An α helix to ß barrel domain switch transforms the transcription factor RfaH into a translation factor.

NusG homologs regulate transcription and coupled processes in all living organisms. The Escherichia coli (E. coli) two-domain paralogs NusG and RfaH have conformationally identical N-terminal domains (NTDs) but dramatically different carboxy-terminal domains (CTDs), a ß barrel in NusG and an α hairpin in RfaH. Both NTDs interact with elongating RNA polymerase (RNAP) to reduce pausing. In NusG, NTD and CTD are completely independent, and NusG-CTD interacts with termination factor Rho or ribosomal protein S10. In contrast, RfaH-CTD makes extensive contacts with RfaH-NTD to mask an RNAP-binding site therein. Upon RfaH interaction with its DNA target, the operon polarity suppressor (ops) DNA, RfaH-CTD is released, allowing RfaH-NTD to bind to RNAP. Here, we show that the released RfaH-CTD completely refolds from an all-α to an all-ß conformation identical to that of NusG-CTD. As a consequence, RfaH-CTD binding to S10 is enabled and translation of RfaH-controlled operons is strongly potentiated. PAPERFLICK:

NMR-Based Metabolite Profiling and the Application of STOCSY toward the Quality and Authentication Assessment of European EVOOs.

Extra virgin olive oil (EVOO) possesses a high-value rank in the food industry, thus making it a common target for adulteration. Hence, several methods have been essentially made available over the years. However, the issue of authentication remains unresolved with national and food safety organizations globally struggling to regulate and control its market. Over the course of this study, the aim was to determine the origin of EVOOs suggesting a high-throughput, state-of-the-art method that could be easily adopted. A rapid, NMR-based untargeted metabolite profiling method was applied and complemented by multivariate analysis (MVA) and statistical total correlation spectroscopy (STOCSY). STOCSY is a valuable statistical tool contributing to the biomarker identification process and was employed for the first time in EVOO analysis. Market samples from three Mediterranean countries of Spain, Italy, and Greece, blended samples from these countries, as well as monocultivar samples from Greece were analyzed. The NMR spectra were collected, with the help of chemometrics acting as "fingerprints" leading to the discovery of certain chemical classes and single biomarkers that were related to the classification of the samples into groups based on their origin.

Quality and potency profile of eight recombinant isoallergens, largely mimicking total Bet v 1-specific IgE binding of birch pollen.

BACKGROUND: To date, only limited information on structure, expression levels and IgE binding of Bet v 1 variants, which are simultaneously expressed in birch pollen, is available. OBJECTIVE: To analyse and compare structure and serum IgE/IgG binding of rBet v 1 variants to Bet v 1.0101. METHODS: Recombinant Bet v 1 variants were studied with sera of 20 subjects allergic to birch pollen. Folding, aggregation and solubility of the rBet v 1 variants were analysed to attribute diverging IgE binding to either allergen structure or methodological features. IgE/IgG binding was studied with rBet v 1 in solution or adsorbed to solid phases. Allergen-mediated cross-linking of FcεRI receptors was determined by mediator release of sensitized humanized rat basophil leukaemia cells. RESULTS: All variants, except for rBet v 1.0113, were monomeric and had Bet v 1-type conformation. Serum IgE binding to variants adsorbed to solid phase was reduced to 6.6%-36.5% compared with Bet v 1.0101. In contrast, inhibition of IgE binding to Bet v 1.0101 by rBet v 1 variants ranged from 62% to 83%. Similarly, mediator release ranged from 30.7% to 55.2% for all variants and was only clearly reduced for rBet v 1.0301 (10.4%). The IgE-binding potency of rBet v 1 variants representing their native quantities in birch pollen was only slightly lower compared to extract. IgG binding to variants was between 50.9% and 134.5% compared with rBet v 1.0101 (100%). CONCLUSION AND CLINICAL RELEVANCE: Bet v 1 variants previously classified as hypoallergenic can exhibit similar functional IgE binding as Bet v 1.0101. Eight rBet v 1 variants largely reproduce total Bet v 1-specific IgE binding of birch pollen extracts. Assay format-dependent variation in IgE-binding properties needs to be considered in the development of diagnostic or therapeutic products.

Thermotoga maritima NusG: domain interaction mediates autoinhibition and thermostability.

