Structural alterations of the tRNA(m1G37)methyltransferase from Salmonella typhimurium affect tRNA substrate specificity

In Salmonella typhimurium, the tRNA(m1G37)methyltransferase (the product of the trmD gene) catalyzes the formation of m1G37, which is present adjacent and 3' of the anticodon (position 37) in seven tRNA species, two of which are tRNA(Pro)CGG and tRN(Pro)GGG. These two tRNA species also exist as +1 frameshift suppressor sufA6 and sufB2, respectively, both having an extra G in the anticodon loop next to and 3' of m1G37. The wild-type form of the tRNA(m1G37)methyltransferase efficiently methylates these mutant tRNAs. We have characterized one class of mutant forms of the tRNA(m1G37)methyltransferase that does not methylate the sufA6 tRNA and thereby induce extensive frameshifting resulting in a nonviable cell. Accordingly, pseudorevertants of strains containing such a mutated trmD allele in conjunction with the sufA6 allele had reduced frameshifting activity caused by either a 9-nt duplication in the sufA6tRNA or a deletion of its structural gene, or by an increased level of m1G37 in the sufA6tRNA. However, the sufB2 tRNA as well as the wild-type counterparts of these two tRNAs are efficiently methylated by this class of structural altered tRNA(m1G37)methyltransferase. Two other mutations (trmD3, trmD10) were found to reduce the methylation of all potential tRNA substrates and therefore primarily affect the catalytic activity of the enzyme. We conclude that all mutations except two (trmD3 and trmD10) do not primarily affect the catalytic activity, but rather the substrate specificity of the tRNA, because, unlike the wild-type form of the enzyme, they recognize and methylate the wild-type but not an altered form of a tRNA. Moreover, we show that the TrmD peptide is present in catalytic excess in the cell.     

Crystal structures of apo wild-type M. jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine

The Methanococcus jannaschii tRNA(Tyr)/TyrRS pair has been engineered to incorporate unnatural amino acids into proteins in E. coli. To reveal the structural basis for the altered specificity of mutant TyrRS for O-methyl-L-tyrosine (OMeTyr), the crystal structures for the apo wild-type and mutant M. jannaschii TyrRS were determined at 2.66 and 3.0 A, respectively, for comparison with the published structure of TyrRS complexed with tRNA(Tyr) and substrate tyrosine. A large conformational change was found for the anticodon recognition loop 257-263 of wild-type TyrRS upon tRNA binding in order to facilitate recognition of G34 of the anticodon loop through pi-stacking and hydrogen bonding interactions. Loop 133-143, which is close to the tRNA acceptor stem-binding site, also appears to be stabilized by interaction with the tRNA(Tyr). Binding of the substrate tyrosine results in subtle and cooperative movements of the side chains within the tyrosine-binding pocket. In the OMeTyr-specific mutant synthetase structure, the signature motif KMSKS loop and acceptor stem-binding loop 133-143 were surprisingly ordered in the absence of bound ATP and tRNA. The active-site mutations result in altered hydrogen bonding and steric interactions which favor binding of OMeTyr over L-tyrosine. The structure of the mutant and wild-type TyrRS now provide a basis for generating new active-site libraries to evolve synthetases specific for other unnatural amino acids.     

Identity elements for N2-dimethylation of guanosine-26 in yeast tRNAs.

N2,N2-dimethylguanosine (m2(2)G) is a characteristic nucleoside that is found in the bend between the dihydro-uridine (D) stem and the anticodon (AC) stem in over 80% of the eukaryotic tRNA species having guanosine at position 26 (G26). However, since a few eukaryotic tRNAs have an unmodified G in that position, G26 is a necessary but not a sufficient condition for dimethylation. In yeast tRNA(Asp) G26 is unmodified. We have successively changed the near surroundings of G26 in this tRNA until G26 became modified to m2(2)G by a tRNA(m2(2)G26)methyltransferase in Xenopus laevis oocytes. In this way we have identified the two D-stem basepairs C11-G24, G10-C25 immediately preceding G26 as major identity elements for the dimethylating enzyme modifying G26. Furthermore, increasing the extra loop in tRNA(Asp) from four to the more usual five bases influenced the global structure of the tRNA such that the m2(2)G26 formation was drastically decreased even if the near region of G26 had the two consensus basepairs. We conclude that not only are the two consensus base pairs in the D-stem a prerequisite for G26 modification, but also is any part of the tRNA molecule that influence the 3D-structure important for the recognition between nuclear coded tRNAs and the tRNA(m2(2)G26)methyltransferase.     

