Manal A. Swairjo

The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA

Threonylcarbamoyladenosine (t6A) is a universal modification found at position 37 of ANN decoding tRNAs, which imparts a unique structure to the anticodon loop enhancing its binding to ribosomes in vitro. Using a combination of bioinformatic, genetic, structural and biochemical approaches, the universal protein family YrdC/Sua5 (COG0009) was shown to be involved in the biosynthesis of this hypermodified base. Contradictory reports on the essentiality of both the yrdC wild-type gene of Escherichia coli and the SUA5 wild-type gene of Saccharomyces cerevisiae led us to reconstruct null alleles for both genes and prove that yrdC is essential in E. coli, whereas SUA5 is dispensable in yeast but results in severe growth phenotypes. Structural and biochemical analyses revealed that the E. coli YrdC protein binds ATP and preferentially binds RNAThr lacking only the t6A modification. This work lays the foundation for elucidating the function of a protein family found in every sequenced genome to date and understanding the role of t6A in vivo.

Structure-specific tRNA-binding protein from the extreme thermophile Aquifex aeolicus

The genome of the bacterium Aquifex aeolicus encodes a polypeptide which is related to a small portion of a sequence found in one prokaryotic and two eukaryotic tRNA synthetases. It also is related to a portion of Arc1p, a tRNA‐binding protein believed to be important for nuclear trafficking of tRNAs. Here we cloned, expressed and purified the 111 amino acid polypeptide (designated Trbp111) and showed by ultracentrifugation analysis that it is a stable dimer in solution. The protein was also crystallized in a monoclinic lattice. X‐ray diffraction analysis at 2.8 Å resolution revealed a prominent non‐crystallographic 2‐fold axis, consistent with the presence of a symmetric homodimeric structure. Band‐shift analysis with polyacrylamide gels showed that the dimer binds tRNAs, but not RNA duplexes, RNA hairpins, single‐stranded RNA nor 5S rRNA. Complex formation with respect to tRNA is non‐specific, with a single tRNA bound per dimer. Thus, Trbp111 is a structure‐specific tRNA‐binding protein. These results and other considerations raise the possibility that Trbp111 is a tRNA‐specific chaperone which stabilizes the native L‐shaped fold in the extreme thermophile and which has been incorporated into much larger tRNA‐binding proteins of higher organisms.

Discovery of a New Prokaryotic Type I GTP Cyclohydrolase Family

GTP cyclohydrolase I (GCYH-I) is the first enzyme of the de novo tetrahydrofolate biosynthetic pathway present in bacteria, fungi, and plants, and encoded in Escherichia coli by the folE gene. It is also the first enzyme of the biopterin (BH4) pathway in Homo sapiens, where it is encoded by a homologous folE gene. A homology-based search of GCYH-I orthologs in all sequenced bacteria revealed a group of microbes, including several clinically important pathogens, that encoded all of the enzymes of the tetrahydrofolate biosynthesis pathway but GCYH-I, suggesting that an alternate family was present in these organisms. A prediction based on phylogenetic occurrence and physical clustering identified the COG1469 family as a potential candidate for this missing enzyme family. The GCYH-I activity of COG1469 family proteins from a variety of sources (Thermotoga maritima, Bacillus subtilis, Acinetobacter baylyi, and Neisseria gonorrhoeae) was experimentally verified in vivo and/or in vitro. Although there is no detectable sequence homology with the canonical GCYH-I, protein fold recognition based on sequence profiles, secondary structure, and solvation potential information suggests that, like GCYH-I proteins, COG1469 proteins are members of the tunnel-fold (T-fold) structural superfamily. This new GCYH-I family is found in ∼20% of sequenced bacteria and is prevalent in Archaea, but the family is to this date absent in Eukarya.