NusG, the only universally conserved transcription factor, comprises an N- and a C-terminal domain (NTD, CTD) that are flexibly connected and move independently in Escherichia coli and other organisms. In NusG from the hyperthermophilic bacterium Thermotoga maritima (tmNusG), however, NTD and CTD interact tightly. This closed state stabilizes the CTD, but masks the binding sites for the interaction partners Rho, NusE and RNA polymerase (RNAP), suggesting that tmNusG is autoinhibited. Furthermore, tmNusG and some other bacterial NusGs have an additional domain, DII, of unknown function. Here we demonstrate that tmNusG is indeed autoinhibited and that binding to RNAP may stabilize the open conformation. We identified two interdomain salt bridges as well as Phe336 as major determinants of the domain interaction. By successive weakening of this interaction we show that after domain dissociation tmNusG-CTD can bind to Rho and NusE, similar to the Escherichia coli NusG-CTD, indicating that these interactions are conserved in bacteria. Furthermore, we show that tmNusG-DII interacts with RNAP as well as nucleic acids with a clear preference for double stranded DNA. We suggest that tmNusG-DII supports tmNusG recruitment to the transcription elongation complex and stabilizes the tmNusG:RNAP complex, a necessary adaptation to high temperatures.

Transcription is regulated by NusA:NusG interaction.

NusA and NusG are major regulators of bacterial transcription elongation, which act either in concert or antagonistically. Both bind to RNA polymerase (RNAP), regulating pausing as well as intrinsic and Rho-dependent termination. Here, we demonstrate by nuclear magnetic resonance spectroscopy that the Escherichia coli NusG amino-terminal domain forms a complex with the acidic repeat domain 2 (AR2) of NusA. The interaction surface of either transcription factor overlaps with the respective binding site for RNAP. We show that NusA-AR2 is able to remove NusG from RNAP. Our in vivo and in vitro results suggest that interaction between NusA and NusG could play various regulatory roles during transcription, including recruitment of NusG to RNAP, resynchronization of transcription:translation coupling, and modulation of termination efficiency.

Development, synthesis and structure-activity-relationships of inhibitors of the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Burkholderia pseudomallei.

The bacteria Burkholderia pseudomallei and Legionella pneumophila cause severe diseases like melioidosis and Legionnaire's disease with high mortality rates despite antibiotic treatment. Due to increasing antibiotic resistances against these and other Gram-negative bacteria, alternative therapeutical strategies are in urgent demand. As a virulence factor, the macrophage infectivity potentiator (Mip) protein constitutes an attractive target. The Mip proteins of B. pseudomallei and L. pneumophila exhibit peptidyl-prolyl cis/trans isomerase (PPIase) activity and belong to the PPIase superfamily. In previous studies, the pipecolic acid moiety proved to be a valuable scaffold for inhibiting this PPIase activity. Thus, a library of pipecolic acid derivatives was established guided by structural information and computational analyses of the binding site and possible binding modes. Stability and toxicity considerations were taken into account in iterative extensions of the library. Synthesis and evaluation of the compounds in PPIase assays resulted in highly active inhibitors. The activities can be interpreted in terms of a common binding mode obtained by docking calculations.

AZT resistance alters enzymatic properties and creates an ATP-binding site in SFVmac reverse transcriptase.

BACKGROUND: The replication of simian foamy virus from macaques can be inhibited by the nucleoside reverse transcriptase inhibitor azidothymidine (AZT, zidovudine). Four substitutions in the protease-reverse transcriptase (PR-RT) protein (K211I, I224T, S345T, E350K) are necessary to obtain highly AZT resistant and fully replication competent virus. AZT resistance is based on the excision of the incorporated AZTMP in the presence of ATP. I224T is a polymorphism which is not essential for AZT resistance per se, but is important for regaining efficient replication of the resistant virus. RESULTS: We constructed PR-RT enzymes harboring one to four amino acid substitutions to analyze them biochemically and to determine their ability to remove the incorporated AZTMP. S345T is the only single substitution variant exhibiting significant AZTMP excision activity. Although K211I alone showed no AZTMP excision activity, excision efficiency doubled when K211I was present in combination with S345T and E350K. K211I also decreased nucleotide binding affinity and increased fidelity. NMR titration experiments revealed that a truncated version of the highly AZT resistant mt4 variant, comprising only the fingers-palm subdomains was able to bind ATP with a KD-value of ca. 7.6 mM, whereas no ATP binding could be detected in the corresponding wild type protein. We could show by NMR spectroscopy that S345T is responsible for ATP binding, probably by making a tryptophan residue accessible. CONCLUSION: Although AZT resistance in SFVmac is based on excision of the incorporated AZTMP like in HIV-1, the functions of the resistance substitutions in SFVmac PR-RT appear to be different. No mutation resulting in an aromatic residue like F/Y215 in HIV, which is responsible for π-π-stacking interactions with ATP, is present in SFVmac. Instead, S345T is responsible for creating an ATP binding site, probably by making an already existing tryptophan more accessible, which in turn can interact with ATP. This is in contrast to HIV-1 RT, in which an ATP binding site is present in the WT RT but differs from that of the AZT resistant enzyme.