Structural specificity of Rn nuclease I as probed on yeast tRNA(Phe) and tRNA(Asp).

A single-strand-specific nuclease from rye germ (Rn nuclease I) was characterized as a tool for secondary and tertiary structure investigation of RNAs. To test the procedure, yeast tRNA(Phe) and tRNA(Asp) for which the tertiary structures are known, as well as the 3'-half of tRNA(Asp) were used as substrates. In tRNA(Phe) the nuclease introduced main primary cuts at positions U33 and A35 of the anticodon loop and G18 and G19 of the D loop. No primary cuts were observed within the double stranded stems. In tRNA(Asp) the main cuts occurred at positions U33, G34, U35, C36 of the anticodon loop and G18 and C20:1 positions in the D loop. No cuts were observed in the T loop in intact tRNA(Asp) but strong primary cleavages occurred at positions psi 55, C56, A57 within that loop in the absence of the tertiary interactions between T and D loops (use of 3'-half tRNA(Asp)). These results show that Rn nuclease I is specific for exposed single-stranded regions.     

Mitochondrial tRNA(thr) mutation in fatal infantile respiratory enzyme deficiency.

The mitochondrial DNA (mtDNA) of two unrelated infants with lethal respiratory chain defects was studied using denaturing gradient gel analysis. This analysis revealed melting behavior differences suggesting a point mutation(s) in a restriction fragment containing the apocytochrome b and tRNA(thr) genes. Sequencing revealed that patient 1 had an A to G mutation at nt 15924 which is the last base pair of the anticodon stem adjacent to the anticodon loop of tRNA(thr). Patient 2 had an A to G mutation at nt 15923 which is the last base of the anticodon loop. The results suggest that mtDNA mutations affecting the anticodon loop structure of tRNA(thr) cause mitochondrial disease that is fatal in infancy.     

19F nuclear magnetic resonance as a probe of anticodon structure in 5-fluorouracil-substituted Escherichia coli transfer RNA

The use of 19F nuclear magnetic resonance (n.m.r.) spectroscopy as a probe of anticodon structure has been extended by investigating the effects of tetranucleotide binding to 5-fluorouracil-substituted Escherichia coli tRNA(Val)1 (anticodon FAC). 19F n.m.r. spectra were obtained in the absence and presence of different concentrations of oligonucleotides having the sequence GpUpApX (X = A,G,C,U), which contain the valine codon GpUpA. Structural changes in the tRNA were monitored via the 5-fluorouracil residues located at positions 33 and 34 in the anticodon loop, as well as in all other loops and stems of the molecule. Binding of GpUpApA, which is complementary to the anticodon and the 5'-adjacent FUra 33, shifts two resonances in the 19F spectrum. One, peak H (3.90 p.p.m.), is also shifted by GpUpA and was previously assigned to FUra 34 at the wobble position of the anticodon. The effects of GpUpApA differ from those of GpUpA in that the tetranucleotide induces the downfield shift of a second resonance, peak F (4.5 p.p.m.), in the 19F spectrum of 19F-labeled tRNA(Val)1. Evidence that the codon-containing oligonucleotides bind to the anticodon was obtained from shifts in the methyl proton spectrum of the 6-methyladenosine residue adjacent to the anticodon and from cleavage of the tRNA at the anticodon by RNase H after binding dGpTpApA, a deoxy analog of the ribonucleotide codon. The association constant for the binding of GpUpApA to fluorinated tRNA(Val)1, obtained by Scatchard analysis of the n.m.r. results, is in good agreement with values obtained by other methods. On the basis of these results, we assign peak F in the 19F n.m.r. spectrum of 19F-labeled tRNA(Val)1 to FUra 33. This assignment and the previous assignment of peak H to FUra 34 are supported by the observation that the intensities of peaks F and H in the 19F spectrum of fluorinated tRNA(Val)1 are specifically decreased after partial hydrolysis with nucleass S1 under conditions leading to cleavage in the anticodon loop. The downfield shift of peak F occurs only with adenosine in the 3'-position of the tetranucleotide; binding of GpUpApG, GpUpApC, or GpUpApU results only in the upfield shift of peak H. The possibility is discussed that this base-specific interaction between the 3'-terminal adenosine and the 5-fluorouracil residue at position 33 involves a 5'-stacked conformation of the anticodon loop. Evidence also is presented for a temperature-dependent conformational change in the anticodon loop below the melting temperature of the tRNA.     

