proteins to reversibly interconvert between two highly dissimilar native states to regulate their protein functions. While about 100 proteins have been identified to undergo fold-switching, unveiling the key residues behind this mechanism for each protein remains challenging. Reasoning that fold-switching in proteins is driven by dynamic changes in local energetic frustration, we combined fold-switching simulations generated using simplified structure-based models with frustration analysis to identify key residues involved in this process based on the change in the density of minimally frustrated contacts during refolding. Using this approach to analyze the fold-switch of the bacterial transcription factor RfaH, we identified 20 residues that significantly change their frustration during its fold-switch, some of which have been experimentally and computationally reported in previous works. Our approach, which we developed as an additional module for the FrustratometeR package, highlights the role of local frustration dynamics in protein fold-switching and offers a robust tool to enhance our understanding of other proteins with significant conformational shifts.

Predicting bacterial transcription factor binding sites through machine learning and structural characterization based on DNA duplex stability.

Transcriptional factors (TFs) in bacteria play a crucial role in gene regulation by binding to specific DNA sequences, thereby assisting in the activation or repression of genes. Despite their central role, deciphering shape recognition of bacterial TFs-DNA interactions remains an intricate challenge. A deeper understanding of DNA secondary structures could greatly enhance our knowledge of how TFs recognize and interact with DNA, thereby elucidating their biological function. In this study, we employed machine learning algorithms to predict transcription factor binding sites (TFBS) and classify them as directed-repeat (DR) or inverted-repeat (IR). To accomplish this, we divided the set of TFBS nucleotide sequences by size, ranging from 8 to 20 base pairs, and converted them into thermodynamic data known as DNA duplex stability (DDS). Our results demonstrate that the Random Forest algorithm accurately predicts TFBS with an average accuracy of over 82% and effectively distinguishes between IR and DR with an accuracy of 89%. Interestingly, upon converting the base pairs of several TFBS-IR into DDS values, we observed a symmetric profile typical of the palindromic structure associated with these architectures. This study presents a novel TFBS prediction model based on a DDS characteristic that may indicate how respective proteins interact with base pairs, thus providing insights into molecular mechanisms underlying bacterial TFs-DNA interaction.

Fundamentals and Exceptions of the LysR-type Transcriptional Regulators.

LysR-type transcriptional regulators (LTTRs) are emerging as a promising group of macromolecules for the field of biosensors. As the largest family of bacterial transcription factors, the LTTRs represent a vast and mostly untapped repertoire of sensor proteins. To fully harness these regulators for transcription factor-based biosensor development, it is crucial to understand their underlying mechanisms and functionalities. In the first part, this Review discusses the established model and features of LTTRs. As dual-function regulators, these inducible transcription factors exude precise control over their regulatory targets. In the second part of this Review, an overview is given of the exceptions to the "classic" LTTR model. While a general regulatory mechanism has helped elucidate the intricate regulation performed by LTTRs, it is essential to recognize the variations within the family. By combining this knowledge, characterization of new regulators can be done more efficiently and accurately, accelerating the expansion of transcriptional sensors for biosensor development. Unlocking the pool of LTTRs would significantly expand the currently limited range of detectable molecules and regulatory functions available for the implementation of novel synthetic genetic circuitry.

A Transient, Excited Species of the Autoinhibited α-State of the Bacterial Transcription Factor RfaH May Represent an Early Intermediate on the Fold-Switching Pathway.

RfaH is a two-domain transcription factor in which the C-terminal domain switches fold from an α-helical hairpin to a β-roll upon binding the ops-paused RNA polymerase. To ascertain the presence of a sparsely populated excited state that may prime the autoinhibited resting state of RfaH for binding ops-paused RNA polymerase, we carried out a series of NMR-based exchange experiments to probe for conformational exchange on the millisecond time scale. Quantitative analysis of these data reveals exchange between major ground (∼95%) and sparsely populated excited (∼5%) states with an exchange lifetime of ∼3 ms involving residues at the interface between the N-terminal and C-terminal domains formed by the β3/β4 hairpin and helix α3 of the N-terminal domain and helices α4 and α5 of the C-terminal domain. The largest 15N backbone chemical shift differences are associated with the β3/β4 hairpin, leading us to suggest that the excited state may involve a rigid body lateral displacement/rotation away from the C-terminal domain to adopt a position similar to that seen in the active RNA polymerase-bound state. Such a rigid body reorientation would result in a reduction in the interface between the N- and C-terminal domains with the possible introduction of a cavity or cavities. This hypothesis is supported by the observation that the population of the excited species and the exchange rate of interconversion between ground and excited states are reduced at a high (2.5 kbar) pressure. Mechanistic implications for fold switching of the C-terminal domain in the context of RNA polymerase binding are discussed.

