A single tRNA base pair mediates bacterial tRNA-dependent biosynthesis of asparagine

In many prokaryotes and in organelles asparagine and glutamine are formed by a tRNA-dependent amidotransferase (AdT) that catalyzes amidation of aspartate and glutamate, respectively, mischarged on tRNA^Asn and tRNA^Gln. These pathways supply the deficiency of the organism in asparaginyl- and glutaminyl-tRNA synthtetases and provide the translational machinery with Asn-tRNA^Asn and Gln-tRNA^Gln. So far, nothing is known about the structural elements that confer to tRNA the role of a specific cofactor in the formation of the cognate amino acid. We show herein, using aspartylated tRNA^Asn and tRNA^Asp variants, that amidation of Asp acylating tRNA^Asn is promoted by the base pair U₁–A₇₂ whereas the G₁–C₇₂ pair and presence of the supernumerary nucleotide U_20A in the D-loop of tRNA^Asp prevent amidation. We predict, based on comparison of tRNA^Gln and tRNA^Glu sequence alignments from bacteria using the AdT-dependent pathway to form Gln-tRNA^Gln, that the same combination of nucleotides also rules specific tRNA-dependent formation of Gln. In contrast, we show that the tRNA-dependent conversion of Asp into Asn by archaeal AdT is mainly mediated by nucleotides G₄₆ and U₄₇ of the variable region. In the light of these results we propose that bacterial and archaeal AdTs use kingdom-specific signals to catalyze the tRNA-dependent formations of Asn and Gln.     

The archaeal transamidosome for RNA-dependent glutamine biosynthesis

Archaea make glutaminyl-tRNA (Gln-tRNA^Gln) in a two-step process; a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) forms Glu-tRNA^Gln, while the heterodimeric amidotransferase GatDE converts this mischarged tRNA to Gln-tRNA^Gln. Many prokaryotes synthesize asparaginyl-tRNA (Asn-tRNA^Asn) in a similar manner using a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) and the heterotrimeric amidotransferase GatCAB. The transamidosome, a complex of tRNA synthetase, amidotransferase and tRNA, was first described for the latter system in Thermus thermophilus [Bailly, M., Blaise, M., Lorber, B., Becker, H.D. and Kern, D. (2007) The transamidosome: a dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine biosynthesis. Mol. Cell, 28, 228–239.]. Here, we show a similar complex for Gln-tRNA^Gln formation in Methanothermobacter thermautotrophicus that allows the mischarged Glu-tRNA^Gln made by the tRNA synthetase to be channeled to the amidotransferase. The association of archaeal ND-GluRS with GatDE (K_D = 100 ± 22 nM) sequesters the tRNA synthetase for Gln-tRNA^Gln formation, with GatDE reducing the affinity of ND-GluRS for tRNA^Glu by at least 13-fold. Unlike the T. thermophilus transamidosome, the archaeal complex does not require tRNA for its formation, is not stable through product (Gln-tRNA^Gln) formation, and has no major effect on the kinetics of tRNA^Gln glutamylation nor transamidation. The differences between the two transamidosomes may be a consequence of the fact that ND-GluRS is a class I aminoacyl-tRNA synthetase, while ND-AspRS belongs to the class II family.     

Crystal structure of a transfer‐ribonucleoprotein particle that promotes asparagine formation

Four out of the 22 aminoacyl‐tRNAs (aa‐tRNAs) are systematically or alternatively synthesized by an indirect, two‐step route requiring an initial mischarging of the tRNA followed by tRNA‐dependent conversion of the non‐cognate amino acid. During tRNA‐dependent asparagine formation, tRNA^Asn promotes assembly of a ribonucleoprotein particle called transamidosome that allows channelling of the aa‐tRNA from non‐discriminating aspartyl‐tRNA synthetase active site to the GatCAB amidotransferase site. The crystal structure of the Thermus thermophilus transamidosome determined at 3 Å resolution reveals a particle formed by two GatCABs, two dimeric ND‐AspRSs and four tRNAs^Asn molecules. In the complex, only two tRNAs are bound in a functional state, whereas the two other ones act as an RNA scaffold enabling release of the asparaginyl‐tRNA^Asn without dissociation of the complex. We propose that the crystal structure represents a transient state of the transamidation reaction. The transamidosome constitutes a transfer‐ribonucleoprotein particle in which tRNAs serve the function of both substrate and structural foundation for a large molecular machine.     

