Terminus Of tRNA Research Articles

Chemistry of aminoacylation and peptide bond formation on the 3′terminus of tRNA

Chemistry of aminoacylation and peptide bond formation on the 3′terminus of tRNA

Participation of the tRNA A76 Hydroxyl Groups throughout Translation

The free 2'-3' cis-diol at the 3'-terminus of tRNA provides a unique juxtaposition of functional groups that play critical roles during protein synthesis. The translation process involves universally conserved chemistry at almost every stage of this multistep procedure, and the 2'- and 3'-OHs are in the immediate vicinity of chemistry at each step. The cis-diol contribution affects steps ranging from tRNA aminoacylation to peptide bond formation. The contributions have been studied in assays related to translation over a period that spans at least three decades. In this review, we follow the 2'- and 3'-OHs through the steps of translation and examine the involvement of these critical functional groups.

On Ribosome Conservation and Evolution

The ribosome is a ribozyme whose active site, the peptidyl transferase center (PTC), is situated within a highly conserved universal symmetrical region that connects all ribosomal functional centers involved in amino acid polymerization. The linkage between this elaborate architecture and A-site tRNA position revealed that the A-> P-site passage of the tRNA terminus in the peptidyl transferase center is performed by a rotatory motion, synchronized with the overall tRNA/mRNA sideways movement. Guided by the PTC, the rotatory motion leads to stereochemistry suitable for peptide bond formation, as well as for substrate-mediated catalysis, consistent with quantum mechanical calculations elucidating the transition state mechanism for peptide bond formation and indicating that the peptide bond is being formed during the rotatory motion. Analysis of substrate binding modes to inactive and active ribosomes illuminated the significant PTC mobility and supported the hypothesis that the ancient ribosome produced single peptide bonds and non-coded chains, utilizing free amino acids. Genetic control of the reaction evolved after poly-peptides capable of enzymatic function were created, and an ancient stable RNA fold was converted into tRNA molecules. As the symmetry relates only the backbone fold and nucleotide orientations, but not nucleotide sequence, it emphasizes the superiority of functional requirement over sequence conservation, and indicates that the PTC has evolved by gene fusion, presumably by taking advantage of similar RNA fold structures.

Exploring Global Motions and Correlations in the Ribosome

We studied slower global coupled motions of the ribosome with half a microsecond of coarse-grained molecular dynamics. A low-resolution anharmonic network model that allows for the evolution of tertiary structure and long-scale sampling was developed and parameterized. Most importantly, we find that functionally important movements of L7/L12 and L1 lateral stalks are anticorrelated. Other principal directions of motions include widening of the tRNA cleft and the rotation of the small subunit which occurs as one block and is in phase with the movement of L1 stalk. The effect of the dynamical correlation pattern on the elongation process is discussed. Small fluctuations of the 3′ tRNA termini and anticodon nucleotides show tight alignment of substrates for the reaction. Our model provides an efficient and reliable way to study the dynamics of large biomolecular systems composed of both proteins and nucleic acids.

On peptide bond formation, translocation, nascent protein progression and the regulatory properties of ribosomes. Derived on 20 October 2002 at the 28th FEBS Meeting in Istanbul.

