Promoting the formation of an active synthetase/tRNA complex by a nonspecific tRNA-binding domain

Previous studies showed that valyl-tRNA synthetase of Saccharomyces cerevisiae contains an N-terminal polypeptide extension of 97 residues, which is absent from its bacterial relatives, but is conserved in its mammalian homologues. We showed herein that this appended domain and its human counterpart are both nonspecific tRNA-binding domains (K(d) approximately 0.5 microm). Deletion of the appended domain from the yeast enzyme severely impaired its tRNA binding, aminoacylation, and complementation activities. This N-domain-deleted yeast valyl-tRNA synthetase mutant could be rescued by fusion of the equivalent domain from its human homologue. Moreover, fusion of the N-domain of the yeast enzyme or its human counterpart to Escherichia coli glutaminyl-tRNA synthetase enabled the otherwise "inactive" prokaryotic enzyme to function as a yeast enzyme in vivo. Different from the native yeast enzyme, which showed different affinities toward mixed tRNA populations, the fusion enzyme exhibited similar binding affinities for all yeast tRNAs. These results not only underscore the significance of nonspecific tRNA binding in aminoacylation, but also provide insights into the mechanism of the formation of aminoacyl-tRNAs.     

Rescuing an essential enzyme-RNA complex with a non-essential appended domain.

Certain protein-RNA complexes, such as synthetase-tRNA complexes, are essential for cell survival. These complexes are formed with a precise molecular fit along the interface of the reacting partners, and mutational analyses have shown that amino acid or nucleotide substitutions at the interface can be used to disrupt functional or repair non-functional complexes. In contrast, we demonstrate here a feature of a eukaryote system that rescues a disrupted complex without directly re-engineering the interface. The monomeric yeast Saccharomyces cerevisiae glutaminyl-tRNA synthetase, like several other class I eukaryote tRNA synthetases, has an active-site-containing ''body'' that is closely homologous to its Escherichia coli relative, but is tagged at its N-terminus with a novel and dispensable appended domain whose role has been obscure. Because of differences between the yeast and E. coli glutamine tRNAs that presumably perturb the enzyme-tRNA interface, E. coli glutaminyl-tRNA synthetase does not charge yeast tRNA. However, linking the novel appended domain of the yeast to the E. coli enzyme enabled the E. coli protein to function as a yeast enzyme, in vitro and in vivo. The appended domain appears to contribute an RNA interaction that compensates for weak or poor complex formation. In eukaryotes, extra appended domains occur frequently in these proteins. These domains may be essential when there are conditions that would otherwise weaken or disrupt formation of a critical RNA-protein complex. They may also be adapted for other, specialized RNA-related functions in specific instances.     

A recurrent general RNA binding domain appended to plant methionyl-tRNA synthetase acts as a cis-acting cofactor for aminoacylation

The cDNA encoding rice methionyl-tRNA synthetase was isolated. The protein exhibited a C-terminal polypeptide appended to a classical MetRS domain. This supplementary domain is related to endothelial monocyte activating polypeptide II (EMAPII), a cytokine produced in mammals after cleavage of p43, a component of the multisynthetase complex. It is also related to Arc1p and Trbp111, two tRNA binding proteins. We expressed rice MetRS and a derivative with a deletion of its EMAPII-like domain. Band-shift analysis showed that this extra-domain provides MetRS with non-specific tRNA binding properties. The EMAPII-like domain contributed a 10-fold decrease in K:(M) for tRNA in the aminoacylation reaction catalyzed by the native enzyme, as compared with the C-terminally truncated MetRS. Consequently, the EMAPII domain provides MetRS with a better catalytic efficiency at the free tRNA concentration prevailing in vivo. This domain binds the acceptor minihelix of tRNA(Met) and facilitates its aminoacylation. These results suggest that the EMAPII module could be a relic of an ancient tRNA binding domain that was incorporated into primordial synthetases for aminoacylation of RNA minihelices taken as the ancestor of modern tRNA.     

Functional redundancy in the nonspecific RNA binding domain of a class I tRNA synthetase

The sequence of a 228-amino acid nonspecific RNA binding domain appended to the N terminus of a eukaryote tRNA synthetase is shown here to have two lysine-rich clusters (LRCs) that are functionally significant in vivo and in vitro. These two LRCs have unrelated sequences and are separated by a spacer of over 100 amino acids. By using a sensitive test for function in vivo, each LRC is shown to be sufficient in the absence of the other. This sufficiency requires fusion of the spacer to either of the LRCs. Experiments in vitro confirmed that the LRCs are each important for RNA binding. Thus, this nonspecific RNA binding domain has two dissimilar lysine-rich sequence elements that are functionally redundant. Further experiments suggest that this redundancy is not used to dock two molecules of RNA but rather to enhance the overall affinity for a single RNA molecule.     