From one amino acid to another: tRNA-dependent amino acid biosynthesis

Aminoacyl-tRNAs (aa-tRNAs) are the essential substrates for translation. Most aa-tRNAs are formed by direct aminoacylation of tRNA catalyzed by aminoacyl-tRNA synthetases. However, a smaller number of aa-tRNAs (Asn-tRNA, Gln-tRNA, Cys-tRNA and Sec-tRNA) are made by synthesizing the amino acid on the tRNA by first attaching a non-cognate amino acid to the tRNA, which is then converted to the cognate one catalyzed by tRNA-dependent modifying enzymes. Asn-tRNA or Gln-tRNA formation in most prokaryotes requires amidation of Asp-tRNA or Glu-tRNA by amidotransferases that couple an amidase or an asparaginase to liberate ammonia with a tRNA-dependent kinase. Both archaeal and eukaryotic Sec-tRNA biosynthesis and Cys-tRNA synthesis in methanogens require O-phosophoseryl-tRNA formation. For tRNA-dependent Cys biosynthesis, O-phosphoseryl-tRNA synthetase directly attaches the amino acid to the tRNA which is then converted to Cys by Sep-tRNA: Cys-tRNA synthase. In Sec-tRNA synthesis, O-phosphoseryl-tRNA kinase phosphorylates Ser-tRNA to form the intermediate which is then modified to Sec-tRNA by Sep-tRNA:Sec-tRNA synthase. Complex formation between enzymes in the same pathway may protect the fidelity of protein synthesis. How these tRNA-dependent amino acid biosynthetic routes are integrated into overall metabolism may explain why they are still retained in so many organisms.     

The assembly of a spliceosomal small nuclear ribonucleoprotein particle

The U1, U2, U4, U5 and U6 small nuclear ribonucleoprotein particles (snRNPs) are essential elements of the spliceosome, the enzyme that catalyzes the excision of introns and the ligation of exons to form a mature mRNA. Since their discovery over a quarter century ago, the structure, assembly and function of spliceosomal snRNPs have been extensively studied. Accordingly, the functions of splicing snRNPs and the role of various nuclear organelles, such as Cajal bodies (CBs), in their nuclear maturation phase have already been excellently reviewed elsewhere. The aim of this review is, then, to briefly outline the structure of snRNPs and to synthesize new and exciting developments in the snRNP biogenesis pathways.     

Three-dimensional reconstruction of a recombinant influenza virus ribonucleoprotein particle

A three-dimensional structural model of an influenza virus ribonucleoprotein particle reconstituted in vivo from recombinant proteins and a model genomic vRNA has been generated by electron microscopy. It shows a circular shape and contains nine nucleoprotein monomers, two of which are connected with the polymerase complex. The nucleoprotein monomers show a curvature that may be responsible for the formation of helical structures in the full-size viral ribonucleoproteins. The monomers show distinct contact boundaries at the two sides of the particle, suggesting that the genomic RNA may be located in association with the nucleoprotein at the base of the ribonucleoprotein complex. Sections of the three-dimensional model show a trilobular morphology in the polymerase complex that is consistent with the presence of its three subunits.     

