tRNA-guanine Transglycosylase Research Articles

It takes two to tRNAgo

In this issue of Structure, Sievers etal.1 gain important insights into the human tRNA guanine transglycosylase QTRT1/2. The study presents a cryo-EM reconstruction of the inhibited human heterodimer in complex with human tRNAAsp, which represents the first snapshot of a eukaryotic TGT in complex with its full-length tRNA substrate.

Structural and functional insights into tRNA recognition by human tRNA guanine transglycosylase

Eukaryotic tRNA guanine transglycosylase (TGT) is an RNA-modifying enzyme which catalyzes the base exchange of the genetically encoded guanine 34 of tRNAsAsp,Asn,His,Tyr for queuine, a hypermodified 7-deazaguanine derivative. Eukaryotic TGT is a heterodimer comprised of a catalytic and a non-catalytic subunit. While binding of the tRNA anticodon loop to the active site is structurally well understood, the contribution of the non-catalytic subunit to tRNA binding remained enigmatic, as no complex structure with a complete tRNA was available. Here, we report a cryo-EM structure of eukaryotic TGT in complex with a complete tRNA, revealing the crucial role of the non-catalytic subunit in tRNA binding. We decipher the functional significance of these additional tRNA-binding sites, analyze solution state conformation, flexibility, and disorder of apo TGT, and examine conformational transitions upon tRNA binding.

TRNATyr has an unusually short half-life in Trypanosoma brucei.

Following transcription, tRNAs undergo a series of processing and modification events to become functional adaptors in protein synthesis. Eukaryotes have also evolved intracellular transport systems whereby nucleus-encoded tRNAs may travel out and into the nucleus. In trypanosomes, nearly all tRNAs are also imported from the cytoplasm into the mitochondrion, which lacks tRNA genes. Differential subcellular localization of the cytoplasmic splicing machinery and a nuclear enzyme responsible for queuosine modification at the anticodon "wobble" position appear to be important quality control mechanisms for tRNATyr, the only intron-containing tRNA in T. brucei Since tRNA-guanine transglycosylase (TGT), the enzyme responsible for Q formation, cannot act on an intron-containing tRNA, retrograde nuclear transport is an essential step in maturation. Unlike maturation/processing pathways, the general mechanisms of tRNA stabilization and degradation in T. brucei are poorly understood. Using a combination of cellular and molecular approaches, we show that tRNATyr has an unusually short half-life. tRNATyr, and in addition tRNAAsp, also show the presence of slow-migrating bands during electrophoresis; we term these conformers: alt-tRNATyr and alt-tRNAAsp, respectively. Although we do not know the chemical or structural nature of these conformers, alt-tRNATyr has a short half-life resembling that of tRNATyr; the same is not true for alt-tRNAAsp We also show that RRP44, which is usually an exosome subunit in other organisms, is involved in tRNA degradation of the only intron-containing tRNA in T. brucei and is partly responsible for its unusually short half-life.

Site-Specific Covalent Labeling of DNA Substrates by an RNA Transglycosylase.

Bacterial tRNA guanine transglycosylases (TGTs) catalyze the exchange of guanine for the 7-deazaguanine queuine precursor, prequeuosine1 (preQ1). While the native nucleic acid substrate for bacterial TGTs is the anticodon loop of queuine-cognate tRNAs, the minimum recognition sequence for the enzyme is a structured hairpin containing the target G nucleobase in a "UGU" loop motif. Previous work has established an RNA modification system, RNA-TAG, in which Escherichia coli TGT exchanges the target G on an RNA of interest for chemically modified preQ1 substrates linked to a small-molecule reporter such as biotin or a fluorophore. While extending the substrate scope of RNA transglycosylases to include DNA would enable numerous applications, it has been previously reported that TGT is incapable of modifying native DNA. Here, we demonstrate that TGT can in fact recognize and label specific DNA substrates. Through iterative testing of rationally mutated DNA hairpin sequences, we determined the minimal sequence requirements for transglycosylation of unmodified DNA by E. coli TGT. Controlling steric constraint in the DNA hairpin dramatically affects labeling efficiency, and, when optimized, can lead to near-quantitative site-specific modification. We demonstrate the utility of our newly developed DNA-TAG system by rapidly synthesizing probes for fluorescent Northern blotting of spliceosomal U6 RNA and RNA FISH visualization of the long noncoding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). The ease and convenience of the DNA-TAG system will provide researchers with a tool for accessing a wide variety of versatile and affordable modified DNA substrates.