BolA-like protein (IbaG) promotes biofilm formation and pathogenicity of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a gram-negative halophilic bacterium widespread in temperate and tropical coastal waters; it is considered to be the most frequent cause of Vibrio-associated gastroenteritis in many countries. BolA-like proteins, which reportedly affect various growth and metabolic processes including flagellar synthesis in bacteria, are widely conserved from prokaryotes to eukaryotes. However, the effects exerted by BolA-like proteins on V. parahaemolyticus remain unclear, and thus require further investigation. In this study, our purpose was to investigate the role played by BolA-like protein (IbaG) in the pathogenicity of V. parahaemolyticus. We used homologous recombination to obtain the deletion strain ΔibaG and investigated the biological role of BolA family protein IbaG in V. parahaemolyticus. Our results showed that IbaG is a bacterial transcription factor that negatively modulates swimming capacity. Furthermore, overexpressing IbaG enhanced the capabilities of V. parahaemolyticus for swarming and biofilm formation. In addition, inactivation of ibaG in V. parahaemolyticus SH112 impaired its capacity for colonizing the heart, liver, spleen, and kidneys, and reduced visceral tissue damage, thereby leading to diminished virulence, compared with the wild-type strain. Finally, RNA-sequencing revealed 53 upregulated and 71 downregulated genes in the deletion strain ΔibaG. KEGG enrichment analysis showed that the two-component system, quorum sensing, bacterial secretion system, and numerous amino acid metabolism pathways had been altered due to the inactivation of ibaG. The results of this study indicated that IbaG exerts a considerable effect on gene regulation, motility, biofilm formation, and pathogenicity of V. parahaemolyticus. To the best of our knowledge, this is the first systematic study on the role played by IbaG in V. parahaemolyticus infections. Thus, our findings may lead to a better understanding of the metabolic processes involved in bacterial infections and provide a basis for the prevention and control of such infections.

Genetically encoded Boolean logic operators to sense and integrate phenylpropanoid metabolite levels in plants.

Synthetic biology has the potential to revolutionize biotechnology, public health, and agriculture. Recent studies have shown the enormous potential of plants as chassis for synthetic biology applications. However, tools to precisely manipulate metabolic pathways for bioproduction in plants are still needed. We used bacterial allosteric transcription factors (aTFs) that control gene expression in a ligand-specific manner and tested their ability to repress semi-synthetic promoters in plants. We also tested the modulation of their repression activity in response to specific plant metabolites, especially phenylpropanoid-related molecules. Using these aTFs, we also designed synthetic genetic circuits capable of computing Boolean logic operations. Three aTFs, CouR, FapR, and TtgR, achieved c. 95% repression of their respective target promoters. For TtgR, a sixfold de-repression could be triggered by inducing its ligand accumulation, showing its use as biosensor. Moreover, we designed synthetic genetic circuits that use AND, NAND, IMPLY, and NIMPLY Boolean logic operations and integrate metabolite levels as input to the circuit. We showed that biosensors can be implemented in plants to detect phenylpropanoid-related metabolites and activate a genetic circuit that follows a predefined logic, demonstrating their potential as tools for exerting control over plant metabolic pathways and facilitating the bioproduction of natural products.