Gln-tRNAGln synthesis in a dynamic transamidosome from Helicobacter pylori, where GluRS2 hydrolyzes excess Glu-tRNAGln

In many bacteria and archaea, an ancestral pathway is used where asparagine and glutamine are formed from their acidic precursors while covalently linked to tRNA(Asn) and tRNA(Gln), respectively. Stable complexes formed by the enzymes of these indirect tRNA aminoacylation pathways are found in several thermophilic organisms, and are called transamidosomes. We describe here a transamidosome forming Gln-tRNA(Gln) in Helicobacter pylori, an ε-proteobacterium pathogenic for humans; this transamidosome displays novel properties that may be characteristic of mesophilic organisms. This ternary complex containing the non-canonical GluRS2 specific for Glu-tRNA(Gln) formation, the tRNA-dependent amidotransferase GatCAB and tRNA(Gln) was characterized by dynamic light scattering. Moreover, we observed by interferometry a weak interaction between GluRS2 and GatCAB (K(D) = 40 ± 5 μM). The kinetics of Glu-tRNA(Gln) and Gln-tRNA(Gln) formation indicate that conformational shifts inside the transamidosome allow the tRNA(Gln) acceptor stem to interact alternately with GluRS2 and GatCAB despite their common identity elements. The integrity of this dynamic transamidosome depends on a critical concentration of tRNA(Gln), above which it dissociates into separate GatCAB/tRNA(Gln) and GluRS2/tRNA(Gln) complexes. Ester bond protection assays show that both enzymes display a good affinity for tRNA(Gln) regardless of its aminoacylation state, and support a mechanism where GluRS2 can hydrolyze excess Glu-tRNA(Gln), ensuring faithful decoding of Gln codons.     

The vertebrate E1/U17 small nucleolar ribonucleoprotein particle

Each of the many different box H/ACA ribonucleoprotein particles (RNPs) present in eukaryotes and archaea consists of four common core proteins and one specific H/ACA small RNA, which bears the sequence elements H (ANANNA) and ACA. Most of the H/ACA RNPs are small nucleolar RNPs (snoRNPs), which are localized in nucleoli, and are one of the two major classes of snoRNPs. Most H/ACA RNPs direct pseudouridine synthesis in pre-rRNA and other RNAs. One H/ACA small nucleolar RNA (snoRNA), vertebrate E1/U17 (snR30 in yeast), is required for pre-rRNA cleavage processing that generates mature 18S rRNA. E1 snoRNA is encoded in introns of protein-coding genes, and the evidence suggests that human E1 RNA undergoes uridine insertional RNA editing. The vertebrate E1 RNA consensus secondary structure shows several features that are absent in other box H/ACA snoRNAs. The available UV-induced RNA-protein crosslinking results suggest that the E1 snoRNP is asymmetrical in vertebrate cells, in contrast to other H/ACA snoRNPs. The vertebrate E1 snoRNP in cells is surprisingly complex: (i) E1 RNA contacts directly and specifically several proteins which do not appear to be any of the H/ACA RNP four core proteins; and (ii) multiple E1 RNA sites are needed for E1 snoRNP formation, E1 RNA stability, and E1 RNA-protein direct interactions.     

E. coli 4.5S RNA is part of a ribonucleoprotein particle that has properties related to signal recognition particle

E. coli 4.5S RNA and P48 have been shown to be homologous to SRP7S RNA and SRP54, respectively. Here we report that expression of human SRP7S in E. coli can suppress the lethality caused by depletion of 4.5S RNA. In E. coli, both RNAs are associated with P48. In vitro, both E. coli P48 and SRP54 specifically bind to 4.5S RNA. Strains depleted of 4.5S RNA strongly accumulate pre-beta-lactamase and fail to accumulate maltose binding protein. These effects commence well before any growth defect is observed and are suppressed by expression of human SRP7S. Strains overproducing P48 also accumulate pre-beta-lactamase. 4.5S RNA and P48 are components of a ribonucleoprotein particle that we propose to be required for the secretion of some proteins.     