Frameshift suppressor mutations outside the anticodon in yeast proline tRNAs containing an intervening sequence.

Extragenic suppressors of +1 frameshift mutations in proline codons map in genes encoding two major proline tRNA isoacceptors. We have shown previously that one isoacceptor encoded by the SUF2 gene (chromosome 3) contains no intervening sequence. SUF2 suppressor mutations result from the base insertion of a G within a 3'-GGA-5' anticodon, allowing the tRNA to read a 4-base code word. In this communication we describe suppressor mutations in genes encoding a second proline tRNA isoacceptor (wild-type anticodon 3'-GGU-5') that result in a novel mechanism for translation of a 4-base genetic code word. The genes that encode this isoacceptor include SUF7 (chromosome 13), SUF8 (chromosome 8), trn1 (chromosome 1), and at least two additional unmapped genes, all of which contain an intervening sequence. We show that suppressor mutations in the SUF7 and SUF8 genes result in G-to-U base substitutions at position 39 that disrupted the normal G . C base pairing in the last base pair of the anticodon stem adjacent to the anticodon loop. These anticodon stem mutations might alter the size of the anticodon loop and permit the use of a 3'-GGGU-5' sequence within the loop to read 4-base proline codons. Uncertainty regarding the exact structure of the mature suppressor tRNAs results from the possibility that anticodon stem mutations might affect sites of intervening sequence removal. The possible role of the intervening sequence in the generation of mature suppressor tRNA is discussed. Besides an analysis of suppressor tRNA genes, we have extended previous observations of the apparent relationship between tRNA genes and repetitive delta sequences found as solo elements or in association with the transposable element TY1. Hybridization studies and a computer analysis of the DNA sequence surrounding the SUF7 gene revealed two incomplete, inverted delta sequences that form a stem and loop structure located 165 base pairs from the 5' end of the tRNA gene. In addition, sequences beginning 164 base pairs from the 5' end of the trn1 gene also exhibit partial homology to delta. These observations provide further evidence for a nonrandom association between tRNA genes and delta sequences.     

Genetic analysis of structure and function in phage T4 tRNASer

We have determined the nucleotide sequences of 55 spontaneous mutations that inactivate a suppressor gene of phage T4 tRNASer. Most of the mutations caused substitutions or deletions of single nucleotides at 18 different positions in the tRNA. Two of three mutations that allowed the synthesis of mature tRNA had nucleotide substitutions at the junction of the dihydrouridine and anticodon stems, suggesting that this region of tRNASer is important for aminoacylation. The third mutation that synthesized tRNA had a nucleotide deletion in the anticodon loop, which presumably affected the translational capacity of the tRNA. We also sequenced 58 spontaneous reversion mutations derived from strains with the inactive suppressor genes. Some of these regenerated the initial tRNA sequence, while other generated a second-site mutation in the tRNA. These second-site mutations restored helical base-pairings to the tRNA that had been eliminated by the initial mutations. The new base-pairings involved G.C and A.U, and the A.C wobble pair at certain positions in the tRNA. This finding establishes the existence of A.C wobble pair in tRNA helices.     