Effects of OxyR regulator on oxidative stress, Apx toxin secretion and virulence of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, poses a significant threat to global swine populations due to its high prevalence, mortality rates, and substantial economic ramifications. Understanding the pathogen's defense mechanisms against host-produced reactive oxygen species is crucial for its survival, with OxyR, a conserved bacterial transcription factor, being pivotal in oxidative stress response. This study investigated the presence and role of OxyR in A. pleuropneumoniae serovar 1-12 reference strains. Transcriptomic analysis was conducted on an oxyR disruption mutant to delineate the biological activities influenced by OxyR. Additionally, specific assays were employed to assess urease activity, catalase expression, ApxI toxin secretion, as well as adhesion and invasion abilities of the oxyR disruption mutant on porcine 3D4/21 and PT cells. A mice challenge experiment was also conducted to evaluate the impact of oxyR inactivation on A. pleuropneumoniae virulence. OxyR was identified as a conserved regulator present in A. pleuropneumoniae serovar 1-12 reference strains. Transcriptomic analysis revealed the involvement of OxyR in multiple biological activities. The oxyR disruption resulted in decreased urease activity, elevated catalase expression, enhanced ApxI toxin secretion-attributed to OxyR binding to the apxIBD promoter-and reduced adhesion and invasion abilities on porcine cells. Furthermore, inactivation of oxyR reduced the virulence of A. pleuropneumoniae in a mice challenge experiment. The findings highlight the pivotal role of OxyR in influencing the virulence mechanisms of A. pleuropneumoniae. The observed effects on various biological activities underscore OxyR as an essential factor contributing to the pathogenicity of this bacterium.

Bacteriophage SPO1 protein Gp46 suppresses functions of HU protein in Francisella tularensis.

The nucleoid-associated protein HU is a common bacterial transcription factor, whose role in pathogenesis and virulence has been described in many bacteria. Our recent studies showed that the HU protein is an indispensable virulence factor in the human pathogenic bacterium Francisella tularensis, a causative agent of tularemia disease, and that this protein can be a key target in tularemia treatment or vaccine development. Here, we show that Francisella HU protein is inhibited by Gp46, a protein of Bacillus subtilis bacteriophage SPO1. We predicted that Gp46 could occupy the F. tularensis HU protein DNA binding site, and subsequently confirmed the ability of Gp46 to abolish the DNA-binding capacity of HU protein. Next, we showed that the growth of Francisella wild-type strain expressing Gp46 in trans corresponded to that of a deletion mutant strain lacking the HU protein. Similarly, the efficiency of intracellular proliferation in mouse macrophages resembled that of the deletion mutant strain, but not that of the wild-type strain. These results, in combination with findings from a recent study on Gp46, enabled us to confirm that Gp46 could be a universal inhibitor of HU proteins among bacterial species.

The Lrp transcriptional factor of an entomopathogenic bacterium, Xenorhabdus hominickii, activates non-ribosomal peptide synthetases to suppress insect immunity

Two bacterial genera, Xenorhabdus and Photorhabdus, are mutually symbiotic to the entomopathogenic nematodes, Steinernema and Heterorhabditis, respectively. The infective juveniles deliver the symbiotic bacteria to the hemocoel of target insects, in which the bacteria proliferate and help the development of the host nematode. The successful parasitism of the nematode-bacterial complex depends on host immunosuppression by the bacteria via their secondary metabolites. Leucine-responsive regulatory protein (Lrp) is a global bacterial transcriptional factor that plays a crucial role in parasitism. However, its regulatory targets to suppress insect immunity are not clearly understood. This study investigated the bacterial genes regulated by Lrp and the subsequent production of secondary metabolites in Xenorhabdus hominickii. Lrp expression occurred at the early infection stage of the bacteria in a target insect, Spodoptera exigua. A preliminary in silico screening indicated that 3.7% genes among 4075 predicted genes encoded in X. hominickii had the Lrp-response element on their promoters, including two non-ribosomal peptide synthetases (NRPSs). Eight NRPS (NRPS1-NRPS8) genes were predicted in the bacterial genome, in which six NRPS (NRPS3-NRPS8) expressions were positively correlated with Lrp expression in the infected larvae of S. exigua. Exchange of the Lrp promoter with an inducible promoter altered the production of the secondary metabolites and the NRPS expression levels. The immunosuppressive activities of X. hominickii were dependent on the Lrp expression level. The metabolites produced by Lrp expression included the eicosanoid-biosynthesis inhibitors and hemolytic factors. A cyclic dipeptide (=cPF) was produced by the bacteria at high Lrp expression and inhibited the phospholipase A2 activity of S. exigua in a competitive inhibitory manner. These results suggest that Lrp is a global transcriptional factor of X. hominickii and plays a crucial role in insect immunosuppression by modulating NRPS expression.