Secret of the major birch pollen allergen Bet v 1: identification of the physiological ligand.

The major birch pollen allergen Bet v 1 is the main elicitor of airborne type I allergies and belongs to the PR-10 family (pathogenesis-related proteins 10). Bet v 1 is the most extensively studied allergen, and is well characterized at a biochemical and immunological level; however, its physiological function remains elusive. In the present study, we identify Q3OS (quercetin-3-O-sophoroside) as the natural ligand of Bet v 1. We isolated Q3OS bound to Bet v 1 from mature birch pollen and confirmed its binding by reconstitution of the Bet v 1-Q3OS complex. Fluorescence and UV-visible spectroscopy experiments, as well as HSQC (heteronuclear single-quantum coherence) titration, and the comparison with model compounds, such as quercetin, indicated the specificity of Q3OS binding. Elucidation of the binding site by NMR combined with a computational model resulted in a more detailed understanding and shed light on the physiological function of Bet v 1. We postulate that the binding of Q3OS to Bet v 1 plays an important, but as yet unclear, role during the inflammation response and Bet v 1 recognition by IgE.

Interdomain contacts control folding of transcription factor RfaH.

Escherichia coli RfaH activates gene expression by tethering the elongating RNA polymerase to the ribosome. This bridging action requires a complete refolding of the RfaH C-terminal domain (CTD) from an α-helical hairpin, which binds to the N-terminal domain (NTD) in the free protein, to a ß-barrel, which interacts with the ribosomal protein S10 following RfaH recruitment to its target operons. The CTD forms a ß-barrel when expressed alone or proteolytically separated from the NTD, indicating that the α-helical state is trapped by the NTD, perhaps co-translationally. Alternatively, the interdomain contacts may be sufficient to drive the formation of the α-helical form. Here, we use functional and NMR analyses to show that the denatured RfaH refolds into the native state and that RfaH in which the order of the domains is reversed is fully functional in vitro and in vivo. Our results indicate that all information necessary to determine its fold is encoded within RfaH itself, whereas accessory factors or sequential folding of NTD and CTD during translation are dispensable. These findings suggest that universally conserved RfaH homologs may change folds to accommodate diverse interaction partners and that context-dependent protein refolding may be widespread in nature.

Structural requirements for enzymatic activities of foamy virus protease-reverse transcriptase.

Reverse transcriptases (RTs) are pivotal in the life cycle of retroviruses and convert the genomic viral RNA into double-stranded DNA. The RT polymerase domain is subdivided into fingers, palm, thumb, and the connection subdomain, which links the polymerase to the C-terminal RNase H domain. In contrast to orthoretroviruses, mature RT of foamy viruses harbors the protease (PR) domain at its N-terminus (PR-RT). Therefore and due to low homology to other RTs, it is difficult to define the boundaries and functions of the (sub)domains. We introduced N- and C-terminal deletions into simian foamy virus PR-RT to investigate the impact of the truncations on the catalytic activities. Both, the RNase H domain and the connection subdomain contribute substantially to polymerase integrity and stability as well as to polymerase activity and substrate binding. The 42 amino acids long region C-terminal of the PR is important for polymerase stability and activity. PR activation via binding of PR-RT to viral RNA requires the presence of the full length PR-RT including the RNase H domain. In vitro, the cleavage efficiencies of FV PR for the Gag and Pol cleavage site are comparable, even though in virus particles only the Pol site is cleaved to completion suggesting that additional factors control PR activity and that virus maturation needs to be strictly regulated.

