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PubMed1986

Topographic modeling of free and methionyl-tRNA synthetase bound tRNAfMet by singlet-singlet energy transfer: bending of the 3'-terminal arm in tRNAfMet.

Conformations of tRNAfMet, free and methionyl-tRNA synthetase bound forms, are analyzed by using singlet-singlet energy transfer as a spectroscopic ruler. tRNAfMet(8-13,3'-Flc), tRNAfMet(8-13,D-Etd), and tRNAfMet(3'-Flc,D-Etd) are prepared by sequential chemical modifications. The methionyl-tRNA synthetase binding affinity of these double-labeled tRNAfMets is similar to those of unmodified tRNAfMet. The fluorescence properties of the individual fluorophore in these tRNAs, including emission spectra, anisotropy, and quenching by methionyl-tRNA synthetase, are similar to those of single-labeled tRNAfMet. The transfer efficiencies of double-labeled tRNAfMets, as determined by both donor quenching and sensitized emission, showed efficient energy transfer in all cases. Random orientation being assumed, the apparent distances are 25 A between 8-13 and D20, 44 A between 8-13 and the 3'-terminus, and 49 A between the 3'-terminus and D20, respectively, in free tRNAfMet. Upon binding of methionyl-tRNA synthetase, the apparent distances are 25 A between 8-13 and D20, 45 A between 8-13 and the 3'-terminus, and 54 A between the 3'-terminus and D20, respectively. These results provide topographic models of these specific locations in free and methionyl-tRNA synthetase bound tRNAfMet and suggest that the immobilized 3'-terminal arm in the amino acid acceptor stem bends toward the inner loop of the L-shaped tRNA upon binding of methionyl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1978

Post-transcriptional modifications of the anticodon loop region: alterations in isoaccepting species of tRNA's during development in Bacillus subtilis.

Structural similarities of tRNA's were compared using three sets of isoaccepting species that had previously been shown to undergo significant changes in chromatographic elution properties as a function of developmental stage in Bacillus subtilis. Comparisons of the structures of the tRNA's were based on the composition of their modified nucleosides, comparisons of oligonucleotide elution profiles from RPC-5 columns, and two-dimensional electrophoretic fingerprint analysis of oligonucleotides. The tRNA's studied were tRNA(Lys) (1) and tRNA(Lys) (3); tRNA(Tyr) (1) and tRNA(Tyr) (2); and tRNA(Trp) (1) and tRNA(Trp) (2). The results suggest that the difference among these pairs of isoaccepting species is a difference in the degree of post-transcriptional modifications of the anticodon loop region. The nucleosides involved were N(6)-(Delta(2)-isopentenyl)adenosine (i(6)A), 2-methylthio-N(6)-(Delta(2)-isopentenyl)adenosine (ms(2)i(6)A), and an unknown nucleoside K, which occurred in a position analogous to N-[9-(beta-d-ribofuranosyl)purin-6-ylcarbamoyl]threonine. The amounts of i(6)A and ms(2)i(6)A, determined using total tRNA from exponential-or stationary-phase cells, suggest that the thiomethylation of i(6)A is a pleiotropic phenomenon affecting several tRNA species. As opposed to the situation in Escherichia coli tRNA, where ms(2)i(6)A constitutes about 90% of the total hydrophobic nucleosides at all growth stages, B. subtilis tRNA's have i(6)A as the predominant hydrophobic nucleoside in exponential growth and ms(2)i(6)A as the predominant nucleoside in stationary phase. Thus, the enzyme system which forms i(6)A and the enzyme system which thiomethylates i(6)A are not coordinated during growth in B. subtilis as they are in E. coli. It is suggested that these changes in anticodon loop modifications in B. subtilis may be related to changes in the translational apparatus which occur during sporulation.

Bacillus subtilis↗
PubMed1976

Undermethylated transfer RNA does not support phage RNA-directed in vitro protein synthesis.