7-Deazaguanines in DNA: functional and structural elucidation of a DNA modification system.

The modified nucleosides 2'-deoxy-7-cyano- and 2'-deoxy-7-amido-7-deazaguanosine (dPreQ0 and dADG, respectively) recently discovered in DNA are the products of the bacterial queuosine tRNA modification pathway and the dpd gene cluster, the latter of which encodes proteins that comprise the elaborate Dpd restriction-modification system present in diverse bacteria. Recent genetic studies implicated the dpdA, dpdB and dpdC genes as encoding proteins necessary for DNA modification, with dpdD-dpdK contributing to the restriction phenotype. Here we report the in vitro reconstitution of the Dpd modification machinery from Salmonella enterica serovar Montevideo, the elucidation of the roles of each protein and the X-ray crystal structure of DpdA supported by small-angle X-ray scattering analysis of DpdA and DpdB, the former bound to DNA. While the homology of DpdA with the tRNA-dependent tRNA-guanine transglycosylase enzymes (TGT) in the queuosine pathway suggested a similar transglycosylase activity responsible for the exchange of a guanine base in the DNA for 7-cyano-7-deazaguanine (preQ0), we demonstrate an unexpected ATPase activity in DpdB necessary for insertion of preQ0 into DNA, and identify several catalytically essential active site residues in DpdA involved in the transglycosylation reaction. Further, we identify a modification site for DpdA activity and demonstrate that DpdC functions independently of DpdA/B in converting preQ0-modified DNA to ADG-modified DNA.

Queuosine salvage in fission yeast by Qng1-mediated hydrolysis to queuine

Queuosine (Q) is a hypermodified 7-deaza-guanosine nucleoside that is found at position 34, also known as the wobble position, of tRNAs with a GUN anticodon, and Q ensures faithful translation of the respective C- and U-ending codons. While Q is present in tRNAs in most eukaryotes, only bacteria can synthesize it denovo. In contrast, eukaryotes rely on external sources like their food and the gut microbiome in order to Q-modify their tRNAs, and Q therefore can be regarded as a micronutrient. The eukaryotic tRNA guanine transglycosylase (eTGT) uses the base queuine (q) as a substrate to replace G34 by Q in the tRNAs. Eukaryotic cells can uptake both q and Q, raising the question how the Q nucleoside is converted to q for incorporation into the tRNAs. Here, we identified Qng1 (also termed Duf2419) as a queuosine nucleoside glycosylase in Schizosaccharomyces pombe. S. pombe cells with a deletion of qng1+ contained Q-modified tRNAs only when cultured in the presence of the nucleobase q, but not with the nucleoside Q, indicating that the cells are proficient at q incorporation, but not in Q hydrolysis. Furthermore, purified recombinant Qng1 hydrolyzed Q to q in vitro. Qng1 displays homology to DNA glycosylases and has orthologs across eukaryotes, including flies, mice and humans. Qng1 therefore plays an essential role in allowing eukaryotic cells to salvage Q from bacterial sources and to recycle Q from endogenous tRNAs.

Structural and Biochemical Investigation of the Heterodimeric Murine tRNA-Guanine Transglycosylase.

In tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis of most bacteria and eukaryotes, the anticodon wobble position may be occupied by the modified nucleoside queuosine, which affects the speed and the accuracy of translation. Since eukaryotes are not able to synthesize queuosine de novo, they have to salvage queuine (the queuosine base) as a micronutrient from food and/or the gut microbiome. The heterodimeric Zn2+ containing enzyme tRNA-guanine transglycosylase (TGT) catalyzes the insertion of queuine into the above-named tRNAs in exchange for the genetically encoded guanine. This enzyme has attracted medical interest since it was shown to be potentially useful for the treatment of multiple sclerosis. In addition, TGT inactivation via gene knockout leads to the suppressed cell proliferation and migration of certain breast cancer cells, which may render this enzyme a potential target for the design of compounds supporting breast cancer therapy. As a prerequisite to fully exploit the medical potential of eukaryotic TGT, we have determined and analyzed a number of crystal structures of the functional murine TGT with and without bound queuine. In addition, we have investigated the importance of two residues of its non-catalytic subunit on dimer stability and determined the Michaelis-Menten parameters of murine TGT with respect to tRNA and several natural and artificial nucleobase substrates. Ultimately, on the basis of available TGT crystal structures, we provide an entirely conclusive reaction mechanism for this enzyme, which in detail explains why the TGT-catalyzed insertion of some nucleobases into tRNA occurs reversibly while that of others is irreversible.