Identity elements for specific aminoacylation of a tRNA by mammalian lysyl-tRNA synthetase bearing a nonspecific tRNA-interacting factor

Mammalian lysyl-tRNA synthetase (LysRS) has an N-terminal polypeptide chain extension appended to a prokaryotic-like synthetase domain. This extension, termed a tRNA-interacting factor (tIF), possesses a RNA-binding motif [KxxxK(K/R)xxK] that binds nonspecifically the acceptor TPsiC stem-loop domain of tRNA and provides a potent tRNA binding capacity to this enzyme. Consequently, native LysRS aminoacylates a RNA minihelix mimicking the amino acid acceptor stem-loop domain of tRNA(3)(Lys). Here, examination of minihelix recognition showed that mammalian LysRS aminoacylates RNA minihelices without specificity of sequence, revealing that none of the nucleotides from the acceptor TPsiC stem-loop domain are essential determinants of tRNA(Lys) acceptor identity. To test whether the tIF domain reduces the specificity of the synthetase with regard to complete tRNA molecules, aminoacylation of wild-type and mutant noncognate tRNAs by wild-type or N-terminally truncated LysRS was examined. The presence of the UUU anticodon of tRNA(Lys) appeared to be necessary and sufficient to transform yeast tRNA(Asp) or tRNA(i)(Met) into potent lysine acceptor tRNAs. Thus, nonspecific RNA-protein interactions between the acceptor stem of tRNA and the tIF domain do not relax the tRNA specificity of mammalian LysRS. The possibility that interaction of the full-length cognate tRNA with the synthetase is required to induce the catalytic center of the enzyme into a productive conformation is discussed.     

Complementation of yeast Arc1p by the p43 component of the human multisynthetase complex does not require its association with yeast MetRS and GluRS

Yeast Arc1p, human p43 and plant methionyl-tRNA synthetase (MetRS) possess an EMAPII-like domain capable of non-specific interactions with tRNA. Arc1p interacts with MetRS (MES1) and GluRS and operates as a tRNA-interacting factor (tIF) in trans of these two synthetases. In plant MetRS, the EMAPII-like domain is fused to the catalytic core of the synthetase and acts as a cis-acting tIF for aminoacylation. We observed that the catalytic core of plant MetRS expressed from a centromeric plasmid cannot complement a yeast arc1(-) mes1(-) strain. Overexpression of the mutant enzyme from a high-copy number plasmid restored cell growth, suggesting that deletion of its C-terminal tIF domain was responsible for the poor aminoacylation efficiency of that enzyme in vivo. Accordingly, expression of full-size plant MetRS from a centromeric plasmid, but also of fusion proteins between its catalytic core and the EMAPII-like domains of yeast Arc1p or of human p43 restored cell viability. These data showed that homologous tIF domains from different origins are interchangeable and may act indifferently in trans or in cis of the catalytic domain of a synthetase. Unexpectedly, co-expression of Arc1p with the catalytic core of plant MetRS restored cell viability as well, even though Arc1p did not associate with plant MetRS. Because Arc1p also interacts with yeast GluRS, restoration of cell growth could be due at least in part to its role of cofactor for that enzyme. However, co-expression of human p43, a tIF that did not associate with plant MetRS or with yeast GluRS and MetRS, also restored cell viability of a yeast strain that expressed the catalytic core of plant MetRS. These results show that p43 and Arc1p are able to facilitate tRNA aminoacylation in vivo even if they do not interact physically with the synthetases. We propose that p43/Arc1p may be involved in sequestering tRNAs in the cytoplasm of eukaryotic cells, thereby increasing their availability for protein synthesis.     

