End Of tRNA Research Articles

Mechanistic Insights into the Kinetics of HIV-1 Nucleocapsid Protein-facilitated tRNA Annealing to the Primer Binding Site

HIV-1 reverse transcriptase uses human tRNALys,3 as a primer to initiate reverse transcription. Prior to initiation, the 3′ 18 nucleotides of this tRNA are annealed to a complementary sequence on the RNA genome known as the primer binding site (PBS). Here, we show that the HIV-1 nucleocapsid protein (NC) enhances this annealing by approximately five orders of magnitude in vitro, decreasing the transition state enthalpy from approximately 20kcalmol−1 for the uncatalyzed reaction to 13kcalmol−1 for the NC-catalyzed process. Moreover, the annealing follows second-order kinetics, consistent with the nucleation of the intermolecular duplex being the rate-limiting step. This nucleation is preceded by melting of a small duplex region within the original structure, and is followed by much faster zipping of the rest of the 18 base-pair (bp) duplex. A tRNA mutational analysis shows that destabilization of the tRNA acceptor stem has only a minor effect on the annealing rate. In contrast, addition of bases to the 5′ end of tRNA that are complementary to its single-stranded 3′ end interferes with duplex nucleation and therefore has a much larger effect on the net reaction rate. Assuming that the apparent transition free energy of the annealing reaction, ΔG‡, is a sum of the melting (ΔGm) and nucleation (ΔGnuc) free energies, we show that NC affects both ΔGm and ΔGnuc. We estimate that ten to 100-fold of the overall rate enhancement is due to NC-induced destabilization of a 4 bp helix in the PBS, while the additional factor of 103–104 rate enhancement is a result of NC-facilitated duplex nucleation. The apparently similar effectiveness of wild-type and SSHS NC, a mutant that lacks the zinc finger structures, in facilitating the tRNA annealing reaction is most likely the result of the mutual cancellation of two factors: SSHS NC is less effective than wild-type NC as a duplex destabilizer, but more effective as a duplex nucleating agent.

Trans-editing of mischarged tRNAs.

Aminoacyl-tRNA synthetases (aaRSs) are multidomain proteins that specifically attach amino acids to their cognate tRNAs. Their most conserved, and presumably evolutionarily oldest, domains are the catalytic cores, which activate amino acids and transfer them to the 3' ends of tRNAs. Additional domains appended to or inserted in the body of aaRSs increase efficiency and specificity of the aminoacylation process, either by providing additional tRNA contacts, or by hydrolyzing noncognate amino acid products (cis-editing). Here, we report specific tRNA-dependent trans-editing by aaRS-like proteins that reciprocate the editing domains of aaRSs, but not the remainder of the corresponding enzyme. A freestanding homologue of the prolyl-tRNA synthetase-editing domain, the PrdX protein from Clostridium sticklandii, efficiently and specifically hydrolyzes Ala-tRNAPro. Similarly, autonomous alanyl-tRNA synthetase-editing domain homologues (AlaX proteins) from Methanosarcina barkeri and Sulfolobus solfataricus hydrolyze Ser-tRNAAla and Gly-tRNAAla substrates. The discovery of autonomous editing proteins efficient in hydrolyzing misacylated products provides a direct link between ancestral aaRSs consisting solely of the catalytic core and extant enzymes to which functionally independent modules are appended.

The Streptomyces coelicolor polynucleotide phosphorylase homologue, and not the putative poly(A) polymerase, can polyadenylate RNA.

A protein containing a nucleotidyltransferase motif characteristic of poly(A) polymerases has been proposed to polyadenylate RNA in Streptomyces coelicolor (P. Bralley and G. H. Jones, Mol. Microbiol. 40:1155-1164, 2001). We show that this protein lacks poly(A) polymerase activity and is instead a tRNA nucleotidyltransferase that repairs CCA ends of tRNAs. In contrast, a Streptomyces coelicolor polynucleotide phosphorylase homologue that exhibits polyadenylation activity may account for the poly(A) tails found in this organism.

Divergent evolutions of trinucleotide polymerization revealed by an archaeal CCA-adding enzyme structure.

CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase], a template-independent RNA polymerase, adds the defined 'cytidine-cytidine-adenosine' sequence onto the 3' end of tRNA. The archaeal CCA-adding enzyme (class I) and eubacterial/eukaryotic CCA-adding enzyme (class II) show little amino acid sequence homology, but catalyze the same reaction in a defined fashion. Here, we present the crystal structures of the class I archaeal CCA-adding enzyme from Archaeoglobus fulgidus, and its complexes with CTP and ATP at 2.0, 2.0 and 2.7 A resolutions, respectively. The geometry of the catalytic carboxylates and the relative positions of CTP and ATP to a single catalytic site are well conserved in both classes of CCA-adding enzymes, whereas the overall architectures, except for the catalytic core, of the class I and class II CCA-adding enzymes are fundamentally different. Furthermore, the recognition mechanisms of substrate nucleotides and tRNA molecules are distinct between these two classes, suggesting that the catalytic domains of class I and class II enzymes share a common origin, and distinct substrate recognition domains have been appended to form the two presently divergent classes.

Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis.

In contrast to Escherichia coli, where the 3' ends of tRNAs are primarily generated by exoribonucleases, maturation of the 3' end of tRNAs is catalysed by an endoribonuclease, known as RNase Z (or 3' tRNase), in many eukaryotic and archaeal systems. RNase Z cleaves tRNA precursors 3' to the discriminator base. Here we show that this activity, previously unsuspected in bacteria, is encoded by the yqjK gene of Bacillus subtilis. Decreased yqjK expression leads to an accumulation of a population of B.subtilis tRNAs in vivo, none of which have a CCA motif encoded in their genes, and YqjK cleaves tRNA precursors with the same specificity as plant RNase Z in vitro. We have thus renamed the gene rnz. A CCA motif downstream of the discriminator base inhibits RNase Z activity in vitro, with most of the inhibition due to the first C residue. Lastly, tRNAs with long 5' extensions are poor substrates for cleavage, suggesting that for some tRNAs, processing of the 5' end by RNase P may have to precede RNase Z cleavage.

Use of Analogues of Methionine and Methionyl Adenylate to Sample Conformational Changes During Catalysis in Escherichia coli Methionyl-tRNA Synthetase

Binding of methionine to methionyl-tRNA synthetase (MetRS) is known to promote conformational changes within the active site. However, the contribution of these rearrangements to enzyme catalysis is not fully understood. In this study, several methionine and methionyl adenylate analogues were diffused into crystals of the monomeric form of Escherichia coli methionyl-tRNA synthetase. The structures of the corresponding complexes were solved at resolutions below 1.9 Å and compared to those of the enzyme free or complexed with methionine. Residues Y15 and W253 play key roles in the strength of the binding of the amino acid and of its analogues. Indeed, full motions of these residues are required to recover the maximum in free energy of binding. Residue Y15 also controls the size of the hydrophobic pocket where the amino acid side-chain interacts. H301 appears to participate to the specific recognition of the sulphur atom of methionine. Complexes with methionyl adenylate analogues illustrate the shielding by MetRS of the region joining the methionine and adenosine moieties. Finally, the structure of MetRS complexed to a methionine analogue mimicking the tetrahedral carbon of the transition state in the aminoacylation reaction was solved. On the basis of this model, we propose that, in response to the binding of the 3′-end of tRNA, Y15 moves again in order to deshield the anhydride bond in the natural adenylate.

Solution Structure of the Bacillus subtilis T-box Antiterminator RNA: Seven Nucleotide Bulge Characterized by Stacking and Flexibility

The T-box transcription antitermination regulatory system is an important mechanism for regulation of expression of aminoacyl-tRNA synthetase, amino acid biosynthesis and transporter gene expression in Gram-positive bacteria. Antitermination is dependent on a complex set of interactions between uncharged tRNA and the leader region of the mRNA of the regulated gene. Here, we report the solution structure of a model RNA, based on the Bacillus subtilis tyrS antiterminator, determined to an rmsd of 3.47 Å for all nine converged structures and 2.66 Å for the seven structures representing the consensus family. The antiterminator is comprised of two short helices with an intervening 7 nt bulge. The bulge region of the antiterminator, which ultimately interacts with the acceptor end of tRNA, exhibits extensive stacking at the 3′ end (encompassing the highly conserved ACC residues) and is the site of a pronounced kink between the two flanking helices. The 5′ end of the bulge exhibits evidence of conformational flexibility. On the basis of the structural studies, there is no indication that the bases at the 5′ end of the bulge that ultimately base-pair with tRNA are pre-organized for binding. Instead, the data are consistent with a model in which the stacking-induced structure at the 3′ end of the bulge may facilitate the pre-selection of a set of conformations for the tRNA to sample during binding.