Two conformations of a crystalline human tRNA synthetase–tRNA complex: implications for protein synthesis

Aminoacylation of tRNA is the first step of protein synthesis. Here, we report the co‐crystal structure of human tryptophanyl‐tRNA synthetase and tRNATrp. This enzyme is reported to interact directly with elongation factor 1α, which carries charged tRNA to the ribosome. Crystals were generated from a 50/50% mixture of charged and uncharged tRNATrp. These crystals captured two conformations of the complex, which are nearly identical with respect to the protein and a bound tryptophan. They are distinguished by the way tRNA is bound. In one, uncharged tRNA is bound across the dimer, with anticodon and acceptor stem interacting with separate subunits. In this cross‐dimer tRNA complex, the class I enzyme has a class II‐like tRNA binding mode. This structure accounts for biochemical investigations of human TrpRS, including species‐specific charging. In the other conformation, presumptive aminoacylated tRNA is bound only by the anticodon, the acceptor stem being free and having space to interact precisely with EF‐1α, suggesting that the product of aminoacylation can be directly handed off to EF‐1α for the next step of protein synthesis.

Crystal structure of Trbp111: a structure-specific tRNA-binding protein

Trbp111 is a 111 amino acid Aquifex aeolicus structure‐specific tRNA‐binding protein that has homologous counterparts distributed throughout evolution. A dimer is the functional unit for binding a single tRNA. Here we report the 3D structures of the A.aeolicus protein and its Escherichia coli homolog at resolutions of 2.50 and 1.87 Å, respectively. The structure shows a symmetrical dimer of two core domains and a central dimerization domain where the N‐ and C‐terminal regions of Trbp111 form an extensive dimer interface. The core of the monomer is a classical oligonucleotide/oligosaccharide‐binding (OB) fold with a five‐stranded β‐barrel and a small capping helix. This structure is similar to that seen in the anticodon‐binding domain of three class II tRNA synthetases and several other proteins. Mutational analysis identified sites important for interactions with tRNA. These residues line the inner surfaces of two clefts formed between the β‐barrel of each monomer and the dimer interface. The results are consistent with a proposed model for asymmetrical docking of the convex side of tRNA to the dimer.

Zinc-Independent Folate Biosynthesis: Genetic, Biochemical, and Structural Investigations Reveal New Metal Dependence for GTP Cyclohydrolase IB

GTP cyclohydrolase I (GCYH-I) is an essential Zn(2+)-dependent enzyme that catalyzes the first step of the de novo folate biosynthetic pathway in bacteria and plants, the 7-deazapurine biosynthetic pathway in Bacteria and Archaea, and the biopterin pathway in mammals. We recently reported the discovery of a new prokaryotic-specific GCYH-I (GCYH-IB) that displays no sequence identity to the canonical enzyme and is present in approximately 25% of bacteria, the majority of which lack the canonical GCYH-I (renamed GCYH-IA). Genomic and genetic analyses indicate that in those organisms possessing both enzymes, e.g., Bacillus subtilis, GCYH-IA and -IB are functionally redundant, but differentially expressed. Whereas GCYH-IA is constitutively expressed, GCYH-IB is expressed only under Zn(2+)-limiting conditions. These observations are consistent with the hypothesis that GCYH-IB functions to allow folate biosynthesis during Zn(2+) starvation. Here, we present biochemical and structural data showing that bacterial GCYH-IB, like GCYH-IA, belongs to the tunneling-fold (T-fold) superfamily. However, the GCYH-IA and -IB enzymes exhibit significant differences in global structure and active-site architecture. While GCYH-IA is a unimodular, homodecameric, Zn(2+)-dependent enzyme, GCYH-IB is a bimodular, homotetrameric enzyme activated by a variety of divalent cations. The structure of GCYH-IB and the broad metal dependence exhibited by this enzyme further underscore the mechanistic plasticity that is emerging for the T-fold superfamily. Notably, while humans possess the canonical GCYH-IA enzyme, many clinically important human pathogens possess only the GCYH-IB enzyme, suggesting that this enzyme is a potential new molecular target for antibacterial development.

Diversity of archaeosine synthesis in crenarchaeota.

