Robert T. Byrne

The crystal structure of unmodified tRNAPhe from Escherichia coli

Post-transcriptional nucleoside modifications fine-tune the biophysical and biochemical properties of transfer RNA (tRNA) so that it is optimized for participation in cellular processes. Here we report the crystal structure of unmodified tRNAPhe from Escherichia coli at a resolution of 3 Å. We show that in the absence of modifications the overall fold of the tRNA is essentially the same as that of mature tRNA. However, there are a number of significant structural differences, such as rearrangements in a triplet base pair and a widened angle between the acceptor and anticodon stems. Contrary to previous observations, the anticodon adopts the same conformation as seen in mature tRNA.

Major reorientation of tRNA substrates defines specificity of dihydrouridine synthases

Significance RNA-binding proteins use diverse mechanisms for generating specificity toward distinct RNA molecules. Different subfamilies of bacterial dihydrouridine synthases (Dus) modify specific uridines in tRNA, but the mechanism for selection of the target nucleotide is unknown. We solved crystal structures of the U16-specific Dus from Escherichia coli complexed with two different tRNAs. These structures reveal that the tRNA is bound in a completely different orientation from that observed in a U20-specific enzyme. The major reorientation of the substrate tRNA, driven by unique amino acid “binding signatures” and plasticity in the position of the C-terminal recognition domain, appears to be an evolutionary innovation to the known strategies that define specificity of enzymes toward tRNA. The reduction of specific uridines to dihydrouridine is one of the most common modifications in tRNA. Increased levels of the dihydrouridine modification are associated with cancer. Dihydrouridine synthases (Dus) from different subfamilies selectively reduce distinct uridines, located at spatially unique positions of folded tRNA, into dihydrouridine. Because the catalytic center of all Dus enzymes is conserved, it is unclear how the same protein fold can be reprogrammed to ensure that nucleotides exposed at spatially distinct faces of tRNA can be accommodated in the same active site. We show that the Escherichia coli DusC is specific toward U16 of tRNA. Unexpectedly, crystal structures of DusC complexes with tRNAPhe and tRNATrp show that Dus subfamilies that selectively modify U16 or U20 in tRNA adopt identical folds but bind their respective tRNA substrates in an almost reverse orientation that differs by a 160° rotation. The tRNA docking orientation appears to be guided by subfamily-specific clusters of amino acids (“binding signatures”) together with differences in the shape of the positively charged tRNA-binding surfaces. tRNA orientations are further constrained by positional differences between the C-terminal “recognition” domains. The exquisite substrate specificity of Dus enzymes is therefore controlled by a relatively simple mechanism involving major reorientation of the whole tRNA molecule. Such reprogramming of the enzymatic specificity appears to be a unique evolutionary solution for altering tRNA recognition by the same protein fold.

Structure and Activity of a Novel Archaeal β-CASP Protein with N-Terminal KH Domains

Summary MTH1203, a β-CASP metallo-β-lactamase family nuclease from the archaeon Methanothermobacter thermautotrophicus, was identified as a putative nuclease that might contribute to RNA processing. The crystal structure of MTH1203 reveals that, in addition to the metallo-β-lactamase nuclease and the β-CASP domains, it contains two contiguous KH domains that are unique to MTH1203 and its orthologs. RNA-binding experiments indicate that MTH1203 preferentially binds U-rich sequences with a dissociation constant in the micromolar range. In vitro nuclease activity assays demonstrated that MTH1203 is a zinc-dependent nuclease. MTH1203 is also shown to be a dimer and, significantly, this dimerization enhances the nuclease activity. Transcription termination in archaea produces mRNA transcripts with U-rich 3′ ends that could be degraded by MTH1203 considering its RNA-binding specificity. We hypothesize that this nuclease degrades mRNAs of proteins targeted for degradation and so regulates archaeal RNA turnover, possibly in concert with the exosome.

S-Adenosyl-S-carboxymethyl-L-homocysteine: a novel cofactor found in the putative tRNA-modifying enzyme CmoA.

The putative methyltransferase CmoA is involved in the nucleoside modification of transfer RNA. X-ray crystallography and mass spectrometry are used to show that it contains a novel SAM derivative, S-adenosyl-S-carboxymethyl-l-homocysteine, in which the donor methyl group is replaced by a carboxymethyl group.

Molecular architecture of the HerA-NurA DNA double-strand break resection complex.

DNA double‐strand breaks can be repaired by homologous recombination, during which the DNA ends are long‐range resected by helicase–nuclease systems to generate 3′ single strand tails. In archaea, this requires the Mre11–Rad50 complex and the ATP‐dependent helicase–nuclease complex HerA–NurA. We report the cryo‐EM structure of Sulfolobus solfataricus HerA–NurA at 7.4 Å resolution and present the pseudo‐atomic model of the complex. HerA forms an ASCE hexamer that tightly interacts with a NurA dimer, with each NurA protomer binding three adjacent HerA HAS domains. Entry to NurAs nuclease active sites requires dsDNA to pass through a 23 Å wide channel in the HerA hexamer. The structure suggests that HerA is a dsDNA translocase that feeds DNA into the NurA nuclease sites.