High-resolution crystal structures of large ribosomal subunits from Deinococcus radiodurans complexed with tRNA-mimics indicate that precise substrate positioning, mandatory for efficient protein biosynthesis with no further conformational rearrangements, is governed by remote interactions of the tRNA helical features. Based on the peptidyl transferase center (PTC) architecture, on the placement of tRNA mimics, and on the existence of a two-fold related region consisting of about 180 nucleotides of the 23S RNA, we proposed a unified mechanism integrating peptide bond formation, A-to-P site translocation, and the entrance of the nascent protein into its exit tunnel. This mechanism implies sovereign, albeit correlated, motions of the tRNA termini and includes a spiral rotation of the A-site tRNA-3' end around a local two-fold rotation axis, identified within the PTC. PTC features, ensuring the precise orientation required for the A-site nucleophilic attack on the P-site carbonyl-carbon, guide these motions. Solvent mediated hydrogen transfer appears to facilitate peptide bond formation in conjunction with the spiral rotation. The detection of similar two-fold symmetry-related regions in all known structures of the large ribosomal subunit, indicate the universality of this mechanism, and emphasizes the significance of the ribosomal template for the precise alignment of the substrates as well as for accurate and efficient translocation. The symmetry-related region may also be involved in regulatory tasks, such as signal transmission between the ribosomal features facilitating the entrance and the release of the tRNA molecules. The protein exit tunnel is an additional feature that has a role in cellular regulation. We showed by crystallographic methods that this tunnel is capable of undergoing conformational oscillations and correlated the tunnel mobility with sequence discrimination, gating and intracellular regulation.

Transfer RNA determinants for translational editing by Escherichia coli valyl-tRNA synthetase.

Valyl-tRNA synthetase (ValRS) has difficulty differentiating valine from structurally similar non-cognate amino acids, most prominently threonine. To minimize errors in aminoacylation and translation the enzyme catalyzes a proofreading (editing) reaction that is dependent on the presence of cognate tRNA(Val). Editing occurs at a site functionally distinct from the aminoacylation site of ValRS and previous results have shown that the 3'-terminus of tRNA(Val) is recognized differently at the two sites. Here, we extend these studies by comparing the contribution of aminoacylation identity determinants to productive recognition of tRNA(Val) at the aminoacylation and editing sites, and by probing tRNA(Val) for editing determinants that are distinct from those required for aminoacylation. Mutational analysis of Escherichia coli tRNA(Val) and identity switch experiments with non-cognate tRNAs reveal a direct relationship between the ability of a tRNA to be aminoacylated and its ability to stimulate the editing activity of ValRS. This suggests that at least a majority of editing by the enzyme entails prior charging of tRNA and that misacylated tRNA is a transient intermediate in the editing reaction.

U2 Small Nuclear RNA Is a Substrate for the CCA-adding Enzyme (tRNA Nucleotidyltransferase)

The CCA-adding enzyme builds and repairs the 3' terminus of tRNA. Approximately 65% of mature human U2 small nuclear RNA (snRNA) ends in 3'-terminal CCA, as do all mature tRNAs; the other 35% ends in 3' CC or possibly 3' C. The 3'-terminal A of U2 snRNA cannot be encoded because the 3' end of the U2 snRNA coding region is CC/CC, where the slash indicates the last encoded nucleotide. The first detectable U2 snRNA precursor contains 10-16 extra 3' nucleotides that are removed by one or more 3' exonucleases. Thus, if 3' exonuclease activity removes the encoded 3' CC during U2 snRNA maturation, as appears to be the case in vitro, the cell may need to build or rebuild the 3'-terminal A, CA, or CCA of U2 snRNA. We asked whether homologous and heterologous class I and class II CCA-adding enzymes could add 3'-terminal A, CA, or CCA to human U2 snRNA lacking 3'-terminal A, CA, or CCA. The naked U2 snRNAs were good substrates for the human CCA-adding enzyme but were inactive with the Escherichia coli enzyme; activity was also observed on native U2 snRNPs. We suggest that the 3' stem/loop of U2 snRNA resembles a tRNA minihelix, the smallest efficient substrate for class I and II CCA-adding enzymes, and that CCA addition to U2 snRNA may take place in vivo after snRNP assembly has begun.

PGp as the main product of bovine tRNA kinase.