The nucleolus: a site of ribonucleoprotein maturation

The nucleolus is the site of ribosomal RNA synthesis, processing and ribosome maturation. Various small ribonucleoproteins also undergo maturation in the nucleolus, involving RNA modification and RNA-protein assembly. Such steps and other activities of small ribonucleoproteins also take place in Cajal (coiled) bodies. Events of ribosome biogenesis are found solely in the nucleolus, which is the final destination of small nucleolar RNAs after their traffic through Cajal bodies. However, nucleoli are just a stopping point in the intricate cellular traffic for small nuclear RNAs and other ribonucleoproteins.     

GroEL1: a dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in mycobacteria

Mycobacteria are unusual in encoding two GroEL paralogs, GroEL1 and GroEL2. GroEL2 is essential--presumably providing the housekeeping chaperone functions--while groEL1 is nonessential, contains the attB site for phage Bxb1 integration, and encodes a putative chaperone with unusual structural features. Inactivation of the Mycobacterium smegmatis groEL1 gene by phage Bxb1 integration allows normal planktonic growth but prevents the formation of mature biofilms. GroEL1 modulates synthesis of mycolates--long-chain fatty acid components of the mycobacterial cell wall--specifically during biofilm formation and physically associates with KasA, a key component of the type II Fatty Acid Synthase involved in mycolic acid synthesis. Biofilm formation is associated with elevated synthesis of short-chain (C56-C68) fatty acids, and strains with altered mycolate profiles--including an InhA mutant resistant to the antituberculosis drug isoniazid and a strain overexpressing KasA--are defective in biofilm formation.     

Identification of a nuclear ribonucleoprotein particle which contains incompletely spliced HIV-1 RNAs

Unspliced and partially spliced HIV RNAs are transported to the cytoplasm by the HIV encoded Rev protein. In the present study, a ribonucleoprotein complex which contains such incompletely spliced HIV RNA is identified. Soluble nuclear extracts were prepared from the lymphocyte cell line H9/IIIB that constitutively produces HIV-1 from a stably integrated provirus. Sucrose gradient centrifugation of the extracts and subsequent analysis of the gradient fractions by a ribonuclease protection assay revealed a population of incompletely spliced HIV-1 RNAs which accumulates in the 100S region of the gradient. Similar analysis of cellular mRNAs including beta-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) revealed that these RNA molecules also exhibit characteristic sedimentation profiles in sucrose gradients. This study suggests that nuclear ribonucleoprotein particles containing incompletely spliced HIV-1 RNAs are amenable for biochemical characterisation.     

Isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small RNA

Rat liver coated vesicle preparations were frequently found to contain small ovoid bodies, which resembled coated vesicles in morphology. We have purified these bodies to homogeneity using sucrose density gradients and preparative agarose gel electrophoresis. When negatively stained and viewed by electron microscopy, the purified structures display a very distinct and complex morphology, resembling the multiple arches which form cathedral vaults. They measure 35 X 65 nm and are therefore considerably larger than ribosomes. When subjected to SDS PAGE, these structures, which we refer to as vaults, appear to contain several minor and five major species: Mr 210,000, 192,000, 104,000, 54,000, and 37,000. One of these (Mr 104,000) greatly predominates, accounting for greater than 70% of the total Coomassie Brilliant Blue-staining protein. Another major species of Mr 37,000 has been identified as a species of small RNA of unusual base composition (adenosine 12.0%, guanosine 29.7%, uridine 30.9%, and 27.4% cytidine), which migrates as a single species in urea PAGE between the 5S and 5.8S ribosomal standards, containing approximately 140 bases. Although the RNA constitutes only 4.6% of the entire structure, the large size of the particle requires that each one contains approximately 9 molecules of this RNA. Antibodies prepared against the entire particle are largely specific for the major (Mr 104,000) polypeptide species. Although they do not directly react with the RNA constituent on Western blots, these antibodies immunoprecipitate a 32P-labeled RNA of identical size from metabolically-labeled rat hepatoma cells. Vaults are observed in partially purified fractions from human fibroblasts, murine 3T3 cells, glial cells, and rabbit alveolar macrophages. It therefore appears that these novel ribonucleoprotein structures are broadly distributed among different cell types. The function of vaults is at present unknown.