The 5'UTR of HCoV-OC43 adopts a topologically constrained structure to intrinsically repress translation.

The emergence of SARS-CoV-2, which is responsible for the COVID-19 pandemic, has highlighted the need for rapid characterization of viral mechanisms associated with cellular pathogenesis. Viral UTRs represent conserved genomic elements that contribute to such mechanisms. Structural details of most CoV UTRs are not available, however. Experimental approaches are needed to allow for the facile generation of high-quality viral RNA tertiary structural models, which can facilitate comparative mechanistic efforts. By integrating experimental and computational techniques, we herein report the efficient characterization of conserved RNA structures within the 5'UTR of the HCoV-OC43 genome, a lab-tractable model coronavirus. We provide evidence that the 5'UTR folds into a structure with well-defined stem-loops (SLs) as determined by chemical probing and direct detection of hydrogen bonds by NMR. We combine experimental base-pair restraints with global structural information from SAXS to generate a 3D model that reveals that SL1-4 adopts a topologically constrained structure wherein SLs 3 and 4 coaxially stack. Coaxial stacking is mediated by short linker nucleotides and allows SLs 1 to 2 to sample different cojoint orientations by pivoting about the SL3,4 helical axis. To evaluate the functional relevance of the SL3,4 coaxial helix, we engineered luciferase reporter constructs harboring the HCoV-OC43 5'UTR with mutations designed to abrogate coaxial stacking. Our results reveal that the SL3,4 helix intrinsically represses translation efficiency since the destabilizing mutations correlate with increased luciferase expression relative to wildtype without affecting reporter mRNA levels, thus highlighting how the 5'UTR structure contributes to the viral mechanism.

A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells.

The positive-sense, single-stranded RNA genome SARS-CoV-2 harbors functionally important cis-acting elements governing critical aspects of viral gene expression. However, insights on how these elements sense various signals from the host cell and regulate viral protein synthesis are lacking. Here, we identified two novel cis-regulatory elements in SARS-CoV-2 ORF1a and S RNAs and describe their role in translational control of SARS-CoV-2. These elements are sequence-unrelated but form conserved hairpin structures (validated by NMR) resembling gamma activated inhibitor of translation (GAIT) elements that are found in a cohort of human mRNAs directing translational suppression in myeloid cells in response to IFN-γ. Our studies show that treatment of human lung cells with receptor-binding S1 subunit, S protein pseudotyped lentivirus, and S protein-containing virus-like particles triggers a signaling pathway involving DAP-kinase1 that leads to phosphorylation and release of the ribosomal protein L13a from the large ribosomal subunit. Released L13a forms a virus activated inhibitor of translation (VAIT) complex that binds to ORF1a and S VAIT elements, causing translational silencing. Translational silencing requires extracellular S protein (and its interaction with host ACE2 receptor), but not its intracellular synthesis. RNA-protein interaction analyses and in vitro translation experiments showed that GAIT and VAIT elements do not compete with each other, highlighting differences between the two pathways. Sequence alignments of SARS-CoV-2 genomes showed a high level of conservation of VAIT elements, suggesting their functional importance. This VAIT-mediated translational control mechanism of SARS-CoV-2 may provide novel targets for small molecule intervention and/or facilitate development of more effective mRNA vaccines. IMPORTANCE Specific RNA elements in the genomes of RNA viruses play important roles in host-virus interaction. For SARS-CoV-2, the mechanistic insights on how these RNA elements could sense the signals from the host cell are lacking. Here we report a novel relationship between the GAIT-like SARS-CoV-2 RNA element (called VAITs) and the signal generated from the host cell. We show that for SARS-CoV-2, the interaction of spike protein with ACE2 not only serves the purpose for viral entry into the host cell, but also transduces signals that culminate into the phosphorylation and the release of L13a from the large ribosomal subunit. We also show that this event leads to the translational arrest of ORF1a and S mRNAs in a manner dependent on the structure of the RNA elements. Translational control of viral mRNA by a host-cell generated signal triggered by viral protein is a new paradigm in the host-virus relationship.