A cell-free protein synthesis system from Escherichia coli Q13 was depleted of mRNA and tRNA so that restoration of maximum activity was dependent of the addition of these components. Protein synthesis, directed by either MS2 or Qbeta phage RNA, was stimulated significantly by the addition of normal tRNA from either E. coli Q13 or B. In contrast, undermethylated tRNA from methionine-starved E. coli RCrel did not cause this stimulation. It is concluded that undermethylated tRNA Lacks sufficient base modifications to function in protein synthesis.

Coliphages↗
PubMed1995

Inactivation of arginyl-tRNA protein transferase by a bifunctional arsenoxide: identification of residues proximal to the arsenoxide site.

Aminoacyl-tRNA protein transferases catalyze (posttranslational) aminoacylation of specific protein N-termini, using aminoacyl-tRNA as substrate. This modification targets the protein for ATP-dependent degradation; in eukaryotes, degradation occurs in the ubiquitin-mediated pathway. The eukaryotic transferase, which catalyzes Arg transfer to N-terminal Glu or Asp residues, is potently inhibited by phenylarsenoxides. The gene encoding Arg-tRNA protein transferase from the yeast Saccharomyces cerevisiae was subcloned and overexpressed in Escherichia coli to provide large amounts of homogeneous protein for a molecular analysis of this inhibition. The bifunctional reagent para-[(bromoacetyl)amino]-phenylarsenoxide is a potent and irreversible inactivator of the yeast transferase; the arsenoxide moiety of the reagent directs binding to the enzyme, while the alkyl halide moiety alkylates a residue(s) proximal to the arsenoxide site. One mole of 14C-labeled reagent was covalently incorporated during inactivation, with the side chain of Cys-315 representing the major site of alkylation. Mutation of Cys-315 to Ala yielded a fully active enzyme which was still subject to stoichiometric, irreversible inactivation by the bifunctional arsenoxide. With the C315A-enzyme, the major fraction of the 14C-labeled bifunctional reagent was associated with the side chain(s) of one or more of a stretch of Glu residues (Glu 339-341). These results show that phenylarsenoxides inhibit Arg-tRNA protein transferase by binding to a site that is either itself essential, or regulates an essential site. Inhibition appears to occur through a steric blockade mechanism.

Acyltransferases↗
PubMed2001

The polypeptide chain release factor eRF1 specifically contacts the s(4)UGA stop codon located in the A site of eukaryotic ribosomes.

It has been shown previously [Brown, C.M. & Tate, W.P. (1994) J. Biol. Chem. 269, 33164-33170.] that the polypeptide chain release factor RF2 involved in translation termination in prokaryotes was able to photocrossreact with mini-messenger RNAs containing stop signals in which U was replaced by 4-thiouridine (s4U). Here, using the same strategy we have monitored photocrosslinking to eukaryotic ribosomal components of 14-mer mRNA in the presence of tRNA(f)(Met), and 42-mer mRNA in the presence of tRNA(Asp) (tRNA(Asp) gene transcript). We show that: (a) both 14-mer and 42-mer mRNAs crossreact with ribosomal RNA and ribosomal proteins. The patterns of the crosslinked ribosomal proteins are similar with both mRNAs and sensitive to ionic conditions; (b) the crosslinking patterns obtained with 42-mer mRNAs show characteristic modification upon addition of tRNA(Asp) providing evidence for appropriate mRNA phasing onto the ribosome. Similar changes are not detected with the 14-mer mRNA.tRNA(f)(Met) pairs; (c) when eukaryotic polypeptide chain release factor 1 (eRF1) is added to the ribosome.tRNA(Asp) complex it crossreacts with the 42-mer mRNA containing the s(4)UGA stop codon located in the A site, but not with the s(4)UCA sense codon; this crosslink involves the N-terminal and middle domains of eRF1 but not the C domain which interacts with eukaryotic polypeptide chain release factor 3 (eRF3); (d) addition of eRF3 has no effect on the yield of eRF1-42-mer mRNA crosslinking and eRF3 does not crossreact with 42-mer mRNA. These experiments delineate the in vitro conditions allowing optimal phasing of mRNA on the eukaryotic ribosome and demonstrate a direct and specific contact of 'core' eRF1 and s(4)UGA stop codon within the ribosomal A site.

