[Show abstract][Hide abstract] ABSTRACT: Ribotoxins cleave essential RNAs for cell killing, and RNA repair neutralizes the damage inflicted by ribotoxins for cell survival. Here we report a new bacterial RNA repair complex that performs RNA repair linked to immunity. This new RNA repair complex is a 270-kDa heterohexamer composed of three proteins-Pnkp1, Rnl and Hen1-that are required to repair ribotoxin-cleaved RNA in vitro. The crystal structure of the complex reveals the molecular architecture of the heterohexamer as two rhomboid-shaped ring structures of Pnkp1-Rnl-Hen1 heterotrimer fused at the Pnkp1 dimer interface. The four active sites required for RNA repair are located on the inner rim of each ring. The architecture and the locations of the active sites of the Pnkp1-Rnl-Hen1 heterohexamer suggest an ordered series of repair reactions at the broken RNA ends that confer immunity to recurrent damage.
[Show abstract][Hide abstract] ABSTRACT: Approximately 25% of cytoplasmic tRNAs in eukaryotic organisms have the wobble uridine (U34) modified at C5 through a process that, according to genetic studies, is carried out by the eukaryotic Elongator complex. Here we show that a single archaeal protein, the homolog of the third subunit of the eukaryotic Elongator complex (Elp3), is able to catalyze the same reaction. The mechanism of action by Elp3 described here represents unprecedented chemistry performed on acetyl-CoA.
Full-text · Article · Aug 2014 · Nature Chemical Biology
[Show abstract][Hide abstract] ABSTRACT: Ribotoxins cleave essential RNAs for cell killing in vivo, and the bacterial polynucleotide kinase-phosphatase (Pnkp)/hua enhancer 1 (Hen1) complex has been shown to repair ribotoxin-cleaved RNAs in vitro. Bacterial Pnkp/Hen1 is distinguished from other RNA repair systems by performing 3'-terminal 2'-O-methylation during RNA repair, which prevents the repaired RNA from repeated cleavage at the same site. To ensure the opportunity of 2'-O-methylation by bacterial Hen1 during RNA repair and, therefore, maintain the quality of the repaired RNA, Pnkp/Hen1 has evolved to require the participation of Hen1 in RNA ligation, because Pnkp alone is unable to carry out the reaction despite possessing all signature motifs of an RNA ligase. However, the precise role of Hen1 in RNA ligation is unknown. Here, we present the crystal structure of an active RNA ligase consisting of the C-terminal half of Pnkp (Pnkp-C) and the N-terminal half of Hen1 (Hen1-N) from Clostridium thermocellum. The structure reveals that the N-terminal domain of Clostridium thermocellum (Cth) Hen1, shaped like a left hand, grabs the flexible insertion module of CthPnkp and locks its conformation via further interaction with the C-terminal addition module of CthPnkp. Formation of the CthPnkp-C/Hen1-N heterodimer creates a ligation pocket with a width for two strands of RNA, depth for two nucleotides, and the adenosine monophosphate (AMP)-binding pocket at the bottom. The structure, combined with functional analyses, provides insight into the mechanism of how Hen1 activates the RNA ligase activity of Pnkp for RNA repair.
Preview · Article · Jul 2012 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: In an RNA transcript, the 2'-OH group at the 3'-terminal nucleotide is unique as it is the only 2'-OH group that is adjacent to a 3'-OH group instead of a phosphate backbone. The 2'-OH group at the 3'-terminal nucleotide of certain RNAs is methylated in vivo, which is acheived by a methyltransferase named Hen1 that is mechanistically distinct from other known RNA 2'-O-methyltransferases. In eukaryotic organisms, 3'-terminal 2'-O-methylation of small RNAs stabilizes these small RNAs for RNA interference (RNAi). In bacteria, the same methylation during RNA repair results in repaired RNA resisting future damage at the site of repair. Although the chemistry performed by the eukaryotic and bacterial Hen1 is the same, the mechanisms of how RNA is stabilized as a result of the 3'-terminal 2'-O-methylation are different between the eukaryotic RNAi and the bacterial RNA repair. In this review, I will discuss the distribution of Hen1 in living organisms, the classification of Hen1 into four subfamilies, the structure and mechanism of Hen1 that allows it to conduct RNA 3'-terminal 2'-O-methylation, and the possible evolutionary origin of Hen1 present in bacterial and eukaryotic organisms.