TRNA transfers to the limelight.

tRNA has a central role in biology as the adaptor between mRNA and protein, in which anticodon interactions with the mRNA codon at one end of the L-shaped tRNA allow an attached amino acid at the other end of the tRNA to condense with the peptide chain on a second tRNA through the action of the ribosome. This familiar role of tRNA requires mature tRNAs to be recognized by the aminoacyl tRNA synthetase to add the amino acid to its 3 end, by EF1 to form a ternary complex with the aminoacylated tRNA and GTP, and by elements of the ribosome and associated components to allow binding, peptide synthesis, and translocation. Before fulfilling this role, all tRNA transcripts have to be extensively processed. Maturation of tRNAs requires five major steps: (1) removal of the 5 leader by RNase P,

TRNAHis maturation: an essential yeast protein catalyzes addition of a guanine nucleotide to the 5' end of tRNAHis.

All tRNAHis molecules are unusual in having an extra 5' GMP residue (G(-1)) that, in eukaryotes, is added after transcription and RNase P cleavage. Incorporation of this G(-1) residue is a rare example of nucleotide addition occurring at an RNA 5' end in a normal phosphodiester linkage. We show here that the essential Saccharomyces cerevisiae ORF YGR024c (THG1) is responsible for this guanylyltransferase reaction. Thg1p was identified by survey of a genomic collection of yeast GST-ORF fusion proteins for addition of [alpha-32P]GTP to tRNAHis. End analysis confirms the presence of G(-1). Thg1p is required for tRNAHis guanylylation in vivo, because cells depleted of Thg1p lack G(-1) in their tRNAHis. His6-Thg1p purified from Escherichia coli catalyzes the guanylyltransferase step of G(-1) addition using a ppp-tRNAHis substrate, and appears to catalyze the activation step using p-tRNAHis and ATP. Thg1p is highlye conserved in eukaryotes, where G(-1) addition is necessary, and is not found in eubacteria, where G(-1) is genome-encoded. Thus, Thg1p is the first member of a new family of enzymes that can catalyze phosphodiester bond formation at the 5' end of RNAs, formally in a 3'-5' direction. Surprisingly, despite its varied activities, Thg1p contains no recognizable catalytic or functional domains.

The essential role of the invariant GGQ motif in the function and stability in vivo of bacterial release factors RF1 and RF2.

Release factors RF1 and RF2 are required in bacteria for the cleavage of peptidyl-tRNA. A single sequence motif, GGQ, is conserved in all eubacterial, archaebacterial and eukaryotic release factors and may mimic the CCA end of tRNA, although the position of the motif in the crystal structures of human eRF1 and Escherichia coli RF2 is strikingly different. Mutations have been introduced at each of the three conserved positions. Changing the Gln residue to Ala or Glu allowed the factors to retain about 22% of tetrapeptide release activity in vitro, but these mutants could not complement thermosensitive RF mutants in vivo. None of several mutants with altered Gly residues retained activity in vivo or in vitro. Many GGQ mutants were poorly expressed and are presumably unstable; many were also toxic to the cell. The toxic mutant factors or their degradation products may bind to ribosomes inhibiting the action of the normal factor. These data are consistent with a common role for the GGQ motif in bacterial and eukaryotic release factors, despite strong divergence in primary, secondary and tertiary structure, but are difficult to reconcile with the hypothesis that the amide nitrogen of the Gln plays a vital role in peptidyl-tRNA hydrolysis.

Outersphere and innersphere coordinated metal ions in an aminoacyl-tRNA synthetase ribozyme.