Toxicological Properties of 7-Methylguanine, and Preliminary Data on its Anticancer Activity.

7-Methylguanine (7-MG) competitively inhibits the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) and RNA-modifying enzyme tRNA-guanine transglycosylase (TGT) and represents a potential anticancer drug candidate. Furthermore, as a natural compound, it could escape the serious side effects characteristic for approved synthetic PARP inhibitors. Here we present a comprehensive study of toxicological and carcinogenic properties of 7-MG. It was demonstrated that 7-MG does not induce mutations or structural chromosomal abnormalities, and has no blastomogenic activity. A treatment regimen with 7-MG has been established in mice (50 mg/kg per os, 3 times per week), exerting no adverse effects or changes in morphology. Preliminary data on the 7-MG anticancer activity obtained on transplantable tumor models support our conclusions that 7-MG can become a promising new component of chemotherapy.

19F-NMR Unveils the Ligand-Induced Conformation of a Catalytically Inactive Twisted Homodimer of tRNA-Guanine Transglycosylase.

Understanding the structural arrangements of protein oligomers can support the design of ligands that interfere with their function in order to develop new therapeutic concepts for disease treatment. Recent crystallographic studies have elucidated a novel twisted and functionally inactive form of the homodimeric enzyme tRNA-guanine transglycosylase (TGT), a putative target in the fight against shigellosis. Active-site ligands have been identified that stimulate the rearrangement of one monomeric subunit by 130° against the other one to form an inactive twisted homodimer state. To assess whether the crystallographic observations also reflect the conformation in solution and rule out effects from crystal packing, we performed 19F-NMR spectroscopy with the introduction of 5-fluorotryptophans at four sites in TGT. The inhibitor-induced conformation of TGT in solution was assessed based on 19F-NMR chemical shift perturbations. We investigated the effect of C(4) substituted lin-benzoguanine ligands and identified a correlation between dynamic protein rearrangements and ligand-binding features in the corresponding crystal structures. These involve the destabilization of a helix next to the active site and the integrity of a flexible loop-helix motif. Ligands that either completely lack an attached C(4) substituent or use it to stabilize the geometry of the functionally competent dimer state do not indicate the presence of the twisted dimer form in the NMR spectra. The perturbation of crucial structural motifs in the inhibitors correlates with an increasing formation of the inactive twisted dimer state, suggesting these ligands are able to shift a conformational equilibrium from active C2-symmetric to inactive twisted dimer conformations. These findings suggest a novel concept for the design of drug candidates for further development.

Modeling the Structure of Human tRNA-Guanine Transglycosylase in Complex with 7-Methylguanine and Revealing the Factors that Determine the Enzyme Interaction with Inhibitors

tRNA-guanine transglycosylase, an enzyme catalyzing replacement of guanine with queuine in human tRNA and participating in the translation mechanism, is involved in the development of cancer. However, information on the small-molecule inhibitors that can suppress activity of this enzyme is very limited. Molecular dynamics simulations were used to determine the amino acid residues that provide efficient binding of inhibitors in the active site of tRNA-guanine transglycosylase. It was demonstrated using 7-methylguanine molecule as a probe that the ability of the inhibitor to adopt a charged state in the environment of hydrogen bond acceptors Asp105 and Asp159 plays a key role in complex formation. Formation of the hydrogen bonds and hydrophobic contacts with Gln202, Gly229, Phe109, and Met259 residues are also important. It has been predicted that introduction of the substituents would have a different effect on the ability to inhibit tRNA-guanine transglycosylase, as well as the DNA repair protein poly(ADP-ribose) polymerase 1, which can contribute to the development of more efficient and selective compounds.

Dynamic queuosine changes in tRNA couple nutrient levels to codon choice in Trypanosoma brucei.