Animals↗
PubMed2000

Modification defect at anticodon wobble nucleotide of mitochondrial tRNAs(Leu)(UUR) with pathogenic mutations of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes.

The mitochondrial tRNA(Leu)(UUR) (R = A or G) gene possesses several hot spots for pathogenic mutations. A point mutation at nucleotide position 3243 or 3271 is associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes and maternally inherited diabetes with deafness. Detailed studies on two tRNAs(Leu)(UUR) with the 3243 or 3271 mutation revealed some common characteristics in cybrid cells: (i) a decreased life span, resulting in a 70% decrease in the amounts of the tRNAs in the steady state, (ii) a slight decrease in the ratios of aminoacyl-tRNAs(Leu)(UUR) versus uncharged tRNAs(Leu)(UUR), and (iii) accurate aminoacylation with leucine without any misacylation. As a marked result, both of the mutant tRNA molecules were deficient in a modification of uridine that occurs in the normal tRNA(Leu)(UUR) at the first position of the anticodon. The lack of this modification may lead to the mistranslation of leucine into non-cognate phenylalanine codons by mutant tRNAs(Leu)(UUR), according to the mitochondrial wobble rule, and/or a decrease in the rate of mitochondrial protein synthesis. This finding could explain why two different mutations (3243 and 3271) manifest indistinguishable clinical features.

Anticodon↗
PubMed1995

Phage T4-coded Stp: double-edged effector of coupled DNA and tRNA-restriction systems.

The optional Escherichia coli prr locus encodes two physically associated restriction systems: the type IC DNA restriction-modification enzyme EcoprrI and the tRNA(Lys)-specific anticodon nuclease, specified by the PrrC polypeptide. Anticodon nuclease is kept latent as a result of this interaction. The activation of anticodon nuclease, upon infection by phage T4, may cause depletion of tRNA(Lys) and, consequently, abolition of T4 protein synthesis. However, this effect is counteracted by the repair of tRNA(Lys) in consecutive reactions catalysed by the phage enzymes polynucleotide kinase and RNA ligase. Stp, a short polypeptide encoded by phage T4, has been implicated with activation of the anticodon nuclease. Here we confirm this notion and also demonstrate a second function of Stp: inhibition of EcoprrI restriction. Both effects depend, in general, on the same residues within the N-proximal 18 residue region of Stp. We propose that Stp alters the conformation of EcoprrI and, consequently, of PrrC, allowing activation of the latent anticodon nuclease. Presumably, Stp evolved to offset a DNA restriction system of the host cell but was turned, eventually, against the phage as an activator of the appended tRNA restriction enzyme.

Amino Acid Sequence↗
PubMed1984

A cloning strategy for the bacteriophage T4 vs gene.

The product of the bacteriophage T4 vs gene, the tau peptide, has been shown to thermally stabilize temperature-sensitive valyl-tRNA synthetase (EC 6.1.1.9). To clone the bacteriophage T4 vs gene, recombinant pBR322-T4 DNA molecules were used to transform Escherichia coli CP 790302 (valSts). Transformants that grew at 43.5 degrees C on selective medium exhibited some properties indicative of a phage-modified valyl-tRNA synthetase. The data are consistent with successful cloning of the vs gene and atypical modification of the valyl-tRNA synthetase in CP 790302.

Centrifugation, Density Gradient↗
PubMed2005

Comparison of the global structure and dynamics of native and unmodified tRNAval.

The effects of post-transcriptional modifications on the structure and dynamics of Escherichia coli tRNA(val) were studied by NMR spectroscopy. NMR chemical shift data and residual dipolar couplings were used to show that the local secondary and tertiary structures are very similar in native and unmodified tRNA(val). Rigid body restrained molecular dynamics calculations showed that the global structure of tRNA is unchanged by the post-transcriptional modifications. Deuterium exchange NMR experiments were used to probe the dynamics and flexibility of native and unmodified tRNA(val). A similar set of slowly exchanging (t(1/2) > 3 min) imino protons were observed in both tRNAs, but the rates of exchange for the slowest exchanging imino protons were approximately 20 times faster in unmodified than in native tRNA. These results demonstrate that the dynamics and flexibility of tRNA(val), but not the local or global structure, are significantly affected by post-transcriptional modifications.