[Show abstract][Hide abstract] ABSTRACT: The Cmr complex carries out target RNA degradation in organisms possessing the CRISPR-Cas system. In this issue of Structure, Cocozaki et al. present the crystal structure of Cmr2, providing insight into the architecture of the Cmr complex.
[Show abstract][Hide abstract] ABSTRACT: Ribotoxins cleave essential RNAs involved in protein synthesis as a strategy for cell killing. RNA repair systems exist in nature to counteract the lethal actions of ribotoxins, as first demonstrated by the RNA repair system from bacteriophage T4 25 yr ago. Recently, we found that two bacterial proteins, named Pnkp and Hen1, form a stable complex and are able to repair ribotoxin-cleaved tRNAs in vitro. However, unlike the well-studied T4 RNA repair system, the natural RNA substrates of the bacterial Pnkp/Hen1 RNA repair system are unknown. Here we present comprehensive RNA repair assays with the recombinant Pnkp/Hen1 proteins from Anabaena variabilis using a total of 33 different RNAs as substrates that might mimic various damaged forms of RNAs present in living cells. We found that unlike the RNA repair system from bacteriophage T4, the bacterial Pnkp/Hen1 RNA repair system exhibits broad substrate specificity. Based on the experimental data presented here, a model of preferred RNA substrates of the Pnkp/Hen1 repair system is proposed.
[Show abstract][Hide abstract] ABSTRACT: Ribotoxins kill cells by endonucleotically cleaving essential RNAs involved in protein translation. We report here that a stable heterotetramer composed of two bacterial proteins, Pnkp and Hen1, was able to repair transfer RNAs cleaved by ribotoxins in vitro. Before the broken RNAs were ligated by the heterotetramer, a methyl group was added to the 2'-OH group that participated in the original RNA cut. Because of the methylation, RNAs repaired by bacterial Pnkp/Hen1 heterotetramer could not be cleaved again by the ribotoxins. Thus, unlike eukaryotic Hen1 involved in RNA interference, the bacterial Hen1 is part of an RNA repair and modification system.
[Show abstract][Hide abstract] ABSTRACT: Small RNAs of approximately 20-30 nt have diverse and important biological roles in eukaryotic organisms. After being generated by Dicer or Piwi proteins, all small RNAs in plants and a subset of small RNAs in animals are further modified at their 3'-terminal nucleotides via 2'-O-methylation, carried out by the S-adenosylmethionine-dependent methyltransferase (MTase) Hen1. Methylation at the 3' terminus is vital for biological functions of these small RNAs. Here, we report four crystal structures of the MTase domain of a bacterial homolog of Hen1 from Clostridium thermocellum and Anabaena variabilis, which are enzymatically indistinguishable from the eukaryotic Hen1 in their ability to methylate small single-stranded RNAs. The structures reveal that, in addition to the core fold of the MTase domain shared by other RNA and DNA MTases, the MTase domain of Hen1 possesses a motif and a domain that are highly conserved and are unique to Hen1. The unique motif and domain are likely to be involved in RNA substrate recognition and catalysis. The structures allowed us to construct a docking model of an RNA substrate bound to the MTase domain of bacterial Hen1, which is likely similar to that of the eukaryotic counterpart. The model, supported by mutational studies, provides insight into RNA substrate specificity and catalytic mechanism of Hen1.