Inhibition of foamy virus reverse transcriptase by human immunodeficiency virus type 1 RNase H inhibitors.

RNase H plays an essential role in the replication of human immunodeficiency virus type 1 (HIV-1). Therefore, it is a promising target for drug development. However, the identification of HIV-1 RNase H inhibitors (RHIs) has been hampered by the open morphology of its active site, the limited number of available RNase H crystal structures in complex with inhibitors, and the fact that, due to the high concentrations of Mg(2+) needed for protein stability, HIV-1 RNase H is not suitable for nuclear magnetic resonance (NMR) inhibitor studies. We recently showed that the RNase H domains of HIV-1 and prototype foamy virus (PFV) reverse transcriptases (RTs) exhibit a high degree of structural similarity. Thus, we examined whether PFV RNase H can serve as an HIV-1 RNase H model for inhibitor interaction studies. Five HIV-1 RHIs inhibited PFV RNase H activity at low-micromolar concentrations similar to those of HIV-1 RNase H, suggesting pocket similarity of the RNase H domains. NMR titration experiments with the PFV RNase H domain and the RHI RDS1643 (6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester) were performed to determine its binding site. Based on these results and previous data, in silico docking analysis showed a putative RDS1643 binding region that reaches into the PFV RNase H active site. Structural overlays were performed with HIV-1 and PFV RNase H to propose the RDS1643 binding site in HIV-1 RNase H. Our results suggest that this approach can be used to establish PFV RNase H as a model system for HIV-1 RNase H in order to identify putative inhibitor binding sites in HIV-1 RNase H.

Diagnosing vascular variability anomalies, not only MESOR-hypertension.

Chronobiology is the study of biological rhythms. Chronomics investigates interactions with environmental cycles in a genetically coded autoresonance of the biosphere with wrangling space and terrestrial weather. Analytical global and local methods applied to human blood pressure records of around-the-clock measurements covering decades detect physiological-physical interactions, a small yet measurable response to solar and terrestrial magnetism. The chronobiological and chronomic interpretation of ambulatory blood pressure monitoring (C-ABPM) records in the light of time-specified reference values derived from healthy peers matched by sex and age identify vascular variability anomalies (VVAs) for an assessment of cardio-, cerebro-, and renovascular disease risk. Even within the conventionally accepted normal range, VVAs have been associated with a statistically significant increase in risk. Long-term C-ABPM records help to "know ourselves," serving for relief of psychological and other strain once transient VVAs are linked to the source of a load, prompting adjustment of one's lifestyle for strain reduction. Persistent circadian VVAs can be treated, sometimes by no more than a change in timing of the daily administration of antihypertensive medication. Circadian VVA assessment is an emergency worldwide, prompted in the United States by 1,000 deaths per day every day from problems related to blood pressure. While some heads of state met under United Nation and World Health Organization sponsorship to declare that noncommunicable diseases are a slow-motion disaster, a resolution has been drafted to propose C-ABPM as an added tool complementing purely physical environmental monitoring to contribute also to the understanding of social and natural as well as personal cataclysms.

Molecular Mechanism of Sirtuin 1 Inhibition by Human Immunodeficiency Virus 1 Tat Protein.

Sirtuins are NAD+-dependent protein lysine deacylases implicated in metabolic regulation and aging-related dysfunctions. The nuclear isoform Sirt1 deacetylates histones and transcription factors and contributes, e.g., to brain and immune cell functions. Upon infection by human immunodeficiency virus 1 (HIV1), Sirt1 deacetylates the viral transactivator of transcription (Tat) protein to promote the expression of the viral genome. Tat, in turn, inhibits Sirt1, leading to the T cell hyperactivation associated with HIV infection. Here, we describe the molecular mechanism of Tat-dependent sirtuin inhibition. Using Tat-derived peptides and recombinant Tat protein, we mapped the inhibitory activity to Tat residues 34-59, comprising Tat core and basic regions and including the Sirt1 deacetylation site Lys50. Tat binds to the sirtuin catalytic core and inhibits Sirt1, Sirt2, and Sirt3 with comparable potencies. Biochemical data and crystal structures of sirtuin complexes with Tat peptides reveal that Tat exploits its intrinsically extended basic region for binding to the sirtuin substrate binding cleft through substrate-like ß-strand interactions, supported by charge complementarity. Tat Lys50 is positioned in the sirtuin substrate lysine pocket, although binding and inhibition do not require prior acetylation and rely on subtle differences to the binding of regular substrates. Our results provide mechanistic insights into sirtuin regulation by Tat, improving our understanding of physiological sirtuin regulation and the role of this interaction during HIV1 infection.