Every type of nucleic acid in cells undergoes programmed chemical post-transcriptional modification. Generally, modification enzymes use substrates derived from intracellular metabolism, one exception is queuine (q)/queuosine (Q), which eukaryotes obtain from their environment; made by bacteria and ultimately taken into eukaryotic cells via currently unknown transport systems. Here, we use a combination of molecular, cell biology and biophysical approaches to show that in Trypanosoma brucei tRNA Q levels change dynamically in response to concentration variations of a sub-set of amino acids in the growth media. Most significant were variations in tyrosine, which at low levels lead to increased Q content for all the natural tRNAs substrates of tRNA-guanine transglycosylase (TGT). Such increase results from longer nuclear dwell time aided by retrograde transport following cytoplasmic splicing. In turn high tyrosine levels lead to rapid decrease in Q content. Importantly, the dynamic changes in Q content of tRNAs have negligible effects on global translation or growth rate but, at least, in the case of tRNATyr it affected codon choice. These observations have implications for the occurrence of other tunable modifications important for ‘normal’ growth, while connecting the intracellular localization of modification enzymes, metabolites and tRNAs to codon selection and implicitly translational output.

Unraveling a Ligand-Induced Twist of a Homodimeric Enzyme by Pulsed Electron-Electron Double Resonance.

Mechanistic insights into protein–ligand interactions can yield chemical tools for modulating protein function and enable their use for therapeutic purposes. For the homodimeric enzyme tRNA‐guanine transglycosylase (TGT), a putative virulence target of shigellosis, ligand binding has been shown by crystallography to transform the functional dimer geometry into an incompetent twisted one. However, crystallographic observation of both end states does neither verify the ligand‐induced transformation of one dimer into the other in solution nor does it shed light on the underlying transformation mechanism. We addressed these questions in an approach that combines site‐directed spin labeling (SDSL) with distance measurements based on pulsed electron–electron double resonance (PELDOR or DEER) spectroscopy. We observed an equilibrium between the functional and twisted dimer that depends on the type of ligand, with a pyranose‐substituted ligand being the most potent one in shifting the equilibrium toward the twisted dimer. Our experiments suggest a dissociation–association mechanism for the formation of the twisted dimer upon ligand binding.

Structural and functional insights into human tRNA guanine transgylcosylase

The eukaryotic tRNA guanine transglycosylase (TGT) is an RNA modifying enzyme incorporating queuine, a hypermodified guanine derivative, into the tRNAsAsp,Asn,His,Tyr. While both subunits of the functional heterodimer have been crystallized individually, much of our understanding of its dimer interface or recognition of a target RNA have been inferred from its more thoroughly studied bacterial homolog. However, since bacterial TGT, by incorporating queuine precursor preQ1, deviates not only in function, but, as a homodimer, also in its subunit architecture, any inferences regarding the subunit association of the eukaryotic heterodimer or the significance of its unique catalytically inactive subunit are based on unstable footing. Here, we report the crystal structure of human TGT in its heterodimeric form and in complex with a 25-mer stem loop RNA, enabling detailed analysis of its dimer interface and interaction with a minimal substrate RNA. Based on a model of bound tRNA we addressed a potential functional role of the catalytically inactive subunit QTRT2 by UV-crosslinking and mutagenesis experiments, identifying the two-stranded βEβF-sheet of the QTRT2 subunit as an additional RNA-binding motif.

Targeting a Cryptic Pocket in a Protein-Protein Contact by Disulfide-Induced Rupture of a Homodimeric Interface.

Interference with protein-protein interfaces represents an attractive as well as challenging option for therapeutic intervention and drug design. The enzyme tRNA-guanine transglycosylase, a target to fight Shigellosis, is only functional as a homodimer. Although we previously produced monomeric variants by site-directed mutagenesis, we only crystallized the functional dimer, simply because upon crystallization the local protein concentration increases and favors formation of the dimer interface, which represents an optimal and highly stable packing of the protein in the solid state. Unfortunately, this prevents access to structural information about the interface geometry in its monomeric state and complicates the development of modulators that can interfere with and prevent dimer formation. Here, we report on a cysteine-containing protein variant in which, under oxidizing conditions, a disulfide linkage is formed. This reinforces a novel packing geometry of the enzyme. In this captured quasi-monomeric state, the monomer units arrange in a completely different way and, thus, expose a loop-helix motif, originally embedded into the old interface, now to the surface. The motif adopts a geometry incompatible with the original dimer formation. Via the soaking of fragments into the crystals, we identified several hits accommodating a cryptic binding site next to the loop-helix motif and modulated its structural features. Our study demonstrates the druggability of the interface by breaking up the homodimeric protein using an introduced disulfide cross-link. By rational concepts, we increased the potency of these fragments to a level where we confirmed their binding by NMR to a nondisulfide-linked TGT variant. The idea of intermediately introducing a disulfide linkage may serve as a general concept of how to transform a homodimer interface into a quasi-monomeric state and give access to essential structural and design information.