One of the Ser-tRNAs, Ser-tRNA(Sec), is converted to Sec-tRNA(Sec) by Sec synthase. This Ser-tRNA(Sec) is also converted to phosphoser-tRNA(Sec) by tRNA kinase. In this study, we analyzed of the products of phosphorylation with tRNA kinase. [3H]Ser-tRNA(Sec) purified on Sephacryl S-200 was phosphorylated with [gamma-32P]ATP by tRNA kinase. The product [32P][3H]phosphoser-tRNA was purified on Sephacryl S-200 and hydrolyzed with ribonuclease T2. The chromatogram of this hydrolyzate on DEAE-cellulose in 7 M urea buffer showed four peaks. The first peak of the pass-through fraction was seryl-adenosine liberated from the 3'-terminal of the tRNA. The second peak, eluted before the third peak containing inorganic phosphate, was phosphoseryl-adenosine. The major compound in the fourth peak was pGp. As a control experiment, non-acylated tRNA(Sec) was used as a substrate of phosphorylation and the product was analyzed. The chromatogram of the digest with ribonuclease T2 showed no peak of phosphoseryl-adenosine, but a peak of pGp was seen with the peak of inorganic phosphate. Thus, the major product in the presence of tRNA kinase was pGp, and a small but significant proportion of the radioactivity was found as phosphoserine in the presence of seryl residue on the 3'-CCA terminal of tRNA(Sec). These results indicated that tRNA kinase phosphorylates not only Ser-tRNA to phosphoser-tRNA but also Gp of the 5'-termini of tRNA to pGp. This study gives a new role to mammalian tRNA kinase.

Fluorescence-monitored Conformational Change on the 3′-end of tRNA Upon Aminoacylation

Fluorescent tRNAs species with formycine in the 3′-terminal position (tRNA-CCF) were derived from Escherichia coli tRNAVal, Thermus thermophilus tRNAAsp and Thermus thermophilus tRNAPhe. The fluorescence of formycine was used to monitor the conformational changes at the 3′-terminus of tRNA caused by aminoacylation and hydrolysis of aminoacyl residue from aminoacyl-tRNAs. An increase of about 15% in the fluorescence intensity was observed after aminoacylation of the three tRNA-CCF. This change in fluorescence amplitude that is reversed by hydrolysis of the aminoacyl residue, does not depend on the structure of the amino acid or tRNA sequence. A local conformational change at the 3′-terminal formycine probably involving a partial destacking of the base moiety in the ACCF end takes place as a consequence of aminoacylation. A structural change at the 3′-terminus of tRNA induced by attachment and detachment of the acyl residue may be important in controlling the substrate/product relationship in reactions in which tRNA participates during protein biosynthesis.

A Novel Interaction of tRNALys,3 with the Feline Immunodeficiency Virus RNA Genome Governs Initiation of Minus Strand DNA Synthesis

Complementarity between nucleotides at the 5' terminus of tRNA(Lys,3) and the U5-IR loop of the feline immunodeficiency virus RNA genome suggests a novel intermolecular interaction controls initiation of minus strand synthesis in a manner analogous to other retroviral systems. Base pairing of this tRNA-viral RNA duplex was confirmed by nuclease mapping of the RNA genome containing full-length or 5'-deleted variants of tRNA(Lys,3) hybridized to the primer-binding site. A major pause in RNA-dependent DNA synthesis occurred 14 nucleotides ahead of the primer-binding site with natural and synthetic tRNA(Lys,3) primers, indicating it was not a consequence of tRNA base modifications. The majority of the paused complexes resulted in dissociation of the reverse transcriptase from the template/primer, as demonstrated by an assay limited to a single binding event. Hybridization of a tRNA mutant whose 5' nucleotides are deleted relieved pausing at this position and subsequently allowed high level DNA synthesis. Additional experiments with tRNA-DNA chimeric primers were used to localize the stage of minus strand synthesis at which the tRNA-viral RNA interaction was disrupted. Finally, replacing nucleotides of the feline immunodeficiency virus U5-IR loop with the (A)(4) sequence of its human immunodeficiency virus (HIV)-1 counterpart also relieved pausing, but did not induce pausing immediately downstream of the primer-binding site previously noted during initiation of HIV-1 DNA synthesis. These combined observations provide further evidence of cis-acting sequences immediately adjacent to the primer-binding site controlling initiation of minus strand DNA synthesis in retroviruses and retrotransposons.