Base Sequence↗
PubMed1981

[Phenylalanyl-tRNA synthetase from E. coli MRE-600. Effect of chemical modification of lysine residues on the enzyme interaction with substrates].

The effect of modification of Phe-RSase from E. coli MRE-600 by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP and L-phenylalanynyl-5'-adenylate obtained by periodate oxidation on the enzyme interaction with substrates was investigated. It was shown that modification of Phe-RSase by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP leads to a decrease of the aminoacylation rate without changing the rate of the ATP-[32P]-pyrophosphate exchange reaction. The substrate analogs L-phenylalanynol and L-phenyl-alanynyladenylate increase the degree of Phe-RSase inactivation in the aminoacylation reaction. tRNAphe strongly protects the enzyme against inactivation. ATP, both in the absence (in case of modification with pyridoxal-5'-phosphate) and in- the presence of Mg2+ and phenylalanine (in case of modification with o-ATP) exhibits a pronounced protective effect. L-Phe does not protect the enzyme against the inactivation by pyridoxal-5'-phosphate or o-ATP. The dissociation constant of the Phe-RSase[14C]-Phe-tRNAphe complex increases 2.5 -- 5-fold after the enzyme modification by pyridoxal-5'-phosphate, while the Km value for tRNAphe decreases approximately two times in the aminoacylation reaction. There are no changes in the Km values for amino acid and ATP and the Hill coefficients for all substrates tested. Modification of Phe-RSase by pyridoxal-5'-phosphate leads to a decrease of stability of the aminoacyladenylate -- enzyme complex. Oxidized L-phenylalanynyladenylate does not produce enzyme inactivation either by aminoacylation or in the isotropic ATP-PP iota exchange reaction. It is assumed that Phe-RSase from E. coli MRE-600 contains some lysine residues essential for binding and aminoacylation of tRNA, which do not occur in the ATP-binding subsite and aminoacyladenylate formation center.

Adenosine Triphosphate↗
PubMed1976

A functional requirement for modification of the wobble nucleotide in tha anticodon of a T4 suppressor tRNA.

Temperature-sensitive mutants of E. coli have been isolated which restrict the growth of strains of bacteriophage T4 which are dependent upon the function of a T4-coded amber or ochre suppressor transfer RNA. One such mutant restricts the growth of certain ochre but not amber suppressor-requiring phage. Analysis of the T4 tRNAs synthesized in this host revealed that many nucleotide modifications are significantly reduced. The modifications most strongly affected are located in the anticodon regions of the tRNA'S. The T4 ochre suppressor tRNAs normally contain a modified U residue in the wobble position of the anticodon; it has been possible to correlate tha absence of this specific modification in the mutant host with the restriction of suppressor activity. Furthermore, the extent of this restriction varies dramatically with the site of the nonsense codon, indicating that the modification requirement is strongly influenced by the local context of the mRNA. An analysis of spontaneous revertants of the E. coli ts mutant indicates that temperature sensitivity, restriction of phage suppressor function, and undermodification of tRNA are the consequences of a single genetic lesion. The isolation of a class of partial revertants to temperature insensitivity which have simultaneously become sensitive to streptomycin suggests that the translational requirement for the anticodon modification can be partially overcome by a change in the structure of the ribosome.

Anticodon↗
PubMed2003

The Saccharomyces cerevisiae U2 snRNA:pseudouridine-synthase Pus7p is a novel multisite-multisubstrate RNA:Psi-synthase also acting on tRNAs.