Idiosyncrasies of hnRNP A1-RNA recognition: Can binding mode influence function.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a diverse family of RNA binding proteins that function in most stages of RNA metabolism. The prototypical member, hnRNP A1, is composed of three major domains; tandem N-terminal RNA Recognition Motifs (RRMs) and a C-terminal mostly intrinsically disordered region. HnRNP A1 is broadly implicated in basic cellular RNA processing events such as splicing, stability, nuclear export and translation. Due to its ubiquity and abundance, hnRNP A1 is also frequently usurped to control viral gene expression. Deregulation of the RNA metabolism functions of hnRNP A1 in neuronal cells contributes to several neurodegenerative disorders. Because of these roles in human pathologies, the study of hnRNP A1 provides opportunities for the development of novel therapeutics, with disruption of its RNA binding capabilities being the most promising target. The functional diversity of hnRNP A1 is reflected in the complex nature by which it interacts with various RNA targets. Indeed, hnRNP A1 binds both structured and unstructured RNAs with binding affinities that span several magnitudes. Available structures of hnRNP A1-RNA complexes also suggest a degree of plasticity in molecular recognition. Given the reinvigoration in hnRNP A1, the goal of this review is to use the available structural biochemical developments as a framework to interpret its wide-range of RNA functions.

Thermodynamic stability of hnRNP A1 low complexity domain revealed by high-pressure NMR.

We have investigated the pressure- and temperature-induced conformational changes associated with the low complexity domain of hnRNP A1, an RNA-binding protein able to phase separate in response to cellular stress. Solution NMR spectra of the hnRNP A1 low-complexity domain fused with protein-G B1 domain were collected from 1 to 2500 bar and from 268 to 290 K. While the GB1 domain shows the typical pressure-induced and cold temperature-induced unfolding expected for small globular domains, the low-complexity domain of hnRNP A1 exhibits unusual pressure and temperature dependences. We observed that the low-complexity domain is pressure sensitive, undergoing a major conformational transition within the prescribed pressure range. Remarkably, this transition has the inverse temperature dependence of a typical folding-unfolding transition. Our results suggest the presence of a low-lying extended and fully solvated state(s) of the low-complexity domain that may play a role in phase separation. This study highlights the exquisite sensitivity of solution NMR spectroscopy to observe subtle conformational changes and illustrates how pressure perturbation can be used to determine the properties of metastable conformational ensembles.

Integrated approaches to reveal mechanisms by which RNA viruses reprogram the cellular environment.

RNA viruses are major threats to global society and mass outbreaks can cause long-lasting damage to international economies. RNA and related retro viruses represent a large and diverse family that contribute to the onset of human diseases such as AIDS; certain cancers like T cell lymphoma; severe acute respiratory illnesses as seen with COVID-19; and others. The hallmark of this viral family is the storage of genetic material in the form of RNA, and upon infecting host cells, their RNA genomes reprogram the cellular environment to favor productive viral replication. RNA is a multifunctional biomolecule that not only stores and transmits heritable information, but it also has the capacity to catalyze complex biochemical reactions. It is therefore no surprise that RNA viruses use this functional diversity to their advantage to sustain chronic or lifelong infections. Efforts to subvert RNA viruses therefore requires a deep understanding of the mechanisms by which these pathogens usurp cellular machinery. Here, we briefly summarize several experimental techniques that individually inform on key physicochemical features of viral RNA genomes and their interactions with proteins. Each of these techniques provide important vantage points to understand the complexities of virus-host interactions, but we attempt to make the case that by integrating these and similar methods, more vivid descriptions of how viruses reprogram the cellular environment emerges. These vivid descriptions should expedite the identification of novel therapeutic targets.

Genome-Wide Analysis of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) Binding to HIV-1 RNA Reveals a Key Role for hnRNP H1 in Alternative Viral mRNA Splicing.