This is the end: processing, editing and repair at the tRNA 3'-terminus.

The generation of a mature tRNA 3'-end is an important step in the processing pathways leading to functional tRNA molecules. While 5'-end processing by RNase P is similar in all organisms, generation of the mature 3'-terminus seems to be more variable and complex. The first step in this reaction is the removal of 3'-trailer sequences. In bacteria, this is a multistep process performed by endo- and exonucleases. In contrast, the majority of eukaryotes generate the mature tRNA 3'-end in a single step reaction, which consists of an endonucleolytic cut at the tRNA terminus. After removal of the 3'-trailer, a terminal CCA triplet has to be added to allow charging of the tRNA with its cognate amino acid. The enzyme catalyzing this reaction is tRNA nucleotidyltransferase, homologs of which have been found in representatives of all three kingdoms. Furthermore, in metazoan mitochondria, some genes encode 3'-terminally truncated tRNAs, which are restored in an editing reaction in order to yield functional tRNAs. Interestingly, this reaction is not restricted to distinct tRNAs, but seems to act on a variety of tRNA molecules and represents therefore a more general tRNA repair mechanism than a specialized editing reaction. In this review, the current knowledge about these crucial reactions is summarized.

New evidence for the genomic tag hypothesis: archaeal CCA-adding enzymes and tDNA substrates.

The CCA-adding enzyme [ATP(CTP):tRNA nucleotidyl transferase] catalyzes the addition and regeneration of the 3’ terminal CCA sequence of tRNAs. The enzyme adds the sequence CCA one nucleotide at a time to the 3’-terminus of tRNA, using CTP and ATP as precursors, and can utilize tRNA substrates lacking one, two, or all three terminal nucleotides. The 3’ terminal CCA is encoded in all tRNA genes of some eubacteria (Escherichia coli, Mycoplasma genitalium), in about half the tRNA genes of other eubacteria (Bacillus subtilis) and Archaea (l-3), but only very rarely in eukaryotes (4). The CCA-adding enzyme thus functions mainly as a “repair” enzyme in a subset of eubacteria, but as an essential biosynthetic enzyme in most eubacteria, as well as in all known Archaea and eukaryotes. As expected, the enzyme is essential for viability in the budding yeast Saccharomyces cerevisiae where CCA is not encoded (5), but it is partially dispensable in E. coli where CCA is encoded (6). We have argued that molecular structure and function can provide compelling evidence for descent from a common ancestor, and that careful inspection of the structure and function of contemporary biological molecules can yield highly informative, intellectually rigorous, functional phylogenies despite the absence of conventionally quantifiable traits. Based on the many different roles that tRNA is known to play in RNA and DNA replication, we have presented one such functional phylogeny showing how these diverse roles of tRNA in replication are likely to have evolved from an RNA to the DNA world (7-10; and see Weiner and

Ribonuclease P of Tetrahymena thermophila

Ribonuclease P (RNase P) is responsible for the generation of mature 5' termini of tRNA. The RNA component of this complex encodes the enzymatic activity in bacteria and is itself catalytically active under appropriate conditions in vitro. The role of the subunits in eucaryotes has not yet been established. We have partially purified RNase P activity from the ciliate protozoan Tetrahymena thermophila to learn more about the biochemical characteristics of RNase P from a lower eucaryote. The Tetrahymena RNase P displays a pH optimum and temperature optimum characteristic of RNase P enzymes isolated from other organisms. The Km of the T. thermophila enzyme for pre-tRNAGln is 1.6 x 10(-7)M, which is comparable to the values reported for other examples of RNase P. The Tetrahymena RNase P is a ribonucleoprotein complex, as supported by its sensitivity to micrococcal nuclease and proteinase K. The buoyant density of the enzyme in Cs2SO4 is 1.42 g/ml, which suggests that the RNA component of the Tetrahymena enzyme comprises a significantly greater percentage of the holoenzyme than that determined for RNase P of other Eucarya or Archaea. The holoenzyme has a requirement for divalent cations displaying characteristics that are unique for RNase P but closely resemble preferences reported for the Tetrahymena group I intron RNA. Puromycin inhibits pre-tRNA processing by the Tetrahymena complex, and implications of the similarities between recognition of tRNA by ribosomal components and RNase P are discussed.