Plasticity and diversity of tRNA anticodon determinants of substrate recognition by eukaryotic A37 isopentenyltransferases

The N(6)-(isopentenyl)adenosine (i(6)A) modification of some tRNAs at position A37 is found in all kingdoms and facilitates codon-specific mRNA decoding, but occurs in different subsets of tRNAs in different species. Here we examine yeasts' tRNA isopentenyltransferases (i.e., dimethylallyltransferase, DMATase, members of the Δ(2)-isopentenylpyrophosphate transferase, IPPT superfamily) encoded by tit1(+) in Schizosaccharomyces pombe and MOD5 in Saccharomyces cerevisiae, whose homologs are Escherichia coli miaA, the human tumor suppressor TRIT1, and the Caenorhabditis elegans life-span gene product GRO-1. A major determinant of miaA activity is known to be the single-stranded tRNA sequence, A36A37A38, in a stem-loop. tRNA(Trp)(CCA) from either yeast is a Tit1p substrate, but neither is a Mod5p substrate despite the presence of A36A37A38. We show that Tit1p accommodates a broader range of substrates than Mod5p. tRNA(Trp)(CCA) is distinct from Mod5p substrates, which we sort into two classes based on the presence of G at position 34 and other elements. A single substitution of C34 to G converts tRNA(Trp)(CCA) to a Mod5p substrate in vitro and in vivo, consistent with amino acid contacts to G34 in existing Mod5p-tRNA(Cys)(GCA) crystal structures. Mutation of Mod5p in its G34 recognition loop region debilitates it differentially for its G34 (class I) substrates. Multiple alignments reveal that the G34 recognition loop sequence of Mod5p differs significantly from Tit1p, which more resembles human TRIT1 and other DMATases. We show that TRIT1 can also modify tRNA(Trp)(CCA) consistent with broad recognition similar to Tit1p. This study illustrates previously unappreciated molecular plasticity and biological diversity of the tRNA-isopentenyltransferase system of eukaryotes.     

Mitochondrial DNA mutations in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS)

The total sequences of mitochondrial DNA were determined in two patients with juvenile-onset mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) due to Complex I deficiency. Patients 1 and 2 had three and two unique point mutations, respectively, causing replacement of phylogenically conserved amino acids. A transition from G to A was found at nucleotide position 5601 in the alanine tRNA gene of Patient 2, and a transition from A to G was found at 3243 in the leucine (UUR) tRNA gene of both patients. The latter mutation located at the phylogenically conserved 5' end of the dihydrouridine loop of the tRNA molecule, and was present in two patients with adult-onset MELAS and absent in controls. These results indicate that a mass of mtDNA mutations including the A-to-G transition in the tRNA(Leu) gene is a genetic cause of MELAS.     

Anticodon domain methylated nucleosides of yeast tRNA(Phe) are significant recognition determinants in the binding of a phage display selected peptide.

The contributions of the natural modified nucleosides to RNA identity in protein/RNA interactions are not understood. We had demonstrated that 15 amino acid long peptides could be selected from a random phage display library using the criterion of binding to a modified, rather than unmodified, anticodon domain of yeast tRNA(Phe) (ASL(Phe)). Affinity and specificity of the selected peptides for the modified ASL(Phe) have been characterized by fluorescence spectroscopy of the peptides' tryptophans. One of the peptides selected, peptide t(F)2, exhibited the highest specificity and most significant affinity for ASL(Phe) modified with 2'-O-methylated cytidine-32 and guanosine-34 (Cm(32) and Gm(34)) and 5-methylated cytidine-40 (m(5)C(40)) (K(d) = 1.3 +/- 0.4 microM) and a doubly modified ASL(Phe)-Gm(34),m(5)C(40) and native yeast tRNA(Phe) (K(d) congruent with 2.3 and 3.8 microM, respectively) in comparison to that for the unmodified ASL(Phe) (K(d) = 70.1 +/- 12.3 microM). Affinity was reduced when a modification altered the ASL loop structure, and binding was negated by modifications that disfavored hairpin formation. Peptide t(F)2's higher affinity for the ASL(Phe)-Cm(32),Gm(34),m(5)C(40) hairpin and fluorescence resonance energy transfer from its tryptophan to the hypermodified wybutosine-37 in the native tRNA(Phe) placed the peptide across the anticodon loop and onto the 3'-side of the stem. Inhibition of purified yeast phenylalanyl-tRNA synthetase (FRS) catalyzed aminoacylation of cognate yeast tRNA(Phe) corroborated the peptide's binding to the anticodon domain. The phage-selected peptide t(F)2 has three of the four amino acids crucial to G(34) recognition by the beta-structure of the anticodon-binding domain of Thermus thermophilus FRS and exhibited circular dichroism spectral properties characteristic of beta-structure. Thus, modifications as simple as methylations contribute identity elements that a selected peptide specifically recognizes in binding synthetic and native tRNA and in inhibiting tRNA aminoacylation.     