Mechanistic insight into the assembly of the HerA–NurA helicase–nuclease DNA end resection complex

Abstract The HerA–NurA helicase–nuclease complex cooperates with Mre11 and Rad50 to coordinate the repair of double-stranded DNA breaks. Little is known, however, about the assembly mechanism and activation of the HerA–NurA. By combining hybrid mass spectrometry with cryo-EM, computational and biochemical data, we investigate the oligomeric formation of HerA and detail the mechanism of nucleotide binding to the HerA–NurA complex from thermophilic archaea. We reveal that ATP-free HerA and HerA-DNA complexes predominantly exist in solution as a heptamer and act as a DNA loading intermediate. The binding of either NurA or ATP stabilizes the hexameric HerA, indicating that HerA–NurA is activated by substrates and complex assembly. To examine the role of ATP in DNA translocation and processing, we investigated how nucleotides interact with the HerA–NurA. We show that while the hexameric HerA binds six nucleotides in an ‘all-or-none’ fashion, HerA–NurA harbors a highly coordinated pairwise binding mechanism and enables the translocation and processing of double-stranded DNA. Using molecular dynamics simulations, we reveal novel inter-residue interactions between the external ATP and the internal DNA binding sites. Overall, here we propose a stepwise assembly mechanism detailing the synergistic activation of HerA–NurA by ATP, which allows efficient processing of double-stranded DNA.

From bacterial to human dihydrouridine synthase: automated structure determination

The crystal structure of a human dihydrouridine synthase, an enzyme associated with lung cancer, with 18% sequence identity to a T. maritima enzyme, has been determined at 1.9 Å resolution by molecular replacement after extensive molecular remodelling of the template.

Crystallization and preliminary X-ray crystallographic analysis of the catalytic domain of human dihydrouridine synthase.

Dihydrouridine synthases catalyse the reduction of uridine to dihydrouridine in the D-loop and variable loop of tRNA. The human dihydrouridine synthase HsDus2L has been implicated in the development of pulmonary carcinogenesis. Here, the purification, crystallization and preliminary X-ray characterization of the HsDus2L catalytic domain are reported. The crystals belonged to space group P2(1) and contained a single molecule of HsDus2L in the asymmetric unit. A complete data set was collected to 1.9 Å resolution using synchrotron radiation.

The bacterial Mre11–Rad50 homolog SbcCD cleaves opposing strands of DNA by two chemically distinct nuclease reactions

Abstract The Mre11–Rad50 complex is a DNA double-strand break sensor that cleaves blocked DNA ends and hairpins by an ATP-dependent endo/exonuclease activity for subsequent repair. For that, Mre11–Rad50 complexes, including the Escherichia coli homolog SbcCD, can endonucleolytically cleave one or both strands near a protein block and process free DNA ends via a 3′-5′ exonuclease, but a unified basis for these distinct activities is lacking. Here we analyzed DNA binding, ATPase and nuclease reactions on different DNA substrates. SbcCD clips terminal bases of both strands of the DNA end in the presence of ATPγS. It introduces a DNA double-strand break around 20–25 bp from a blocked end after multiple rounds of ATP hydrolysis in a reaction that correlates with local DNA meltability. Interestingly, we find that nuclease reactions on opposing strands are chemically distinct, leaving a 5′ phosphate on one strand, but a 3′ phosphate on the other strand. Collectively, our results identify an unexpected chemical variability of the nuclease, indicating that the complex is oriented at a free DNA end and facing a block with opposite polarity. This suggests a unified model for ATP-dependent endo- and exonuclease reactions at internal DNA near a block and at free DNA ends.

Expression, purification, crystallization and preliminary X-ray studies of the TAN1 orthologue from Methanothermobacter thermautotrophicus

MTH909 is the Methanothermobacter thermautotrophicus orthologue of Saccharomyces cerevisiae TAN1, which is required for N(4)-acetylcytidine formation in tRNA. The protein consists of an N-terminal near-ferredoxin-like domain and a C-terminal THUMP domain. Unlike most other proteins containing the THUMP domain, TAN1 lacks any catalytic domains and has been proposed to form a complex with a catalytic protein that is capable of making base modifications. MTH909 has been cloned, overexpressed and purified. The molecule exists as a monomer in solution. X-ray data were collected to 2.85 A resolution from a native crystal belonging to space group P6(1)22 (or P6(5)22), with unit-cell parameters a = 69.9, c = 408.5 A.