Metal ions are essential cofactors for various ribozymes. Here we dissect the roles of metal ions in an aminoacyl-tRNA synthetase-like ribozyme (ARS ribozyme), which was evolved in vitro. This ribozyme can charge phenylalanine on tRNA in cis, where it is covalently attached to the 5'-end of tRNA (i.e. a form of precursor tRNA), as well as in trans, where it can act as a catalyst. The presence of magnesium ion is essential for this ribozyme to exhibit full catalytic activity. Metal-dependent kinetics, as well as structural mappings using Tb3+ in competition with Mg2+ or Co(NH3)6(3+), identified two potential metal-binding sites which are embedded near the tRNA-binding site. The high affinity metal-binding site can be filled with either Mg2+ or Co(NH3)6(3+) and thus the activity relies on a metal ion that is fully coordinated with water or ammonium ions. This site also overlaps with the amino acid-binding site, suggesting that the metal ion plays a role in constituting the catalytic core. The weak metal-binding site is occupied only by a metal ion(s) that can form innersphere contacts with ligands in the ribozyme and, hence, Mg2+ can enhance ribozyme activity, but Co(NH3)6(3+) cannot. The experiments described in this work establish the roles of metal ions that have distinct coordination properties in the ARS ribozyme.

Direct and Indirect Contributions of RNA Secondary Structure Elements to the Initiation of HIV-1 Reverse Transcription

Initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription requires specific recognition between the viral RNA (vRNA), tRNA(3)(Lys), which acts as primer, and reverse transcriptase (RT). The specificity of this ternary complex is mediated by intricate interactions between the HIV-1 RNA and tRNA(3)(Lys). Here, we compared the relative importance of the secondary structure elements of this complex in the initiation process. To this aim, we used the previously published three-dimensional model of the initiation complex to rationally introduce a series of deletions and substitutions in the vRNA. When necessary, we used chemical probing to check the structure of the tRNA(3)(Lys)-mutant vRNA complexes. For each of them, we measured the binding affinity of RT and the kinetics of initial extension of tRNA(3)(Lys) and of synthesis of the (-) strand strong stop DNA. Our results were overall in keeping with the three-dimensional model of the initiation complex. Surprisingly, we found that disruption of the intermolecular template-primer interactions, which are not directly recognized by RT, more severely affected reverse transcription than deletions or disruption of one of the intramolecular helices to which RT directly binds. Perturbations of the highly constrained junction between the intermolecular helix formed by the primer binding site and the 3' end of tRNA(3)(Lys) and the helix immediately upstream also had dramatic effects on the initiation of reverse transcription. Taken together, our results demonstrate the overwhelming importance of the overall three-dimensional structure of the initiation complex and identify structural elements that constitute promising targets for anti-initiation-specific drugs.

Release of Peptide Promoted by the GGQ Motif of Class 1 Release Factors Regulates the GTPase Activity of RF3

E. coli mutants of RF1 and RF2, in which the universal GGQ motif is changed to GAQ, are slow in peptide release from ribosomes. Other kinetic properties are unchanged, suggesting that the GGQ motif is in contact with the peptidyl-transferase center. Deacylated tRNA terminates protein synthesis codon specifically, indicating that the CCA end of tRNA and the GGQ motif operate similarly. Addition of a mutant factor to a pretermination ribosomal complex stimulates exchange of RF3-bound GDP with free GDP, but binding of GTP to RF3 and GTP hydrolysis requires peptide chain release. Therefore, the sequence of steps during termination of translation is regulated by removal of the polypeptide, an event that might trigger a conformational change in the ribosome.

Discrimination of tRNA(Leu) isoacceptors by the mutants of Escherichia coli leucyl-tRNA synthetase in editing.

Leucyl-tRNA synthetase (LeuRS), one of the class Ia aminoacyl-tRNA synthetases, joins Leu to tRNA(Leu) and excludes noncognate amino acids in protein synthesis. In this study, Escherichia coli LeuRS mutants at amino acid E292, which was located in the connective polypeptide 1 insertion region, were synthesized. Although mutated LeuRS showed little change in structure compared with wild-type LeuRS, the mutants were impaired in activity to varying extents. It was also showed that mutations did not affect the adenylation reaction. However, mutated LeuRS can mischarge tRNA(Leu) isoacceptors tRN or tRN with isoleucine to different extents. Isoleucylation of tRN was more than that of tRN. The mutant LeuRS-E292S, which was picked out as an example for the investigation of the relationship between tRNA(Leu) isoacceptors and editing function, can discriminate the Watson-Crick base pair of the first base pair of tRNA(Leu) from the wobble base pair. The tRNA(Leu) with the Watson-Crick base pair may result in more isoleucylated product than that with the wobble base pair. The same phenomenon happened to another mutant, LeuRS-A293D. It seems that the flexibility of the first base pair affects the editing reaction of LeuRS. The results indicate that the flexibility of the first base pair of tRNA(Leu) may probably affect the mischarged 3'-end of tRNA(Leu) shuttling from synthetic site to editing site and that the transferred acceptor arm of tRNA(Leu) may interact with LeuRS in the region around E292.