A base pair between tRNA and 23S rRNA in the peptidyl transferase centre of the ribosome.

Interaction of the conserved CCA terminus of tRNA with rRNA in the peptidyl transferase P site has been studied by in vitro genetics. A watson-Crick G-C pair between G2252 in a conserved hairpin loop of 23S rRNA and C74 at the acceptor end of tRNA is required for proper functional interaction of the CCA end of tRNA with the ribosomal P site. These findings establish a direct role for 23S rRNA in protein synthesis.

Posttranscriptional Modification of the Central Loop of Domain V in Escherichia coli 23 S Ribosomal RNA

Knowledge of the sites, structures, and functional roles of posttranscriptional modification in rRNAs is limited, despite steadily accumulating evidence that rRNA plays a direct role in the peptidyl transferase reaction and that modified nucleotides are concentrated at the functional center of the ribosome. Using methods based on mass spectrometry, modifications have been mapped in Escherichia coli 23 S rRNA in the central loop of domain V, a region of established interaction between 23 S RNA and tRNA. Two segments of RNA were isolated following protection with oligodeoxynucleotides and nuclease digestion: residues 2423-2473 (51-mer) and 2481-2519 (39-mer). Dihydrouridine was located at position 2449, within the RNase T1 hydrolysis product 2448-ADAACAGp-2454, as evidenced by a molecular mass 2 daltons higher than the gene sequence-predicted mass. This nucleoside, which is nearly ubiquitous in tRNA (where it is involved in maintenance of loop structure), is two bases from A-2551, a previously determined site of interaction between 23 S RNA and the CCA-aminoacyl terminus of tRNA at the ribosomal P-site. The oligonucleotide 2496-CACmCUCGp-2502 was isolated and accurately mass measured, and its nucleoside constituents were characterized by high performance liquid chromatography-mass spectrometry; there was no evidence of modification at position 2501 as implied by earlier work. Using similar techniques, the modified adenosine at position 2503 was unambiguously determined to be 2-methyladenosine in the fragment 2503-m2A psi Gp-2505.

The role of the CCA sequence of tRNA in the peptidyl transfer reaction

Peptidyl transfer is a key step in the process of protein biosynthesis. To examine the role of the universal CCA terminal sequence of tRNA in the process of peptidyl transfer, various mutant transcripts of Escherichia coli valine tRNA were constructed. Peptidyl transferase activity, monitored by the ‘fragment reaction’ with a slight modification, was decreased by mutation at any one base of CCA. The effect of mutation was moderate in the UCA, CUA and CCG mutants. Replacement of A76 by a pyrimidine nucleotide, or replacement of either C74 or C75 by a purine nucleotide caused a marked decrease in the activity. These findings suggested that the universal CCA terminus of tRNA makes a functional interaction with ribosomal RNA by base-pairing.

The importance of conserved nucleotides of 23 S ribosomal RNA and transfer RNA in ribosome catalyzed peptide bond formation.