High-throughput screening system of citrus bacterial canker-associated transcription factors and its application to the regulation of citrus canker resistance

One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker (CBC), caused by the bacteria Xanthomonas citri subsp. citri (Xcc). Response to CBC is a complex process, with both protein-DNA as well as protein–protein interactions for the regulatory network. To detect such interactions in CBC resistant regulation, a citrus high-throughput screening system with 203 CBC-inducible transcription factors (TFs), were developed. Screening the upstream regulators of target by yeast-one hybrid (Y1H) methods was also performed. A regulatory module of CBC resistance was identified based on this system. One TF (CsDOF5.8) was explored due to its interactions with the 1-kb promoter fragment of CsPrx25, a resistant gene of CBC involved in reactive oxygen species (ROS) homeostasis regulation. Electrophoretic mobility shift assay (EMSA), dual-LUC assays, as well as transient overexpression of CsDOF5.8, further validated the interactions and transcriptional regulation. The CsDOF5.8–CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via H2O2 homeostasis. The high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance regulation. In addition, it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.

Neuromodulators as Interdomain Signaling Molecules Capable of Occupying Effector Binding Sites in Bacterial Transcription Factors

Hormones and neurotransmitters are important components of inter-kingdom signaling systems that ensure the coexistence of eukaryotes with their microbial community. Their ability to affect bacterial physiology, metabolism, and gene expression was evidenced by various experimental approaches, but direct penetration into bacteria has only recently been reported. This opened the possibility of considering neuromodulators as potential effectors of bacterial ligand-dependent regulatory proteins. Here, we assessed the validity of this assumption for the neurotransmitters epinephrine, dopamine, and norepinephrine and two hormones (melatonin and serotonin). Using flexible molecular docking for transcription factors with ligand-dependent activity, we assessed the ability of neuromodulators to occupy their effector binding sites. For many transcription factors, including the global regulator of carbohydrate metabolism, CRP, and the key regulator of lactose assimilation, LacI, this ability was predicted based on the analysis of several 3D models. By occupying the ligand binding site, neuromodulators can sterically hinder the interaction of the target proteins with the natural effectors or even replace them. The data obtained suggest that the direct modulation of the activity of at least some bacterial transcriptional factors by neuromodulators is possible. Therefore, the natural hormonal background may be a factor that preadapts bacteria to the habitat through direct perception of host signaling molecules.

A method for assaying DNA flexibility.

The transcription, replication, packaging, and repair of genetic information ubiquitously involves DNA:protein interactions and other biological processes that require local mechanical distortions of DNA. The energetics of such DNA-deforming processes are thus dependent on the local mechanical properties of DNA such as bendability or torsional rigidity. Such properties, in turn, depend on sequence, making it possible for sequence to regulate diverse biological processes by controlling the local mechanical properties of DNA. A deeper understanding of how such a "mechanical code" can encode broad regulatory information has historically been hampered by the absence of technology to measure in high throughput how local DNA mechanics varies with sequence along large regions of the genome. This was overcome in a recently developed technique called loop-seq. Here we describe a variant of the loop-seq protocol, that permits making rapid flexibility measurements in low-throughput, without the need for next-generation sequencing. We use our method to validate a previous prediction about how the binding site for the bacterial transcription factor Integration Host Factor (IHF) might serve as a rigid roadblock, preventing efficient enhancer-promoter contacts in IHF site containing promoters in E. coli, which can be relieved by IHF binding.

Structural basis of transcription factor YhaJ for DNT detection

Detection of landmines without harming personnel is a global issue. The bacterial transcription factor YhaJ selectively detects metabolites of explosives, and it can be used as a key component of DNT biosensors. However, the wild-type YhaJ has a binding affinity that is not sufficient for the detection of trace amounts of explosives leaked from landmines buried in the soil. Here, we report crystal structures of the effector-binding domain of YhaJ in both the apo- and effector-bound forms. A structural comparison of the two forms revealed that the loop above the primary effector-binding site significantly switches its conformation upon effector binding. The primary effector-binding site involves hydrophobic and polar interactions, having specificity to hydroxyl-substituted benzene compounds. The structures explain the mechanism of activity-enhancing mutations and provide information for the rational engineering of YhaJ biosensors for the sensitive detection of explosives.