Expression, purification, crystallization and preliminary diffraction analysis of RNase P protein from Thermotoga maritima.

Ribonuclease P (RNase P), the ubiquitous endonuclease that catalyzes maturation of the 5'-end of tRNA in bacteria, is a ribonucleoprotein particle composed of one large RNA and one small protein. Two major structural types of bacterial RNase P RNA have been identified by phylogenetic comparative analysis: the A (ancestral) and B (Bacillus) types. The RNase P protein from Thermotoga maritima, a hyperthermophilic bacterium with an A-type RNase P RNA, has been expressed in Escherichia coli. A purification strategy was developed to obtain a protein preparation suitable for crystallization. Protein crystals suitable for diffraction studies were obtained and characterized.

Plasticity of Recognition of the 3′-End of Mischarged tRNA by Class I Aminoacyl-tRNA Synthetases

Certain aminoacyl-tRNA synthetases prevent potential errors in protein synthesis through deacylation of mischarged tRNAs. For example, the close homologs isoleucyl-tRNA synthetase (IleRS) and valyl-tRNA synthetase (ValRS) deacylate Val-tRNA(Ile) and Thr-tRNA(Val), respectively. Here we examined the chemical requirements at the 3'-end of the tRNA for these hydrolysis reactions. Single atom substitutions at the 2'- and 3'-hydroxyls of a variety of mischarged RNAs revealed that, while acylation is at the 2'-OH for both enzymes, IleRS catalyzes deacylation specifically from the 3'-OH and not from the 2'-OH. In contrast, ValRS can deacylate non-cognate amino acids from the 2'-OH. Moreover, for IleRS the specificity for a 3'-O location of the scissile ester bond could be forced to the 2'-position by introduction of a 3'-O-methyl moiety. Cumulatively, these and other results suggest that the editing sites of these class I aminoacyl-tRNA synthetases have a degree of inherent plasticity for substrate recognition. The ability to adapt to subtle differences in mischarged RNAs may be important for the high accuracy of aminoacylation.

Characterization of regions of the cyanobacterial tRNA(pro) gene that affect the expression of a beta-glucuronidase reporter gene.

The E3 strong promoter-active fragment harbors the tRNA(pro) (GGG) gene upstream of the promoterless beta-glucuronidase (GUS) reporter gene in plasmid pKG-E3. The 74-bp tRNA(pro) coding sequence contains two regions exhibiting strong homology to blocks A and B which are the split promoter elements of eukaryotic tRNA genes. Results in this study showed that the promoter region of tRNA(pro) gene located upstream of its coding sequence and harbored the putative -10 (TACATT) and -35 (TTGGCA) regions which conformed to the Escherichia coli sigma(70) promoter. Differentiation of the 5' end of tRNA(pro)-GUS transcripts of pKG-E3 revealed that the true transcription initiation sites were located at positions -3, -4, and -6, while the processed sites were located at position +75, +76 and +78 with respect to the first nucleotide of the tRNA(pro) coding sequence. The presence of block A decreased GUS activity about three-fold, whereas block B and the 3' end of tRNA(pro) gene completely abolished GUS expression. However, the presence of full-length tRNA(pro) gene did not affect the GUS expression. Downstream of the tRNA(pro) coding sequence in chromosomal DNA contained a 32-bp stem-loop structure with a predicted DeltaG value of -21.7 kcal x mol(-1). The absence of this stem-loop structure downstream of the tRNA(pro) coding sequence in pKG-E3 resulted in read-through transcription into the adjoining GUS gene.