Primary structure of tRNA Thr 1a and b from brewer's yeast]

One of the two major species of brewer's yeast tRNA threonine (tRNA Thr 1) has been purified by countercurrent distribution followed by two chromatographic steps (respectively on a Sepharose 4B and a BD-cellulose column). Complete digestion with pancreatic and T1 RNases and a partial hydrolysis with T1 RNase followed by the isolation and determination of the nucleotide sequences of the resulting fragments permitted the derivation of its primary structure. tRNA Thr 1 is in fact a mixture of two subspecies differing only by a A49-U65 base pair in 50 per cent of the molecules which is replaced by a G49-C65 pair in the other 50 per cent. These two subspecies consist of 76 nucleotide residues including 14 minor nucleotides. They show a characteristic m3C at the 3'terminal end of the anticodon loop, an anticodon I-G-U followed by t6A and C48, uncompletely modified (50 per cent) to m5C within the 5 nucleotides long extra-arm. The minor nucleotides m2G m2 2G are located at positions in which they generally occur in the tRNA structures as does m1A within the T-psi-C loop.     

Base-pairing between 23S rRNA and tRNA in the ribosomal A site

The aminoacyl (A site) tRNA analog 4-thio-dT-p-C-p-puromycin (s4TCPm) photochemically cross-links with high efficiency and specificity to G2553 of 23S rRNA and is peptidyl transferase reactive in its cross-linked state, establishing proximity between the highly conserved 2555 loop in domain V of 23S rRNA and the universally conserved CCA end of tRNA. To test for base-pairing interactions between 23S rRNA and aminoacyl tRNA, site-directed mutations were made at the universally conserved nucleotides U2552 and G2553 of 23S rRNA in both E. coli and B. stearothermophilus ribosomal RNA and incorporated into ribosomes. Mutations at G2553 resulted in dominant growth defects in E. coli and in decreased levels of peptidyl transferase activity in vitro. Genetic analysis in vitro of U2552 and G2553 mutant ribosomes and CCA end mutant tRNA substrates identified a base-pairing interaction between C75 of aminoacyl tRNA and G2553 of 23S rRNA.     

Role of the three consecutive G:C base pairs conserved in the anticodon stem of initiator tRNAs in initiation of protein synthesis in Escherichia coli.

The three consecutive G:C base pairs, G29:C41, G30:C40, and G31:C39, are conserved in the anticodon stem of virtually all initiator tRNAs from eubacteria, eukaryotes, and archaebacteria. We show that these G:C base pairs are important for function of the tRNA in initiation of protein synthesis in vivo. We changed these base pairs individually and in combinations and analyzed the activities of the mutant Escherichia coli initiator tRNAs in initiation in vivo. For assessment of activity of the mutant tRNAs in vivo, mutations in the G:C base pairs were coupled to mutation in the anticodon sequence from CAU to CUA. Mutations in each of the G:C base pairs reduced activity of the mutant tRNA in initiation, with mutation in the second G:C base pair having the most severe effect. The greatly reduced activity of this C30:G40 mutant tRNA is not due to defects in aminoacylation or formulation of the tRNA or defects in base modification of the A37, next to the anticodon, which we had previously shown to be important for activity of the mutant tRNAs in initiation. The anticodon stem mutants are most likely affected specifically at the step of binding to the ribosomal P site. The pattern of cleavages in the anticodon loop of mutant tRNAs by S1 nuclease indicate that the G:C base pairs may be involved directly in interactions of the tRNA with components of the P site on the ribosome rather than indirectly by inducing a particular conformation of the anticodon loop critical for function of the tRNA in initiation.