TRANSFER RNA QUEUOSINE MODIFICATION ENZYME MANIPULATES TIGHT JUNCTION PROTEINS IN INFLAMMATORY BOWEL DISEASE

Transfer RNA (tRNA) queuosine (Q)-modification occur at the wobble anticodon position of four special cellular tRNAs. In eukaryotes, tRNA-Q modification relies on the intestinal microbial product queuine and eukaryotic tRNA-guanine transglycosylase complex contains of Q tRNA ribosyltransferase catalytic subunit 1 (QTRT1). Q-tRNA modification is critical for the fidelity and accuracy to translate RNA to protein. It is known that dysfunction of Q-tRNA associates with cancer proliferation and malignancy.

TRANSFER RNA QUEUOSINE MODIFICATION ENZYME MANIPULATES TIGHT JUNCTION PROTEINS IN INFLAMMATORY BOWEL DISEASE

Abstract Background Transfer RNA (tRNA) queuosine (Q)-modification occur at the wobble anticodon position of four special cellular tRNAs. In eukaryotes, tRNA-Q modification relies on the intestinal microbial product queuine and eukaryotic tRNA-guanine transglycosylase complex contains of Q tRNA ribosyltransferase catalytic subunit 1 (QTRT1). Q-tRNA modification is critical for the fidelity and accuracy to translate RNA to protein. It is known that dysfunction of Q-tRNA associates with cancer proliferation and malignancy. However, mechanisms of how Q and Q-tRNA modifications influence intestinal epithelial biology and chronic inflammation are unknown. Methods We explored the expression of QTRT1 in patients with IBD by investigating human biopsy tissues and re-analyzing gene expression datasets (with both UC and CD patients). To further investigate the QTRT1 role in IBD, the dextran sodium sulfate (DSS) induced colitis mouse model and IL10-deficient mouse model were used to evaluate the molecular mechanism. We further investigated the impact of QTRT1 reduction on intestinal cells by generating QTRT1 knockdown HCT116 and Caco-2 BBE cells using a Double Nickase Plasmid. Results We found that the QTRT1 expression in IHC staining was significantly downregulated in the colon of both Ulcerative Colitis (UC) and Crohn’s Disease (CD) patients compared with normal controls. After extracting and analyzing the datasets, we found the gene expression of QTRT1 was significantly suppressed in colonic mucosa of UC and intestinal mucosa of CD, compared with controls. Moreover, the four tRNA-Q-related tRNA synthetase (asparaginyl-tRNA synthetase, aspartyl-tRNA synthetase, histidyl-tRNA synthetase and tyrosyl-tRNA synthetase) were also decreased in IBD patients. The decreased QTRT1-expression was further confirmed in both DSS-induced colitis mice and IL10-defecient mice. Meanwhile, the significantly downregulated beta-catenin and Claudin-5, and upregulated Caludin-2 were identified in these two colitis models. All these molecular alterations, which are important in cell adhesion and tight junction formation, were further verified in protein expression and mRNA level using QTRT1 reduction HCT116 and Caco-2 BBE cells in vitro. Conclusion Our results demonstrate that the QTRT1 plays a novel role in human IBD by altering intestinal cell adhesion and junctions. Microbiome-dependent Q supply can be altered during disease development or when a limited variety of food-types are ingested. Investigations on the gut microbiome-derived tRNA modification in IBD will uncover novel molecular mechanisms for potential prevention and therapy.