Alternative splicing of HIV-1 mRNAs increases viral coding potential and controls the levels and timing of gene expression. HIV-1 splicing is regulated in part by heterogeneous nuclear ribonucleoproteins (hnRNPs) and their viral target sequences, which typically repress splicing when studied outside their native viral context. Here, we determined the location and extent of hnRNP binding to HIV-1 mRNAs and their impact on splicing in a native viral context. Notably, hnRNP A1, hnRNP A2, and hnRNP B1 bound to many dispersed sites across viral mRNAs. Conversely, hnRNP H1 bound to a few discrete purine-rich sequences, a finding that was mirrored in vitro hnRNP H1 depletion and mutation of a prominent viral RNA hnRNP H1 binding site decreased the use of splice acceptor A1, causing a deficit in Vif expression and replicative fitness. This quantitative framework for determining the regulatory inputs governing alternative HIV-1 splicing revealed an unexpected splicing enhancer role for hnRNP H1 through binding to its target element.IMPORTANCE Alternative splicing of HIV-1 mRNAs is an essential yet quite poorly understood step of virus replication that enhances the coding potential of the viral genome and allows the temporal regulation of viral gene expression. Although HIV-1 constitutes an important model system for general studies of the regulation of alternative splicing, the inputs that determine the efficiency with which splice sites are utilized remain poorly defined. Our studies provide an experimental framework to study an essential step of HIV-1 replication more comprehensively and in much greater detail than was previously possible and reveal novel cis-acting elements regulating HIV-1 splicing.

Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a multipurpose RNA-binding protein (RBP) involved in normal and pathological RNA metabolism. Transcriptome-wide mapping and in vitro evolution identify consensus hnRNP A1 binding motifs; however, such data do not reveal how surrounding RNA sequence and structural context modulate affinity. We determined the affinity of hnRNP A1 for all possible sequence variants (n = 16,384) of the HIV exon splicing silencer 3 (ESS3) 7-nt apical loop. Analysis of the affinity distribution identifies the optimal motif 5'-YAG-3' and shows how its copy number, position in the loop, and loop structure modulate affinity. For a subset of ESS3 variants, we show that specificity is determined by association rate constants and that variants lacking the minimal sequence motif bind competitively with consensus RNA. Thus, the results reveal general rules of specificity of hnRNP A1 and provide a quantitative framework for understanding how it discriminates between alternative competing RNA ligands in vivo.

Conserved structures formed by heterogeneous RNA sequences drive silencing of an inflammation responsive post-transcriptional operon.

RNA-protein interactions with physiological outcomes usually rely on conserved sequences within the RNA element. By contrast, activity of the diverse gamma-interferon-activated inhibitor of translation (GAIT)-elements relies on the conserved RNA folding motifs rather than the conserved sequence motifs. These elements drive the translational silencing of a group of chemokine (CC/CXC) and chemokine receptor (CCR) mRNAs, thereby helping to resolve physiological inflammation. Despite sequence dissimilarity, these RNA elements adopt common secondary structures (as revealed by 2D-1H NMR spectroscopy), providing a basis for their interaction with the RNA-binding GAIT complex. However, many of these elements (e.g. those derived from CCL22, CXCL13, CCR4 and ceruloplasmin (Cp) mRNAs) have substantially different affinities for GAIT complex binding. Toeprinting analysis shows that different positions within the overall conserved GAIT element structure contribute to differential affinities of the GAIT protein complex towards the elements. Thus, heterogeneity of GAIT elements may provide hierarchical fine-tuning of the resolution of inflammation.

Differential Conformational Dynamics Encoded by the Linker between Quasi RNA Recognition Motifs of Heterogeneous Nuclear Ribonucleoprotein H.

Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) F/H family are multipurpose RNA binding proteins that participate in most stages of RNA metabolism. Despite having similar RNA sequence preferences, hnRNP F/H proteins function in overlapping and, in some cases, distinct cellular processes. The domain organization of hnRNP F/H proteins is modular, consisting of N-terminal tandem quasi-RNA recognition motifs (F/HqRRM1,2) and a third C-terminal qRRM3 embedded between glycine-rich repeats. The tandem qRRMs are connected through a 10-residue linker, with several amino acids strictly conserved between hnRNP H and F. A significant difference occurs at position 105 of the linker, where hnRNP H contains a proline and hnRNP F an alanine. To investigate the influence of P105 on the conformational properties of hnRNP H, we probed the structural dynamics of its HqRRM1,2 domain with X-ray crystallography, NMR spectroscopy, and small-angle X-ray scattering. The collective results best describe that HqRRM1,2 exists in a conformational equilibrium between compact and extended structures. The compact structure displays an electropositive surface formed at the qRRM1-qRRM2 interface. Comparison of NMR relaxation parameters, including Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion, between HqRRM1,2 and FqRRM1,2 indicates that FqRRM1,2 primarily adopts a more extended and flexible conformation. Introducing the P105A mutation into HqRRM1,2 alters its conformational dynamics to favor an extended structure. Thus, our work demonstrates that the linker compositions confer different structural properties between hnRNP F/H family members that might contribute to their functional diversity.

Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1.

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3' acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.

Thermodynamic and phylogenetic insights into hnRNP A1 recognition of the HIV-1 exon splicing silencer 3 element.

Complete expression of the HIV-1 genome requires balanced usage of suboptimal splice sites. The 3' acceptor site A7 (ssA7) is negatively regulated in part by an interaction between the host hnRNP A1 protein and a viral splicing silencer (ESS3). Binding of hnRNP A1 to ESS3 and other upstream silencers is sufficient to occlude spliceosome assembly. Efforts to understand the splicing repressive properties of hnRNP A1 on ssA7 have revealed hnRNP A1 binds specific sites within the context of a highly folded RNA structure; however, biochemical models assert hnRNP A1 disrupts RNA structure through cooperative spreading. In an effort to improve our understanding of the ssA7 binding properties of hnRNP A1, herein we have performed a combined phylogenetic and biophysical study of the interaction of its UP1 domain with ESS3. Phylogenetic analyses of group M sequences (x̅ = 2860) taken from the Los Alamos HIV database reveal the ESS3 stem loop (SL3(ESS3)) structure has been conserved throughout HIV-1 evolution, despite variations in primary sequence. Calorimetric titrations with UP1 clearly show the SL3(ESS3) structure is a critical binding determinant because deletion of the base-paired region reduces the affinity by ∼150-fold (Kd values of 27.8 nM and 4.2 µM). Cytosine substitutions of conserved apical loop nucleobases show UP1 preferentially binds purines over pyrimidines, where site-specific interactions were detected via saturation transfer difference nuclear magnetic resonance. Chemical shift mapping of the UP1-SL3(ESS3) interface by (1)H-(15)N heteronuclear single-quantum coherence spectroscopy titrations reveals a broad interaction surface on UP1 that encompasses both RRM domains and the inter-RRM linker. Collectively, our results describe a UP1 binding mechanism that is likely different from current models used to explain the alternative splicing properties of hnRNP A1.

Regio-selective chemical-enzymatic synthesis of pyrimidine nucleotides facilitates RNA structure and dynamics studies.

Isotope labeling has revolutionized NMR studies of small nucleic acids, but to extend this technology to larger RNAs, site-specific labeling tools to expedite NMR structural and dynamics studies are required. Using enzymes from the pentose phosphate pathway, we coupled chemically synthesized uracil nucleobase with specifically (13) C-labeled ribose to synthesize both UTP and CTP in nearly quantitative yields. This chemoenzymatic method affords a cost-effective preparation of labels that are unattainable by current methods. The methodology generates versatile (13) C and (15) N labeling patterns which, when employed with relaxation-optimized NMR spectroscopy, effectively mitigate problems of rapid relaxation that result in low resolution and sensitivity. The methodology is demonstrated with RNAs of various sizes, complexity, and function: the exon splicing silencer 3 (27 nt), iron responsive element (29 nt), Pro-tRNA (76 nt), and HIV-1 core encapsidation signal (155 nt).

Enterovirus evolution reveals the mechanism of an RNA-targeted antiviral and determinants of viral replication.