Full-text · Article · Oct 2009 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Pseudouridine (Psi) is formed through isomerization of uridine (U) catalyzed by a class of enzymes called pseudouridine synthases (PsiS). TruD is the fifth family of PsiS. Studies of the first four families (TruA, TruB, RsuA, and RluA) of PsiS reveal a conserved Asp and Tyr are critical for catalysis. However, in TruD family, the tyrosine is not conserved. In this study, we measured the enzymatic parameters for TruD in Escherichia coli, and carried out enzymatic assays for a series of single, double, and triple TruD mutants. Our studies indicate that a Glu, strictly conserved in only TruD family is likely to be the general base in TruD. We also proposed a possible distinct mechanism of TruD-catalyzed Psi formation compared to the first four families.
No preview · Article · Sep 2009 · Archives of Biochemistry and Biophysics
[Show abstract][Hide abstract] ABSTRACT: To understand the mechanisms that govern T cell receptor (TCR)-peptide MHC (pMHC) binding and the role that different regions of the TCR play in affinity and antigen specificity, we have studied the TCR from T cell clone 2C. High-affinity mutants of the 2C TCR that bind QL9-L(d) as a strong agonist were generated previously by site-directed mutagenesis of complementarity determining regions (CDRs) 1beta, 2alpha, 3alpha, or 3beta. We performed isothermal titration calorimetry to assess whether they use similar thermodynamic mechanisms to achieve high affinity for QL9-L(d). Four of the five TCRs examined bound to QL9-L(d) in an enthalpically driven, entropically unfavorable manner. In contrast, the high-affinity CDR1beta mutant resembled the wild-type 2C TCR interaction, with favorable entropy. To assess fine specificity, we measured the binding and kinetics of these mutants for both QL9-L(d) and a single amino acid peptide variant of QL9, called QL9-Y5-L(d). While 2C and most of the mutants had equal or higher affinity for the Y5 variant than for QL9, mutant CDR1beta exhibited 8-fold lower affinity for Y5 compared to QL9. To examine possible structural correlates of the thermodynamic and fine specificity signatures of the TCRs, the structure of unliganded QL9-L(d) was solved and compared to structures of the 2C TCR/QL9-L(d) complex and three high-affinity TCR/QL9-L(d) complexes. Our findings show that the QL9-L(d) complex does not undergo major conformational changes upon binding. Thus, subtle changes in individual CDRs account for the diverse thermodynamic and kinetic binding mechanisms and for the different peptide fine specificities.
[Show abstract][Hide abstract] ABSTRACT: Hypermodifications near the anticodon of tRNA are fundamental for the efficiency and fidelity of protein synthesis. Dimethylallyltransferase (DMATase) catalyzes transfer of a dimethylallyl moiety from dimethylallyl pyrophosphate to N6 of A37 in certain tRNAs. Here we present the crystal structures of Saccharomyces cerevisiae DMATase-tRNA(Cys) complex in four distinct forms, which provide snapshots of the RNA modification reaction catalyzed by DMATase. The structures reveal that the enzyme recognizes the tRNA substrate through indirect sequence readout. The targeted nucleotide A37 flips out from the anticodon loop of tRNA and flips into a channel in DMATase, where it meets its reaction partner di methylallyl pyrophosphate, which enters the channel from the opposite end. Structural changes accompanying the transfer reaction taking place in the crystal result in disengagement of DMATase-tRNA interaction near the reaction center. In addition, structural comparison of DMATase in the complex with unliganded bacterial DMATase provides a molecular basis of ordered substrate binding by DMATase.