The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding.

BACKGROUND: The ribonuclease H (RNase H) domains of retroviral reverse transcriptases play an essential role in the replication cycle of retroviruses. During reverse transcription of the viral genomic RNA, an RNA/DNA hybrid is created whose RNA strand needs to be hydrolyzed by the RNase H to enable synthesis of the second DNA strand by the DNA polymerase function of the reverse transcriptase. Here, we report the solution structure of the separately purified RNase H domain from prototype foamy virus (PFV) revealing the so-called C-helix and the adjacent basic loop, which both were suggested to be important in substrate binding and activity. RESULTS: The solution structure of PFV RNase H shows that it contains a mixed five-stranded ß-sheet, which is sandwiched by four α-helices (A-D), including the C-helix, on one side and one α-helix (helix E) on the opposite side. NMR titration experiments demonstrate that upon substrate addition signal changes can be detected predominantly in the basic loop as well as in the C-helix. All these regions are oriented towards the bound substrate. In addition, signal intensities corresponding to residues in the B-helix and the active site decrease, while only minor or no changes of the overall structure of the RNase H are detectable upon substrate binding. Dynamic studies confirm the monomeric state of the RNase H domain. Structure comparisons with HIV-1 RNase H, which lacks the basic protrusion, indicate that the basic loop is relevant for substrate interaction, while the C-helix appears to fulfill mainly structural functions, i.e. positioning the basic loop in the correct orientation for substrate binding. CONCLUSIONS: The structural data of PFV RNase H demonstrate the importance of the basic loop, which contains four positively charged lysines, in substrate binding and the function of the C-helix in positioning of the loop. In the dimeric full length HIV-1 RT, the function of the basic loop is carried out by a different loop, which also harbors basic residues, derived from the connection domain of the p66 subunit. Our results suggest that RNases H which are also active as separate domains might need a functional basic loop for proper substrate binding.

An inhibitory peptide derived from the α-subunit of the epithelial sodium channel (ENaC) shows a helical conformation.

Proteolytic activation of the heteromeric epithelial sodium channel (ENaC) is thought to involve the release of inhibitory peptides from the extracellular domains of its α- and γ-subunit. Recently, we demonstrated that an α-13-mer peptide, corresponding to a putative inhibitory region within the extracellular domain of human αENaC, inhibits human αßγENaC. The aim of the present study was to investigate the structural basis of the inhibitory effect of this α-13-mer peptide. Analysis of the peptide by replica exchange molecular dynamics method, circular dichroism spectroscopy, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations suggested that a helical turn at the carboxy-terminus is the preferred conformational state of the α-13-mer peptide. From this we predicted that a specific mutation (leucine 188 to alanine) should have a strong effect on the conformational preferences of the peptide. To functionally test this, we compared the effect of the wild-type α-13-mer with that of a mutant α-L188A-13-mer on ENaC currents in Xenopus laevis oocytes heterologously expressing human αßγENaC. We demonstrated that replacing the leucine 188 by alanine abolished the inhibitory effect of the α-13-mer peptide on ENaC. These findings suggest that a helical conformation in its carboxyterminal part is functionally important to mediate ENaC inhibition by the α-13-mer peptide. However, high resolution structural information on the complex of the inhibitory αENaC peptide and the channel are needed to confirm this conclusion.

Regulation of foamy virus protease activity by viral RNA: a novel and unique mechanism among retroviruses.