Synthesis of azide congeners of preQ1 as potential substrates for tRNA guanine transglycosylase

AbstractPreQ1 (2) is a precursor of queuine (1) that in eubacteria is incorporated into transfer RNA (tRNA) by tRNA guanine transglycosylase (TGT) before being further elaborated into queuine. The queuine modification is unusual and occurs across all eukaryotes and eubacteria with few exceptions, but its function remains unclear. As the modified nucleotide occurs through incorporation of a specially synthesized nucleotide instead of via modification of a genetically encoded base, a study of the sites of modification by prepared probes is possible. We report the synthesis of two novel azide congeners (3,4) of preQ1 for this purpose. The evaluation of their interaction with TGT shows that both probes act as weak competitive inhibitors of guanine exchange of guanine(34) tRNATyr with a Ki of ~70 μM. However, we could not show that these are substrates for TGT‐catalyzed incorporation into tRNA. We believe the reason for this is a marked loss of binding due to the azide functionality of 3 and 4 abrogating the possibility of two hydrogen bonds to the carbonyl group of Leu231 and Met260 of TGT, previously observed for the terminal methylene amine of preQ1 by x‐ray crystallography.

The Importance of Charge in Perturbing the Aromatic Glue Stabilizing the Protein-Protein Interface of Homodimeric tRNA-Guanine Transglycosylase.

Bacterial tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the modified tRNA nucleoside queuosine present in the anticodon wobble position of tRNAs specific for aspartate, asparagine, histidine, and tyrosine. Inactivation of the tgt gene leads to decreased pathogenicity of Shigella bacteria. Therefore, Tgt constitutes a putative target for Shigellosis drug therapy. Since it is only active as homodimer, interference with dimer-interface formation may, in addition to active-site inhibition, provide further means to disable this protein. A cluster of four aromatic residues seems important to stabilize the homodimer. We mutated residues of this aromatic cluster and analyzed each mutated variant with respect to the dimer and thermal stability or enzyme activity by applying native mass spectrometry, a thermal shift assay, enzyme kinetics, and X-ray crystallography. Our structural studies indicate a strong influence of pH on the homodimer stability. Apparently, protonation of a histidine within the aromatic cluster supports the collapse of an essential structural motif within the dimer interface at slightly acidic pH.

Enzymatic RNA Biotinylation for Affinity Purification and Identification of RNA-Protein Interactions.

Throughout their cellular lifetime, RNA transcripts are bound to proteins, playing crucial roles in RNA metabolism, trafficking, and function. Despite the importance of these interactions, identifying the proteins that interact with an RNA of interest in mammalian cells represents a major challenge in RNA biology. Leveraging the ability to site-specifically and covalently label an RNA of interest using E. coli tRNA guanine transglycosylase and an unnatural nucleobase substrate, we establish the identification of RNA-protein interactions and the selective enrichment of cellular RNA in mammalian systems. We demonstrate the utility of this approach through the identification of known binding partners of 7SK snRNA via mass spectrometry. Through a minimal 4-nucleotide mutation of the long noncoding RNA HOTAIR, enzymatic biotinylation enables identification of putative HOTAIR binding partners in MCF7 breast cancer cells that suggest new potential pathways for oncogenic function. Furthermore, using RNA sequencing and qPCR, we establish that an engineered enzyme variant achieves high levels of labeling selectivity against the human transcriptome allowing for 145-fold enrichment of cellular RNA directly from mammalian cell lysates. The flexibility and breadth of this approach suggests that this system could be routinely applied to the functional characterization of RNA, greatly expanding the toolbox available for studying mammalian RNA biology.

Fragment Screening Hit Draws Attention to a Novel Transient Pocket Adjacent to the Recognition Site of the tRNA-Modifying Enzyme TGT.

Fragment-based lead discovery was applied to tRNA-guanine transglycosylase, an enzyme modifying post-transcriptionally tRNAs in Shigella, the causative agent of shigellosis. TGT inhibition prevents translation of Shigella's virulence factor VirF, hence reducing pathogenicity. One discovered fragment opens a transient subpocket in the preQ1-recognition site by pushing back an aspartate residue. This step is associated with reorganization of further amino acids structurally transforming a loop adjacent to the recognition site by duplicating the volume of the preQ1-recognition pocket. We synthesized 6-carboxamido-, 6-hydrazido-, and 4-guanidino-benzimidazoles to target the opened pocket, including a dihydro-imidazoquinazoline with a propyn-1-yl exit vector pointing into the transient pocket and displacing a conserved water network. MD simulations and hydration-site analysis suggest water displacement to contribute favorably to ligand binding. A cysteine residue, exclusively present in bacterial TGTs, serves as gatekeeper of the transient subpocket. It becomes accessible upon pocket opening for selective covalent attachment of electrophilic ligands in eubacterial TGTs.