The C-terminal domain of T4 RNA ligase 1 confers specificity for tRNA repair

T4 RNA ligase 1 (Rnl1) is a tRNA repair enzyme that thwarts a tRNA-damaging host response to virus infection. The 374-aa Rnl1 protein consists of an N-terminal nucleotidyltransferase domain fused to a unique C-terminal domain composed of 10 alpha helices. We exploited an in vitro tRNA splicing system to demonstrate that Rnl1 has an inherent specificity for sealing tRNA with a break in the anticodon loop. The tRNA specificity is imparted by the C domain, any deletion of which caused the broken tRNA to be sealed as poorly as the linear intron in vitro and also abolished Rnl1 tRNA splicing activity in vivo. Deletion analysis demarcated Rnl1-(1-254) as a minimal catalytic domain of Rnl1, capable of all chemical steps of the nonspecific RNA ligation reaction. Alanine scanning of the N domain identified Ser103, Leu104, Lys117, and Ser118 as important for pRNA ligation in vitro and tRNA repair in vivo.     

An essential protein-binding domain of nuclear RNase P RNA

Eukaryotic RNase P and RNase MRP are endoribonucleases composed of RNA and protein subunits. The RNA subunits of each enzyme share substantial secondary structural features, and most of the protein subunits are shared between the two. One of the conserved RNA subdomains, designated P3, has previously been shown to be required for nucleolar localization. Phylogenetic sequence analysis suggests that the P3 domain interacts with one of the proteins common to RNase P and RNase MRP, a conclusion strengthened by an earlier observation that the essential domain can be interchanged between the two enzymes. To examine possible functions of the P3 domain, four conserved nucleotides in the P3 domain of Saccharomyces cerevisiae RNase P RNA (RPR1) were randomized to create a library of all possible sequence combinations at those positions. Selection of functional genes in vivo identified permissible variations, and viable clones that caused yeast to exhibit conditional growth phenotypes were tested for defects in RNase P RNA and tRNA biosynthesis. Under nonpermissive conditions, the mutants had reduced maturation of the RPR1 RNA precursor, an expected phenotype in cases where RNase P holoenzyme assembly is defective. This loss of RPR1 RNA maturation coincided, as expected, with a loss of pre-tRNA maturation characteristic of RNase P defects. To test whether mutations at the conserved positions inhibited interactions with a particular protein, specific binding of the individual protein subunits to the RNA subunit was tested in yeast using the three-hybrid system. Pop1p, the largest subunit shared by RNases P and MRP, bound specifically to RPR1 RNA and the isolated P3 domain, and this binding was eliminated by mutations at the conserved P3 residues. These results indicate that Pop1p interacts with the P3 domain common to RNases P and MRP, and that this interaction is critical in the maturation of RNase P holoenzyme.     

Footprinting analysis of interactions between the largest eukaryotic RNase P/MRP protein Pop1 and RNase P/MRP RNA components

Ribonuclease (RNase) P and RNase MRP are closely related catalytic ribonucleoproteins involved in the metabolism of a wide range of RNA molecules, including tRNA, rRNA, and some mRNAs. The catalytic RNA component of eukaryotic RNase P retains the core elements of the bacterial RNase P ribozyme; however, the peripheral RNA elements responsible for the stabilization of the global architecture are largely absent in the eukaryotic enzyme. At the same time, the protein makeup of eukaryotic RNase P is considerably more complex than that of the bacterial RNase P. RNase MRP, an essential and ubiquitous eukaryotic enzyme, has a structural organization resembling that of eukaryotic RNase P, and the two enzymes share most of their protein components. Here, we present the results of the analysis of interactions between the largest protein component of yeast RNases P/MRP, Pop1, and the RNA moieties of the enzymes, discuss structural implications of the results, and suggest that Pop1 plays the role of a scaffold for the stabilization of the global architecture of eukaryotic RNase P RNA, substituting for the network of RNA–RNA tertiary interactions that maintain the global RNA structure in bacterial RNase P.     

More than half of yeast U1 snRNA is dispensable for growth.

Yeast U1 snRNA (568 nucleotides) is 3.5-fold larger than its mammalian counterpart (164 nucleotides) and contains apparent sequence homology only at the 5' and 3' ends. We have used deletion analysis to determine whether the yeast-specific U1 sequences play essential roles in vivo. Yeast cells carrying a deletion of more than 60% (355 nucleotides) of the single-copy U1 gene are viable, though slow-growing, while a deletion of 316 nucleotides allows essentially wild-type growth. The boundaries of the viable deletions define a dispensable internal domain which comprises sequences unique to yeast. In contrast, the essential 5' and 3' terminal domains correspond to phylogenetically conserved sequences and/or structures previously implicated in RNA:RNA and RNA:protein interactions. The minimal essential sequences of yeast U1 can be drawn in a secondary structure which resembles metazoan U1 in four of seven structural domains.     