Selective pressures on viruses provide opportunities to establish target site specificity and mechanisms of antivirals. Enterovirus (EV)-A71 with resistant mutations in the stem loop (SL) II internal ribosome entry site (IRES) (SLIIresist) were selected at low doses of the antiviral dimethylamiloride (DMA)-135. The EV-A71 mutants were resistant to DMA-135 at concentrations that inhibit replication of wild-type virus. EV-A71 IRES structures harboring resistant mutations induced efficient expression of Luciferase messenger RNA in the presence of noncytotoxic doses of DMA-135. Nuclear magnetic resonance indicates that the mutations change the structure of SLII at the binding site of DMA-135 and at the surface recognized by the host protein AU-rich element/poly(U)-binding/degradation factor 1 (AUF1). Biophysical studies of complexes formed between AUF1, DMA-135, and either SLII or SLIIresist show that DMA-135 stabilizes a ternary complex with AUF1-SLII but not AUF1-SLIIresist. This work demonstrates how viral evolution elucidates the (DMA-135)-RNA binding site specificity in cells and provides insights into the viral pathways inhibited by the antiviral.

High-affinity interaction of hnRNP A1 with conserved RNA structural elements is required for translation and replication of enterovirus 71.

Human Enterovirus 71 (EV71) is an emerging pathogen of infectious disease and a serious threat to public health. Currently, there are no antivirals or vaccines to slow down or prevent EV71 infections, thus underscoring the urgency to better understand mechanisms of host-enterovirus interactions. EV71 uses a type I internal ribosome entry site (IRES) to recruit the 40S ribosomal subunit via a pathway that requires the cytoplasmic localization of hnRNP A1, which acts as an IRES trans-activating factor. The mechanism of how hnRNP A1 trans activates EV71 RNA translation is unknown, however. Here, we report that the UP1 domain of hnRNP A1 interacts specifically with stem loop II (SLII) of the IRES, via a thermodynamically well-defined biphasic transition that involves conserved bulge 5'-AYAGY-3' and hairpin 5'-RY(U/A)CCA-3' loops. Calorimetric titrations of wild-type and mutant SLII constructs reveal these structural elements are essential to form a high-affinity UP1-SLII complex. Mutations that alter the bulge and hairpin primary or secondary structures abrogate the biphasic transition and destabilize the complex. Notably, mutations within the bulge that destabilize the complex correlate with a large reduction in IRES-dependent translational activity and impair EV71 replication. Taken together, this study shows that a conserved SLII structure is necessary to form a functional hnRNP A1-IRES complex, suggesting that small molecules that target this stem loop may have novel antiviral properties.

Biophysical Analysis of Small Molecule Binding to Viral RNA Structures.

RNA molecules are essential for carrying genetic information and regulating gene expression in most organisms including human pathogenic RNA and relate retro viruses. Targeting viral RNA (vRNA) structures provide broad opportunities to develop chemical tools to probe molecular virology and to discover novel targets for therapeutic intervention. An increasing number of RNA binding small molecules are being identified, stimulating increased interests in small molecule drug discovery for RNA targets. In this chapter, we describe protocols to characterize and robustly validate vRNA-small molecule (vRNA-sm) interactions starting from vRNA sample preparation, followed by small molecule screening against vRNA targets and finally to validating the vRNA-sm interactions via NMR spectroscopy and calorimetric titrations.

Thermodynamic Coupling of the tandem RRM domains of hnRNP A1 underlie its Pleiotropic RNA Binding Functions.

The functional properties of RNA-binding proteins (RBPs) require allosteric regulation through inter-domain communication. Despite the foundational importance of allostery to biological regulation, almost no studies have been conducted to describe the biophysical nature by which inter-domain communication manifests in RBPs. Here, we show through high-pressure studies with hnRNP A1 that inter-domain communication is vital for the unique stability of its N- terminal domain containing a tandem of RNA Recognition Motifs (RRMs). Despite high sequence similarity and nearly identical tertiary structures, the two RRMs exhibit drastically different stability under pressure. RRM2 unfolds completely under high-pressure as an individual domain, but when appended to RRM1, it remains stable. Variants in which inter-domain communication is disrupted between the tandem RRMs show a large decrease in stability under pressure. Carrying these mutations over to the full-length protein for in vivo experiments revealed that the mutations affected the ability of the disordered C-terminus to engage in protein-protein interactions and more importantly, they also influenced the RNA binding capacity. Collectively, this work reveals that thermodynamic coupling between the tandem RRMs of hnRNP A1 accounts for its allosteric regulatory functions.