Structural insights into the DNA recognition mechanism by the bacterial transcription factor PdxR.

Specificity in protein-DNA recognition arises from the synergy of several factors that stem from the structural and chemical signatures encoded within the targeted DNA molecule. Here, we deciphered the nature of the interactions driving DNA recognition and binding by the bacterial transcription factor PdxR, a member of the MocR family responsible for the regulation of pyridoxal 5'-phosphate (PLP) biosynthesis. Single particle cryo-EM performed on the PLP-PdxR bound to its target DNA enabled the isolation of three conformers of the complex, which may be considered as snapshots of the binding process. Moreover, the resolution of an apo-PdxR crystallographic structure provided a detailed description of the transition of the effector domain to the holo-PdxR form triggered by the binding of the PLP effector molecule. Binding analyses of mutated DNA sequences using both wild type and PdxR variants revealed a central role of electrostatic interactions and of the intrinsic asymmetric bending of the DNA in allosterically guiding the holo-PdxR-DNA recognition process, from the first encounter through the fully bound state. Our results detail the structure and dynamics of the PdxR-DNA complex, clarifying the mechanism governing the DNA-binding mode of the holo-PdxR and the regulation features of the MocR family of transcription factors.

Discrete Dynamics of Warhead Modulation on Covalent Inhibition of Oxyr: A QM/MM Study.

The bacterial transcriptional factor OxyR, a peroxide sensor conserved in bacterial virulence pathways, has the capability to exhibit exceptional reactivity toward hydrogen peroxide (H2O2). H2O2 is essential for oxidizing cysteine thiolates to maintain cellular redox homeostasis and is dispensable for bacterial growth that can potentially mitigate drug resistance, thus underlining OxyR as a valuable target. We employ quantum mechanics/molecular mechanics (QM/MM) umbrella sampling (US) simulations at the DFTB3/MM level of theory and propose a reaction mechanism with four potential covalent inhibitors. The potential of mean force reveals the direct role of intrinsic reactivity of inhibitors, for instance, benzothiophenes and modified experimental inhibitors with methyl oxo-enoate warhead-activated carbonyl samples in the first step of reaction, which shed light on the significance of proton transfer indispensable for full inhibition, whereas the nitrile inhibitor undergoes a stepwise mechanism with a small proton-transfer energy barrier and lower imaginary frequencies that materialize instantly after nucleophilic attack. To unveil the molecular determinants of respective binding affinities, transition states along the reaction path are optimized and characterized with B3LYP 6-31+G(d,p). Furthermore, the post-simulation analysis indicates the catalytic triad (His130/Cys199/Thr129), thermodynamically favored for inhibition, which restricts water molecules from acting as the potential source of protonation/deprotonation. This study thus serves as a preamble to add variation in the proposed structures and unveils the impact of functional groups lying in warheads that modulate the kinetics of proton transfer, which will certainly aid to design more selective and efficient irreversible inhibitors of OxyR.

Transcription regulation by CarD in mycobacteria is guided by basal promoter kinetics

Bacterial pathogens like Mycobacterium tuberculosis (Mtb) employ transcription factors to adapt their physiology to the diverse environments within their host. CarD is a conserved bacterial transcription factor that is essential for viability in Mtb. Unlike classical transcription factors that recognize promoters by binding to specific DNA sequence motifs, CarD binds directly to the RNA polymerase to stabilize the open complex intermediate (RPo) during transcription initiation. We previously showed using RNA-sequencing that CarD is capable of both activating and repressing transcription invivo. However, it is unknown how CarD achieves promoter-specific regulatory outcomes in Mtb despite binding indiscriminate of DNA sequence. We propose a model where CarD's regulatory outcome depends on the promoter's basal RPo stability and test this model using invitro transcription from a panel of promoters with varying levels of RPo stability. We show that CarD directly activates full-length transcript production from the Mtb ribosomal RNA promoter rrnAP3 (AP3) and that the degree of transcription activation by CarD is negatively correlated with RPo stability. Using targeted mutations in the extended-10 and discriminator region of AP3, we show that CarD directly represses transcription from promoters that form relatively stable RPo. DNA supercoiling also influenced RPo stability and affected the direction of CarD regulation, indicating that the outcome of CarD activity can be regulated by factors beyond promoter sequence. Our results provide experimental evidence for how RNA polymerase-binding transcription factors like CarD can exert specific regulatory outcomes based on the kinetic properties of a promoter.