Preview · Article · Nov 2008 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Dimethylallyltransferase (DMATase) transfers a five-carbon isoprenoid moiety from dimethylallyl pyrophosphate (DMAPP) to the amino group of adenosine at position 37 of certain tRNAs. Reported here are the crystal structures of Pseudomonas aeruginosa DMATase alone and in complex with pyrophosphate at 1.9 A resolution. Surprisingly, the enzyme possesses a central channel spanning the entire width of the enzyme. Both the accepting substrate tRNA and the donating substrate DMAPP appear to enter the channel from opposite sides in an ordered sequence, with tRNA first and DMAPP second, and the RNA modification reaction occurs in the middle of the channel once the two substrates have met. The structure of DMATase is homologous to a class of small soluble kinases involved in biosynthesis of nucleotide precursors for nucleic acids, indicating its possibly evolutionary origin. Furthermore, specific recognition of the pyrophosphate by a conserved loop in DMATase, similar to the P-loop commonly seen in diverse nucleotide-binding proteins, demonstrates that DMATase is structurally and mechanistically distinct from farnesyltransferase, another family of prenyltransferases involved in protein modification.
[Show abstract][Hide abstract] ABSTRACT: Colicin E5 is a tRNA-specific ribonuclease that recognizes and cleaves four tRNAs in Escherichia coli that contain the hypermodified nucleoside queuosine (Q) at the wobble position. Cells that produce colicin E5 also synthesize the cognate immunity protein (Im5) that rapidly and tightly associates with colicin E5 to prevent it from cleaving its own tRNAs to avoid suicide. We report here the crystal structure of Im5 in a complex with the activity domain of colicin E5 (E5-CRD) at 1.15A resolution. The structure reveals an extruded domain from Im5 that docks into the recessed RNA binding cleft in E5-CRD, resulting in extensive interactions between the two proteins. The interactions are primarily hydrophilic, with an interface that contains complementary surface charges between the two proteins. Detailed interactions in three separate regions of the interface account for specific recognition of colicin E5 by Im5. Furthermore, single-site mutational studies of Im5 confirmed the important role of particular residues in recognition and binding of colicin E5. Structural comparison of the complex reported here with E5-CRD alone, as well as with a docking model of RNA-E5-CRD, indicates that Im5 achieves its inhibition by physically blocking the cleft in colicin E5 that engages the RNA substrate.
No preview · Article · May 2006 · Journal of Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Sequence alignment of the TruA, TruB, RsuA, and RluA families of pseudouridine synthases (PsiS) identifies a strictly conserved aspartic acid, which has been shown to be the critical nucleophile for the PsiS-catalyzed formation of pseudouridine (Psi). However, superposition of the representative structures from these four families of enzymes identifies two additional amino acids, a lysine or an arginine (K/R) and a tyrosine (Y), from a K/RxY motif that are structurally conserved in the active site. We have created a series of Thermotoga maritima and Escherichia coli pseudouridine 55 synthase (Psi55S) mutants in which the conserved Y is mutated to other amino acids. A new crystal structure of the T. maritima Psi55S Y67F mutant in complex with a 5FU-RNA at 2.4 A resolution revealed formation of 5-fluoro-6-hydroxypseudouridine (5FhPsi), the same product previously seen in wild-type Psi55S-5FU-RNA complex structures. HPLC analysis confirmed efficient formation of 5FhPsi by both Psi55S Y67F and Y67L mutants but to a much lesser extent by the Y67A mutant when 5FU-RNA substrate was used. However, both HPLC analysis and a tritium release assay indicated that these mutants had no detectable enzymatic activity when the natural RNA substrate was used. The combined structural and mutational studies lead us to propose that the side chain of the conserved tyrosine in these four families of PsiS plays a dual role within the active site, maintaining the structural integrity of the active site through its hydrophobic phenyl ring and acting as a general base through its OH group for the proton abstraction required in the last step of PsiS-catalyzed formation of Psi.