Foamy viruses (FVs) synthesize the Pol precursor protein from a specific transcript. Thus, in contrast to what was found for orthoretroviruses, e.g., human immunodeficiency virus, no Gag-Pol precursor protein is synthesized. Foamy viral Pol consists of a protease (PR) domain, a reverse transcriptase domain, and an integrase domain and is processed into a mature protease-reverse transcriptase (PR-RT) fusion protein and the integrase. Protease activity has to be strictly regulated in order to avoid premature Gag and Pol processing before virus assembly. We have demonstrated recently that FV protease is an inactive monomer with a very weak dimerization tendency and postulated protease activation through dimerization. Here, we identify a specific protease-activating RNA motif (PARM) located in the pol region of viral RNA which stimulates PR activity in vitro and in vivo, revealing a novel and unique mechanism of retroviral protease activation. This mechanism is strikingly different to that of orthoretroviruses, where the protease can be activated even in the absence of viral RNA during the assembly of virus-like particles. Although it has been shown that the integrase domain is important for Pol uptake, activation of the foamy virus protease is integrase independent. We show that at least two foamy virus PR-RT molecules bind to the PARM and only RNAs containing the PARM result in significant activation of the protease. DNA harboring the PARM is not capable of protease activation. Structure determination of the PARM by selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) revealed a distinct RNA folding, important for protease activation and thus virus maturation.

Transformation: the next level of regulation.

The textbook view that a primary sequence determines the unique fold of a given protein has been challenged by identification of proteins with variant structures, such as prions. Our recent studies revealed that the transcription factor RfaH simultaneously changes its topology and function. RfaH is a two-domain protein whose N-terminal domain binds to transcribing RNA polymerase, stimulating its processivity. The α-helical C-terminal domain masks the RNA polymerase-binding site of the N-terminal domain, preventing unwarranted recruitment to genes lacking a specific DNA sequence. Upon binding to its DNA target, RfaH domains dissociate, and the C-terminal domain refolds into a ß-barrel. This dramatic transformation allows binding to the ribosomal protein S10 and subsequent recruitment of a ribosome, coupling transcription and translation. We define RfaH as first example of "transformer proteins", in which two alternative structural states have distinct cellular functions and hypothesize that transformer proteins may be widespread in nature.

Domain interactions of the transcription-translation coupling factor Escherichia coli NusG are intermolecular and transient.

The bacterial transcription factor NusG (N-utilization substance G) is suggested to act as a key coupling factor between transcription and translation [Burmann, Schweimer, Luo, Wahl, Stitt, Gottesman and Rösch (2010) Science 328, 501-504] and contributes to phage λ-mediated antitermination in Escherichia coli that enables read-through of early transcription termination sites. E. coli NusG consists of two structurally and functionally distinct domains that are connected through a flexible linker. The homologous Aquifex aeolicus NusG, with a secondary structure that is highly similar to E. coli NusG shows direct interaction between its N- and C-terminal domains in a domain-swapped dimer. In the present study, we performed NMR paramagnetic relaxation enhancement measurements and identified interdomain interactions that were concentration dependent and thus probably not only weak and transient, but also predominantly intermolecular. This notion of two virtually independent domains in a monomeric protein was supported by 15N-relaxation measurements. Thus we suggest that a regulatory role of NusG interdomain interactions is highly unlikely.

Fine tuning of the E. coli NusB:NusE complex affinity to BoxA RNA is required for processive antitermination.

Phage lambda propagation in Escherichia coli host cells requires transcription antitermination on the lambda chromosome mediated by lambdaN protein and four host Nus factors, NusA, B, E (ribosomal S10) and G. Interaction of E. coli NusB:NusE heterodimer with the single stranded BoxA motif of lambdanutL or lambdanutR RNA is crucial for this reaction. Similarly, binding of NusB:NusE to a BoxA motif is essential to suppress transcription termination in the ribosomal RNA (rrn) operons. We used fluorescence anisotropy to measure the binding properties of NusB and of NusB:NusE heterodimer to BoxA-containing RNAs differing in length and sequence. Our results demonstrate that BoxA is necessary and sufficient for binding. We also studied the gain-of-function D118N NusB mutant that allows lambda growth in nusA1 or nusE71 mutants. In vivo lambda burst-size determinations, CD thermal unfolding measurements and X-ray crystallography of this as well as various other NusB D118 mutants showed the importance of size and polarity of amino acid 118 for RNA binding and other interactions. Our work suggests that the affinity of the NusB:NusE complex to BoxA RNA is precisely tuned to maximize control of transcription termination.