The Saccharomyces cerevisiae Pus7 protein was recently characterized as a novel RNA:pseudouridine (Psi)-synthase acting at position 35 in U2 snRNA. However, U2 snRNA was the only potential substrate tested for this enzyme. In this work, we demonstrated that although Pus7p is responsible for the formation of only one of the six Psi residues present in yeast UsnRNAs, it catalyzes U to Psi conversion at position 13 in cytoplasmic tRNAs and at position 35 in pre-tRNA(Tyr). Sites of RNA modification by Pus7p were identified by analysis of the in vivo RNA modification defects resulting from the absence of active Pus7p production and by in vitro tests using extracts from WT and genetically modified yeast cells. For demonstration of the direct implication of Pus7p in RNA modification, the activity of the WT and mutated Pus7p recombinant proteins was tested on in vitro produced tRNA and pre-tRNA transcripts. Mutation of an aspartic acid residue (D256) that is conserved in all Pus7 homologs abolishes the enzymatic activity both in vivo and in vitro. This suggests the direct involvement of D256 in catalysis. Target sites of Pus7p in RNAs share a common sequence Pu(G/C)UNPsiAPu (Pu = purine, N = any nucleotide), which is expected to be important for substrate recognition. Modification of tRNAs by Pus7p explains the presence of Pus7p homologs in archaea and some bacteria species, which do not have U2 snRNA, and in vertebrates, where Psi34 (equivalent to Psi35 in yeast) formation in U2 snRNA is an H/ACA snoRNA guided process. Our results increase the number of known RNA modification enzymes acting on different types of cellular RNAs.

Base Sequence↗
PubMed1984

Undermodification in the first position of the anticodon of supG-tRNA reduces translational efficiency.

Two mutants of Escherichia coli, trmC1 and trmC2, which are both defective in the synthesis of 5-methylaminomethyl-2-thiouridine (mnm5s2U) were utilized to study the function of this complex modified nucleoside. Transfer RNAs specific for glutamine, glutamic acid and lysine as well as a specific ochre suppressor derived from lysine tRNA (tRNAUAAlys encoded by the supG allele), contain this modified nucleoside at position 34 (the wobble position). It was found that two different undermodified derivatives of mnm5s2U were present in the two trmC mutants, which suggests that the two mutations affect two different enzymatic activities. Using the lacI-Z fusion system (Miller and Albertini 1983), we found that the efficiency of supG-mediated suppression was reduced to 30%-90% of the wild-type value in the trmC mutants. The modification-deficient supG-tRNA in the mutants showed a higher sensitivity to codon context than the normal tRNAUAAlys.

Anticodon↗
PubMed2001

Interactions between 23S rRNA and tRNA in the ribosomal E site.

Interactions between tRNA or its analogs and 23S rRNA in the large ribosomal subunit were analyzed by RNA footprinting and by modification-interference selection. In the E site, tRNA protected bases G2112, A2392, and C2394 of 23S rRNA. Truncated tRNA, lacking the anticodon stem-loop, protected A2392 and C2394, but not G2112, and tRNA derivatives with a shortened 3' end protected only G2112, but not A2392 or C2394. Modification interference revealed C2394 as the only accessible nucleotide in 23S rRNA whose modification interferes with binding of tRNA in the large ribosomal subunit E site. The results suggest a direct contact between A76 of tRNA A76 and C2394 of 23S rRNA. Protections at G2112 may reflect interaction of this 23S rRNA region with the tRNA central fold.

Anticodon↗
PubMed1988

Codon and amino-acid specificities of a transfer RNA are both converted by a single post-transcriptional modification.

An Escherichia coli isoleucine transfer RNA specific for the codon AUA (tRNA(2Ile) or tRNA(minorIle] has a novel modified nucleoside, lysidine in the first position of the anticodon (position 34), which is essential for the specific recognition of the codon AUA. We isolated the gene for tRNA(2Ile) (ileX) and found that the anticodon is CAT, which is characteristic of the methionine tRNA gene. Replacement of L(34) of tRNA(2Ile) molecule enzymatically with unmodified C(34) resulted in a marked reduction of the isoleucine-accepting activity and, surprisingly, in the appearance of methionine-accepting activity. Thus, both the codon and amino-acid specificity of this tRNA are converted by a single post-transcriptional modification of the first position of the anticodon during tRNA maturation.

Anticodon↗
PubMed2001

Purine N7 groups that are crucial to the interaction of Escherichia coli rnase P RNA with tRNA.