The Repurposing of Cellular Proteins during Enterovirus A71 Infection.

Viruses pose a great threat to people's lives. Enterovirus A71 (EV-A71) infects children and infants all over the world with no FDA-approved treatment to date. Understanding the basic mechanisms of viral processes aids in selecting more efficient drug targets and designing more effective antivirals to thwart this virus. The 5'-untranslated region (5'-UTR) of the viral RNA genome is composed of a cloverleaf structure and an internal ribosome entry site (IRES). Cellular proteins that bind to the cloverleaf structure regulate viral RNA synthesis, while those that bind to the IRES also known as IRES trans-acting factors (ITAFs) regulate viral translation. In this review, we survey the cellular proteins currently known to bind the 5'-UTR and influence viral gene expression with emphasis on comparing proteins' functions and localizations pre- and post-(EV-A71) infection. A comprehensive understanding of how the host cell's machinery is hijacked and reprogrammed by the virus to facilitate its replication is crucial for developing effective antivirals.

Influenza virus mRNAs encode determinants for nuclear export via the cellular TREX-2 complex.

Nuclear export of influenza A virus (IAV) mRNAs occurs through the nuclear pore complex (NPC). Using the Auxin-Induced Degron (AID) system to rapidly degrade proteins, we show that among the nucleoporins localized at the nucleoplasmic side of the NPC, TPR is the key nucleoporin required for nuclear export of influenza virus mRNAs. TPR recruits the TRanscription and EXport complex (TREX)-2 to the NPC for exporting a subset of cellular mRNAs. By degrading components of the TREX-2 complex (GANP, Germinal-center Associated Nuclear Protein; PCID2, PCI domain containing 2), we show that influenza mRNAs require the TREX-2 complex for nuclear export and replication. Furthermore, we found that cellular mRNAs whose export is dependent on GANP have a small number of exons, a high mean exon length, long 3' UTR, and low GC content. Some of these features are shared by influenza virus mRNAs. Additionally, we identified a 45 nucleotide RNA signal from influenza virus HA mRNA that is sufficient to mediate GANP-dependent mRNA export. Thus, we report a role for the TREX-2 complex in nuclear export of influenza mRNAs and identified RNA determinants associated with the TREX-2-dependent mRNA export.

Differential binding between PatS C-terminal peptide fragments and HetR from Anabaena sp. PCC 7120.

Heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120 occurs at regular intervals under nitrogen starvation and is regulated by a host of signaling molecules responsive to availability of fixed nitrogen. The heterocyst differentiation inhibitor PatS contains the active pentapeptide RGSGR (PatS-5) at its C-terminus considered the minimum PatS fragment required for normal heterocyst pattern formation. PatS-5 is known to bind HetR, the master regulator of heterocyst differentiation, with a moderate affinity and a submicromolar dissociation constant. Here we characterized the affinity of HetR for several PatS C-terminal fragments by measuring the relative ability of each fragment to knockdown HetR binding to DNA in electrophoretic mobility shift assays and using isothermal titration calorimetry (ITC). HetR bound to PatS-6 (ERGSGR) >30 times tighter (K(d) = 7 nM) than to PatS-5 (K(d) = 227 nM) and >1200 times tighter than to PatS-7 (DERGSGR) (K(d) = 9280 nM). No binding was detected between HetR and PatS-8 (CDERGSGR). Quantitative binding constants obtained from ITC measurements were consistent with qualitative results from the gel shift knockdown assays. CW EPR spectroscopy confirmed that PatS-6 bound to a MTSL spin-labeled HetR L252C mutant at a 10-fold lower concentration compared to PatS-5. Substituting the PatS-6 N-terminal glutamate to aspartate, lysine, or glycine did not alter binding affinity, indicating that neither the charge nor size of the N-terminal residue's side chain played a role in enhanced HetR binding to PatS-6, but rather increased binding affinity resulted from new interactions with the PatS-6 N-terminal residue peptide backbone.