Characterization of regions of the cyanobacterial tRNA pro gene that affect the expression of a β-glucuronidase reporter gene

The E3 strong promoter-active fragment harbors the tRNA pro (GGG) gene upstream of the promoterless β-glucuronidase ( GUS) reporter gene in plasmid pKG-E3. The 74-bp tRNA pro coding sequence contains two regions exhibiting strong homology to blocks A and B which are the split promoter elements of eukaryotic tRNA genes. Results in this study showed that the promoter region of tRNA pro gene located upstream of its coding sequence and harbored the putative −10 (TACATT) and −35 (TTGGCA) regions which conformed to the Escherichia coli σ 70 promoter. Differentiation of the 5′ end of tRNA pro- GUS transcripts of pKG-E3 revealed that the true transcription initiation sites were located at positions −3, −4, and −6, while the processed sites were located at position +75, +76 and +78 with respect to the first nucleotide of the tRNA pro coding sequence. The presence of block A decreased GUS activity about three-fold, whereas block B and the 3′ end of tRNA pro gene completely abolished GUS expression. However, the presence of full-length tRNA pro gene did not affect the GUS expression. Downstream of the tRNA pro coding sequence in chromosomal DNA contained a 32-bp stem-loop structure with a predicted Δ G value of −21.7 kcal mol −1. The absence of this stem-loop structure downstream of the tRNA pro coding sequence in pKG-E3 resulted in read-through transcription into the adjoining GUS gene.

A Los1p-independent pathway for nuclear export of intronless tRNAs in Saccharomycescerevisiae.

Los1p, the Saccharomyces cerevisiae exportin-t homologue, binds tRNA and functions in pre-tRNA splicing and export of mature tRNA from the nucleus to the cytosol. Because LOS1 is unessential in yeast, other pathways for tRNA nuclear export must exist. We report that Cca1p, which adds nucleotides C, C, and A to the 3' end of tRNAs, is a multicopy suppressor of the defect in tRNA nuclear export caused by los1 null mutations. Mes1p, methionyl-tRNA synthetase, also suppresses the defect in nuclear export of tRNA(Met) in los1 cells. Thus, Cca1p and Mes1p seem to function in a Los1p-independent tRNA nuclear export pathway. Heterokaryon analysis indicates that Cca1p is a nucleus/cytosol-shuttling protein, providing the potential for Cca1p to function as an exporter or an adapter in this tRNA nuclear export pathway. In yeast, most mutations that affect tRNA nuclear export also cause defects in pre-tRNA splicing leading to tight coupling of the splicing and export processes. In contrast, we show that overexpressed Cca1p corrects the nuclear export, but not the pre-tRNA-splicing defects of los1Kan(r) cells, thereby uncoupling pre-tRNA splicing and tRNA nuclear export.

Transit of tRNA through the Escherichia coli ribosome: cross-linking of the 3′ end of tRNA to ribosomal proteins at the P and E sites

Photoreactive derivatives of yeast tRNA Phe containing 2-azidoadenosine at their 3′ termini were used to trace the movement of tRNA across the 50S subunit during its transit from the P site to the E site of the 70S ribosome. When bound to the P site of poly(U)-programmed ribosomes, deacylated tRNA Phe, Phe-tRNA Phe and N-acetyl-Phe-tRNA Phe probes labeled protein L27 and two main sites within domain V of the 23S RNA. In contrast, deacylated tRNA Phe bound to the E site in the presence of poly(U) labeled protein L33 and a single site within domain V of the 23S rRNA. In the absence of poly(U), the deacylated tRNA Phe probe also labeled protein L1. Cross-linking experiments with vacant 70S ribosomes revealed that deacylated tRNA enters the P site through the E site, progressively labeling proteins L1, L33 and, finally, L27. In the course of this process, tRNA passes through the intermediate P/E binding state. These findings suggest that the transit of tRNA from the P site to the E site involves the same interactions, but in reverse order. Moreover, our results indicate that the final release of deacylated tRNA from the ribosome is mediated by the F site, for which protein L1 serves as a marker. The results also show that the precise placement of the acceptor end of tRNA on the 50S subunit at the P and E sites is influenced in subtle ways both by the presence of aminoacyl or peptidyl moieties and, more surprisingly, by the environment of the anticodon on the 30S subunit.