We have detected by nucleotide analog interference mapping (NAIM) purine N7 functional groups in Escherichia coli RNase P RNA that are important for tRNA binding under moderate salt conditions (0.1 M Mg2+, 0.1 M NH4+). The majority of identified positions represent highly or universally conserved nucleotides. Our assay system allowed us, for the first time, to identify c7-deaza interference effects at two G residues (G292, G306). Several c7-deazaadenine interference effects (A62, A65, A136, A249, A334, A351) have also been identified in other studies performed at very different salt concentrations, either selecting for substrate binding in the presence of 0.025 M Ca2+ and 1 M NH4+ or self-cleavage of a ptRNA-RNase P RNA conjugate in the presence of 3 M NH4+ or Na+. This indicates that these N7 functional groups play a key role in the structural organization of ribozyme-substrate and -product complexes. We further observed that a c7-deaza modification at A76 of tRNA interferes with tRNA binding to and ptRNA processing by E. coli RNase P RNA. This finding combined with the strong c7-deaza interference at G292 of RNase P RNA supports a model in which substrate and product binding to E. coli RNase P RNA involves the formation of intermolecular base triples (A258-G292-C75 and G291-G259-A76).

Base Sequence↗
PubMed2002

Synthesis of the tRNA(Lys,3) anticodon stem-loop domain containing the hypermodified ms2t6A nucleoside.

The synthesis of a protected form of the hypermodified nucleoside, N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine, (ms2t6A) is reported. The hypermodified nucleoside was subsequently elaborated to the protected nucleoside phosphophoramidite using a protecting group strategy compatible with standard RNA oligonucleotide chemistry. The phosphoramidite reagent was then used to synthesize the 17-nucleotide RNA hairpin having the sequence of the anticodon stem-loop (ASL) domain of human tRNA(Lys,3), the primer for HIV-1 reverse transcriptase. Introduction of the modification at position 37 of the tRNA ASL modestly decreases the thermodynamic stability of the RNA hairpin as has been seen previously for the prokaryotic t6A nucleoside lacking the 2-methylthio substituent. 2D NOESY NMR spectra of the ms2t6A containing tRNA ASL indicate that the threonyl side chain adopts a conformation similar to that seen in the solution structure of the analogous t6A containing E. coli tRNA(Lys), despite the presence of the bulky methylthio group. This synthetic approach allows for site-specific incorporation of the hypermodified nucleoside and should facilitate future studies directed at understanding the roles of nucleoside modification in modulating the stability and specificity of biologically important RNA-RNA interactions. Our synthesis of the ms2t6A containing RNAs demonstrates that this methodology is suitable for obtaining quantities of RNA required for structural studies of the HIV primer tRNA.

Anticodon↗
PubMed2004

Substrate specificity for 4-thiouridine modification in Escherichia coli.

The biosynthesis of 4-thiouridine (s4U) in Escherichia coli tRNA requires the action of both the thiamin pathway enzyme ThiI and the cysteine desulfurase IscS. IscS catalyzes sulfur transfer from l-cysteine to ThiI, which utilizes Mg-ATP to activate uridine 8 in tRNA and transfers sulfur to give s4U. In this work, we show through deletion analysis of unmodified E. coli tRNA(Phe) that the minimum substrate for s4U modification is a mini-helix comprising the stacked acceptor and T stems containing an internal bulged region. The size of the bulged loop must be at least 4 nucleotides and contain the target uridine as the first nucleotide. Replacement of the T loop sequence with a tetraloop in the deletion substrate increases activity and shows that the TpsiC primary sequence is not a recognition element. An unmodified tRNA(Phe) transcript in which the 3'-terminal ACCA sequence is removed to give a blunt terminus has <0.1% activity, although the addition of a single overhanging base essentially restores activity. In addition, reducing the distance of the 3' terminus relative to U8 by as little as 1 bp severely impairs activity. By dissecting a minimal RNA substrate in the T loop region, a two-piece system consisting of a substrate RNA and a "guide" RNA is efficiently modified. Our results indicate that outside of the modified U8, there is no primary sequence requirement for substrate recognition. However, the secondary and tertiary structure restrictions appear sufficient to explain why s4U modification is limited in the cell to tRNA.

Bacterial Proteins↗