The N-terminal domain of mammalian Lysyl-tRNA synthetase is a functional tRNA-binding domain.

Lysyl-tRNA synthetase from higher eukaryotes possesses a lysine-rich N-terminal polypeptide extension appended to a classical prokaryotic-like LysRS domain. Band shift analysis showed that this extra domain provides LysRS with nonspecific tRNA binding properties. A N-terminally truncated derivative of LysRS, LysRS-DeltaN, displayed a 100-fold lower apparent affinity for tRNA(3)Lys and a 3-fold increase in K(m) for tRNA(3)Lys in the aminoacylation reaction, as compared with the native enzyme. The isolated N-domain of LysRS also displayed weak affinity for tRNA, suggesting that the catalytic and N-domains of LysRS act synergistically to provide a high affinity binding site for tRNA. A more detailed analysis revealed that LysRS binds and specifically aminoacylates an RNA minihelix mimicking the amino acid acceptor stem-loop structure of tRNA(3)Lys, whereas LysRS-DeltaN did not. As a consequence, merging an additional RNA-binding domain into a bacterial-like LysRS increases the catalytic efficiency of the enzyme, especially at the low concentration of deacylated tRNA prevailing in vivo. Our results provide new insights into tRNA(Lys) channeling in eukaryotic cells and shed new light on the possible requirement of native LysRS for triggering tRNA(3)Lys packaging into human immunodeficiency virus, type 1 viral particles.     

Importance of homodimerization for the in vivo function of yeast RNA triphosphatase

Saccharomyces cerevisiae RNA triphosphatase Cet1 is an essential component of the yeast mRNA capping apparatus. The active site of Cet1 resides within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel) that is supported by a globular hydrophobic core. The homodimeric quaternary structure of Cet1 is formed by a network of contacts between the partner protomers. By studying the effects of alanine-cluster mutations, we highlight the contributions of two separate facets of the crystallographic dimer interface to Cet1 function in vivo. One essential facet of the interface entails hydrophobic cross-dimer interactions of Cys(330) and Val(331) and a cross-dimer hydrogen bond of Asp(280) with the backbone amide of Gln(329). The second functionally relevant dimer interface involves hydrophobic side-chain interactions of Phe(272) and Leu(273). Ala-cluster mutations involving these residues elicited lethal or severe temperature-sensitive phenotypes that were suppressed completely by fusion of the mutated triphosphatases to the guanylyltransferase domain of mammalian capping enzyme. The recombinant D279A-D280A and F272A-L273A proteins retained phosphohydrolase activity but sedimented as monomers. These results indicate that a disruption of the dimer interface is uniquely deleterious when the yeast RNA triphosphatase must function in concert with the endogenous yeast guanylyltransferase. We also identify key residue pairs in the hydrophobic core of the Cet1 protomer that support the active site tunnel and stabilize the triphosphatase in vivo.     

RtcB, a novel RNA ligase, can catalyze tRNA splicing and HAC1 mRNA splicing in vivo

RtcB enzymes are novel RNA ligases that join 2',3'-cyclic phosphate and 5'-OH ends. The phylogenetic distribution of RtcB points to its candidacy as a tRNA splicing/repair enzyme. Here we show that Escherichia coli RtcB is competent and sufficient for tRNA splicing in vivo by virtue of its ability to complement growth of yeast cells that lack the endogenous "healing/sealing-type" tRNA ligase Trl1. RtcB also protects yeast trl1Δ cells against a fungal ribotoxin that incises the anticodon loop of cellular tRNAs. Moreover, RtcB can replace Trl1 as the catalyst of HAC1 mRNA splicing during the unfolded protein response. Thus, RtcB is a bona fide RNA repair enzyme with broad physiological actions. Biochemical analysis of RtcB highlights the uniqueness of its active site and catalytic mechanism. Our findings draw attention to tRNA ligase as a promising drug target.     

Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins

Pre-tRNA splicing is an essential process in all eukaryotes. It requires the concerted action of an endonuclease to remove the intron and a ligase for joining the resulting tRNA halves as studied best in the yeast Saccharomyces cerevisiae. Here, we report the first characterization of an RNA ligase protein and its gene from a higher eukaryotic organism that is an essential component of the pre-tRNA splicing process. Purification of tRNA ligase from wheat germ by successive column chromatographic steps has identified a protein of 125 kDa by its potentiality to covalently bind AMP, and by its ability to catalyse the ligation of tRNA halves and the circularization of linear introns. Peptide sequences obtained from the purified protein led to the elucidation of the corresponding proteins and their genes in Arabidopsis and Oryza databases. The plant tRNA ligases exhibit no overall sequence homologies to any known RNA ligases, however, they harbour a number of conserved motifs that indicate the presence of three intrinsic enzyme activities: an adenylyltransferase/ligase domain in the N-terminal region, a polynucleotide kinase in the centre and a cyclic phosphodiesterase domain at the C-terminal end. In vitro expression of the recombinant Arabidopsis tRNA ligase and functional analyses revealed all expected individual activities. Plant RNA ligases are active on a variety of substrates in vitro and are capable of inter- and intramolecular RNA joining. Hence, we conclude that their role in vivo might comprise yet unknown essential functions besides their involvement in pre-tRNA splicing.     

Independent binding sites of small protein B onto transfer-messenger RNA during trans-translation

Stalled bacterial ribosomes are freed by transfer-messenger RNA (tmRNA). With the help of small protein B (SmpB), protein synthesis restarts and tmRNA adds a tag to the stalled protein for destruction. The conformation of a 347 nt long tmRNA from a thermophile and its interactions with SmpB were monitored using structural probes. The RNA is highly folded, including the reading frame, with <30% of unpaired residues. Footprints between SmpB and tmRNA are in the elbow of the tRNA domain, in some pseudoknots including one essential for function and in the lower part of the stem exiting the tRNA domain. The footprints outside the tRNA domain are scattered onto the tmRNA sequence, but form a cluster onto its tertiary structure derived from cryo-EM data. Some footprints flank the first triplet to be translated in tmRNA, suggesting that SmpB participates in the insertion of the tmRNA-encoded reading frame into the decoding center. To discriminate between a conformational rearrangement of tmRNA and independent binding sites, surface plasmon resonance was used and has identified three independent binding sites of SmpB on the RNA, including the site on the tRNA domain. Accordingly, SmpB is proposed to move on the tmRNA scaffold during trans-translation.     

The tRNA aminoacylation co-factor Arc1p is excluded from the nucleus by an Xpo1p-dependent mechanism

Arc1p, a yeast tRNA-binding protein, forms a complex with the aminoacyl-tRNA synthetases, methionyl tRNA synthetase (MetRS) and glutamyl tRNA synthetase (GluRS). Although this complex localizes normally in the cytoplasm, in the absence of Arc1p the two free synthetases are also found inside the nucleus. In this work, in order to localize free Arc1 we abolished complex assembly by deleting the appended domains from both MetRS and GluRS. Surprisingly, free Arc1p remained cytoplasmic even when fitted with a strong nuclear localization signal (NLS). However, NLS-Arc1p accumulated in the nucleus when Xpo1/Crm1, the export receptor for NES-containing cargo proteins, was mutated. Thus, the cytoplasmic location of Arc1p is maintained by Xpo1p-dependent nuclear export and Arc1p could act as an adapter in the nucleocytoplasmic trafficking of tRNA and/or the tRNA-aminoacylation machinery.     

Functional divergence of a unique C-terminal domain of leucyl-tRNA synthetase to accommodate its splicing and aminoacylation roles

Leucyl-tRNA synthetase (LeuRS) performs dual essential roles in group I intron RNA splicing as well as protein synthesis within the yeast mitochondria. Deletions of the C terminus differentially impact the two functions of the enzyme in splicing and aminoacylation in vivo. Herein, we determined that a fiveamino acid C-terminal deletion of LeuRS, which does not complement a null strain, can form a ternary complex with the bI4 intron and its maturase splicing partner. However, the complex fails to stimulate splicing activity. The x-ray co-crystal structure of LeuRS showed that a C-terminal extension of about 60 amino acids forms a discrete domain, which is unique among the LeuRSs and interacts with the corner of the L-shaped tRNALeu. Interestingly, deletion of the entire yeast mitochondrial LeuRS C-terminal domain enhanced its aminoacylation and amino acid editing activities. In striking contrast, deletion of the corresponding C-terminal domain of Escherichia coli LeuRS abolished aminoacylation of tRNALeu and also amino acid editing of mischarged tRNA molecules. These results suggest that the role of the leucine-specific C-terminal domain in tRNA recognition for aminoacylation and amino acid editing has adapted differentially and with surprisingly opposite effects. We propose that the secondary role of yeast mitochondrial LeuRS in RNA splicing has impacted the functional evolution of this critical C-terminal domain.