Archaeosine (G(+)) is found at position 15 of many archaeal tRNAs. In Euryarchaeota, the G(+) precursor, 7-cyano-7-deazaguanine (preQ(0)), is inserted into tRNA by tRNA-guanine transglycosylase (arcTGT) before conversion into G(+) by ARChaeosine Synthase (ArcS). However, many Crenarchaeota known to harbor G(+) lack ArcS homologues. Using comparative genomics approaches, two families that could functionally replace ArcS in these organisms were identified: (1) GAT-QueC, a two-domain family with an N-terminal glutamine amidotransferase class-II domain fused to a domain homologous to QueC, the enzyme that produces preQ(0) and (2) QueF-like, a family homologous to the bacterial enzyme catalyzing the reduction of preQ(0) to 7-aminomethyl-7-deazaguanine. Here we show that these two protein families are able to catalyze the formation of G(+) in a heterologous system. Structure and sequence comparisons of crenarchaeal and euryarchaeal arcTGTs suggest the crenarchaeal enzymes have broader substrate specificity. These results led to a new model for the synthesis and salvage of G(+) in Crenarchaeota.

Discovery and Characterization of an Amidinotransferase Involved in the Modification of Archaeal tRNA

The presence of the 7-deazaguanosine derivative archaeosine (G+) at position 15 in tRNA is one of the diagnostic molecular characteristics of the Archaea. The biosynthesis of this modified nucleoside is especially complex, involving the initial production of 7-cyano-7-deazaguanine (preQ0), an advanced precursor that is produced in a tRNA-independent portion of the biosynthesis, followed by its insertion into the tRNA by the enzyme tRNA-guanine transglycosylase (arcTGT), which replaces the target guanine base yielding preQ0-tRNA. The enzymes responsible for the biosynthesis of preQ0 were recently identified, but the enzyme(s) catalyzing the conversion of preQ0-tRNA to G+-tRNA have remained elusive. Using a comparative genomics approach, we identified a protein family implicated in the late stages of archaeosine biosynthesis. Notably, this family is a paralog of arcTGT and is generally annotated as TgtA2. Structure-based alignments comparing arcTGT and TgtA2 reveal that TgtA2 lacks key arcTGT catalytic residues and contains an additional module. We constructed a Haloferax volcanii ΔtgtA2 derivative and demonstrated that tRNA from this strain lacks G+ and instead accumulates preQ0. We also cloned the corresponding gene from Methanocaldococcus jannaschii (mj1022) and characterized the purified recombinant enzyme. Recombinant MjTgtA2 was shown to convert preQ0-tRNA to G+-tRNA using several nitrogen sources and to do so in an ATP-independent process. This is the only example of the conversion of a nitrile to a formamidine known in biology and represents a new class of amidinotransferase chemistry.

Crystallization and preliminary X-ray characterization of the nitrile reductase QueF: a queuosine-biosynthesis enzyme

QueF (MW = 19.4 kDa) is a recently characterized nitrile oxidoreductase which catalyzes the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of the modified tRNA nucleoside queuosine. Initial crystals of homododecameric Bacillus subtilis QueF diffracted poorly to 8.0 A. A three-dimensional model based on homology with the tunnel-fold enzyme GTP cyclohydrolase I suggested catalysis at intersubunit interfaces and a potential role for substrate binding in quaternary structure stabilization. Guided by this insight, a second crystal form was grown that was strictly dependent on the presence of preQ0. This crystal form diffracted to 2.25 A resolution.

Positional recognition of a tRNA determinant dependent on a peptide insertion.

The crystal structure of a catalytic fragment of Aquifex aeolicus AlaRS and additional data suggest how the critical G3:U70 identity element of its cognate tRNA acceptor stem is recognized. Though this identity element is conserved from bacteria to the cytoplasm of eukaryotes, Drosophila melanogaster mitochondrial (Dm mt) tRNA(Ala) contains a G:U base pair that has been translocated to the adjacent 2:71 position. This G2:U71 is the major determinant for identity of Dm mt tRNA(Ala). Sequence alignments showed that Dm mt AlaRS is differentiated from G3:U70-recognizing AlaRSs by an insertion of 27 amino acids in the region of the protein that contacts the acceptor stem. Precise deletion of this insertion from Dm mt AlaRS gave preferential recognition to a G3:U70-containing substrate. Larger or smaller deletions were ineffective. The crystal structure of the orthologous A. aeolicus protein places this insertion on the surface, where it can act as a hinge that provides positional switching of G:U recognition.