[Show abstract][Hide abstract] ABSTRACT: Initial RNA transcription produces several tRNAs (one in prokaryotes and plant chloroplasts and seven or eight in eukaryotes) that contain an adenosine (A) at the wobble position (position 34). However, in all cases, adenosine at position 34 is post-transcriptionally converted to inosine (I), producing mature tRNAs without adenosine at the wobble position. The enzymes responsible for this A-to-I conversion in tRNA are tadA (acting as a homodimer) in prokaryotes and the heterodimeric ADAT2-ADAT3 complex in eukaryotes. The genes encoding these proteins are essential for cell viability, illustrating the biological importance of A-to-I editing at the wobble position of tRNA. In this study, recombinant tadA proteins from Escherichia coli, Agrobacterium tumefaciens, and Aquifex aeolicus, as well as the ADAT2-ADAT3 proteins from Saccharomyces cerevisiae, were overexpressed in E. coli and purified to homogeneity by chromatography. Crystallization of a proteolytically cleaved A. tumefaciens tadA (missing the last eight amino acids at the C-terminus) produced high-quality crystals, and the structure was determined at 1.6 A resolution. In addition, enzymatic assays of the wild-type proteins as well as several mutants were carried out using both the full-length E. coli tRNA(arg2) and the truncated anticodon stem-loop motif as substrates. Our biochemical and structural studies, in combination with sequence and structural comparisons with other deaminases, allow us to propose a model of tadA-tRNA interaction that explains the molecular basis of tRNA recognition by tadA. In particular, a conserved FFxxxR motif at the C-terminus, which is unique to tadA, has been identified, and its critical role in tRNA substrate recognition is proposed. Furthermore, the structural study of prokaryotic tadA presented here also sheds light on tRNA substrate recognition and the possible evolutionary origin of the eukaryotic ADAT2-ADAT3 heterodimer.
[Show abstract][Hide abstract] ABSTRACT: Colicin E5 specifically cleaves four tRNAs in Escherichia coli that contain the modified nucleotide queuosine (Q) at the wobble position, thereby preventing protein synthesis and ultimately resulting in cell death. Here, the crystal structure of the catalytic domain of colicin E5 (E5-CRD) from E. coli was determined at 1.5 A resolution. Unexpectedly, E5-CRD adopts a core folding with a four-stranded beta-sheet packed against an alpha-helix, seen in the well-studied ribonuclease T1 despite a lack of sequence similarity. Beyond the core catalytic domain, an N-terminal helix, a C-terminal beta-strand and loop, and an extended internal loop constitute an RNA binding cleft. Mutational analysis identified five amino acids that were important for tRNA substrate binding and cleavage by E5-CRD. The structure, together with the mutational study, allows us to propose a model of colicin E5-tRNA interactions, suggesting the molecular basis of tRNA substrate recognition and the mechanism of tRNA cleavage by colicin E5.
[Show abstract][Hide abstract] ABSTRACT: Crystal structure of HIV-RT in complex with a DNA template:primer and a dTTP leads us to design and synthesize a new class of nucleoside analog inhibitors containing a branched 3'-group against HIV-RT. An in vitro primer extension assay indicates that three out of five compounds are effective HIV-RT inhibitors.
[Show abstract][Hide abstract] ABSTRACT: Pseudouridine 55 synthase (Ψ55S) catalyzes isomerization of uridine (U) to pseudouridine (Ψ) at position 55 in transfer RNA.
The crystal structures of Thermotoga maritima Ψ55S, and its complex with RNA, have been determined at 2.9 and 3.0 Å resolutions, respectively. Structural comparisons with
other families of pseudouridine synthases (ΨS) indicate that Ψ55S may acquire its ability to recognize a stem–loop RNA substrate
by two insertions of polypeptides into the ΨS core. The structure of apo‐Ψ55S reveals that these two insertions interact with
each other. However, association with RNA substrate induces substantial conformational change in one of the insertions, resulting
in disruption of interaction between insertions and association of both insertions with the RNA substrate. Specific interactions
between two insertions, as well as between the insertions and the RNA substrate, account for the molecular basis of the conformational
Preview · Article · Feb 2004 · Nucleic Acids Research