We have constructed the double mutant G2252C/G2253C in Escherichia coli 23 S rRNA by site-directed mutagenesis. These phylogenetically conserved residues are protected from chemical modification by the 3' CCA terminus of the peptidyl-tRNA site (P site)-bound tRNA. Expression of C2252/C2253 23 S rRNA in E. coli severely compromises cell growth. Mutant rRNA is assembled into 50 S subunits and 70 S ribosomes but is discriminated against in polysomes. Mutant ribosomes function at lower rates in peptidyltransferase assays than wild type ribosomes. To test whether this defect derives from disruption of base pairing with the 2 cytidines of the invariant 3' CCA terminus of tRNA, a mutant E. coli tRNAPhe gene was constructed, with the CCA sequence changed to GGA. As deacylated species, mutant and wild type tRNAPhe inhibit peptidyl transfer identically. Mutant tRNAPhe was aminoacylated in vitro but failed to react as a P site substrate, with either mutant or wild type ribosomes. These results support a role for G2252 and G2253 of 23 S rRNA in peptidyltransferase function and a role for the 3' residues of peptidyl-tRNA in catalytically productive P site interaction; but they fail to provide evidence supporting canonical base pairing between these 23 S residues and the 3' end of peptidyl-tRNA.

On the function of the 2′-hydroxyl group in the peptidyl tRNA terminus during elongation in ribosomal peptide biosynthesis

Intramolecular ester amminolysis in 1,2-diglycyloxyethane, which was used in the present study to represent a bis(acyloxy)-tRNA adenylate terminus, was found to be so slow (5–10% yield after 8–10 hours at 195°) that the likelihood of bis(acyloxy)-tRNA as an intermediate in the ribosomal peptide biosynthetic process appears to be highly improbable.

Glutamyl-tRNA synthetase from Thermus thermophilus HB8. Molecular cloning of the gltX gene and crystallization of the overproduced protein.

The gene for the Glu-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8, was isolated using a synthetic oligonucleotide probe coding for the N-terminal amino acid sequence of Glu-tRNA synthetase. Nucleotide-sequence analysis revealed an open reading frame coding for a protein composed of 468 amino acid residues (Mr 53,901). Codon usage in the T. thermophilus Glu-tRNA synthetase gene was in fact similar to the characteristic usages in the genes for proteins from bacteria of genus Thermus: the G + C content in the third position of the codons was as high as 94%. In contrast, the amino acid sequence of T. thermophilus Glu-tRNA synthetase showed high similarity with bacterial Glu-tRNA synthetases (35-45% identity); the sequences of the binding sites for ATP and for the 3' terminus of tRNA(Glu) are highly conserved. The Glu-tRNA synthetase gene was efficiently expressed in Escherichia coli under the control of the tac promoter. The recombinant T. thermophilus Glu-tRNA synthetase was extremely thermostable and was purified to homogeneity by heat treatment and three-step column chromatography. Single crystals of T. thermophilus Glu-tRNA synthetase were obtained from poly(ethylene glycol) 6000 solution by a vapor-diffusion technique. The crystals diffract X-rays beyond 0.35 nm. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters of a = 8.64 nm, b = 8.86 nm and c = 8.49 nm.

Sites of interaction of the CCA end of peptidyl-tRNA with 23S rRNA.

Oligonucleotide fragments derived from the 3' CCA terminus of acylated tRNA, such as CACCA-(AcPhe), UACCA-(AcLeu), and CAACCA-(fMet), bind specifically to ribosomes in the presence of sparsomycin and methanol [Monro, R. E., Celma, M. L. & Vazquez, D. (1969) Nature (London) 222, 356-358]. All three oligonucleotides protect a characteristic set of bases in 23S rRNA from chemical probes: G2252, G2253, A2439, A2451, U2506, and U2585. A2602 shows enhanced reactivity. These account for most of the same bases that are protected when peptidyl-tRNA analogues such as AcPhe-tRNA are bound to the ribosomal P site, and correspond precisely to those bases whose protection is abolished by removal of the 3'-CA end of tRNA. We conclude that most of the observed interactions between tRNA and 23S rRNA in the 50S ribosomal P site involve the conserved CCA terminus of tRNA. Sparsomycin may inhibit protein synthesis by stabilizing interaction between the peptidyl-CCA and the 23S P site, preventing formation of the intermediate A/P hybrid state.