Three-dimensional chromosome re-modelling: The integral mechanism of transcription regulation in bacteria.

Nucleoid-associated proteins (NAPs) are architectural proteins of the bacterial chromosome and transcription factors that dynamically organise the chromosome and regulate gene expression in response to physicochemical environmental signals. While the architectural and regulatory functions of NAPs have been verified independently, the coupling between these functions in vivo has not been conclusively proven. Here we describe a model NAP - histone-like nucleoid structuring protein (H-NS) - as a coupled sensor-effector that directly regulates gene expression by chromatin re-modelling in response to physicochemical environmental signals. We outline how H-NS-binding partners and post-translational modifications modulate the role of H-NS as a transcription factor by influencing its DNA structuring properties. We consolidate our ideas in models of how H-NS may regulate the expression of the proVWX and hlyCABD operons by chromatin re-modelling. The interplay between chromosome structure and gene expression may be a common - but, at present, under-appreciated - concept of transcription regulation in bacteria.

Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone, and side-chains in EL222.

Photoreceptors containing the light-oxygen-voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light. The mechanism and kinetics of dark-state recovery are not well understood. Here we incorporated the non-canonical amino acid p-cyanophenylalanine (CNF) by genetic code expansion technology at 45 positions of the bacterial transcription factor EL222. Screening of light-induced changes in infrared (IR) absorption frequency, electric field and hydration of the nitrile groups identified residues CNF31 and CNF35 as reporters of monomer/oligomer and caged/decaged equilibria, respectively. Time-resolved multi-probe UV/visible and IR spectroscopy experiments of the lit-to-dark transition revealed four dynamical events. Predominantly, rearrangements around the A'α helix interface (CNF31 and CNF35) precede FMN-cysteinyl adduct scission, folding of α-helices (amide bands), and relaxation of residue CNF151. This study illustrates the importance of characterizing all parts of a protein and suggests a key role for the N-terminal A'α extension of the LOV domain in controlling EL222 photocycle length.

Combinatorial protein dimerization enables precise multi-input synthetic computations

Bacterial transcription factors (TFs) with helix-turn-helix (HTH) DNA-binding domains have been widely explored to build orthogonal transcriptional regulation systems in mammalian cells. Here we capitalize on the modular structure of these proteins to build a framework for multi-input logic gates relying on serial combinations of inducible protein–protein interactions. We found that for some TFs, their HTH domain alone is sufficient for DNA binding. By fusing the HTH domain to TFs, we established dimerization dependent rather than DNA-binding-dependent activation. This enabled us to convert gene switches from OFF-type into more widely applicable ON-type systems and to create mammalian gene switches responsive to new inducers. By combining both OFF and ON modes of action, we built a compact, high-performance bandpass filter. Furthermore, we were able to show cytosolic and extracellular dimerization. Cascading up to five pairwise fusion proteins yielded robust multi-input AND logic gates. Combinations of different pairwise fusion proteins afforded a variety of 4-input 1-output AND and OR logic gate configurations.

CAMP Activation of the cAMP Receptor Protein, a Model Bacterial Transcription Factor.

The active and inactive structures of the Escherichia coli cAMP receptor protein (CRP), a model bacterial transcription factor, are compared to generate a paradigm in the cAMP-induced activation of CRP. The resulting paradigm is shown to be consistent with numerous biochemical studies of CRP and CRP*, a group of CRP mutants displaying cAMP-free activity. The cAMP affinity of CRP is dictated by two factors: (i) the effectiveness of the cAMP pocket and (ii) the protein equilibrium of apo-CRP. How these two factors interplay in determining the cAMP affinity and cAMP specificity of CRP and CRP* mutants are discussed. Both the current understanding and knowledge gaps of CRP-DNA interactions are also described. This review ends with a list of several important CRP issues that need to be addressed in the future.