Structure of the yeast tRNA m7G methylation complex.

Loss of N7-methylguanosine (m7G) modification is involved in the recently discovered rapid tRNA degradation pathway. In yeast, this modification is catalyzed by the heterodimeric complex composed of a catalytic subunit Trm8 and a noncatalytic subunit Trm82. We have solved the crystal structure of Trm8 alone and in complex with Trm82. Trm8 undergoes subtle conformational changes upon Trm82 binding which explains the requirement of Trm82 for activity. Cocrystallization with the S-adenosyl-methionine methyl donor defines the putative catalytic site and a guanine binding pocket. Small-angle X-ray scattering in solution of the Trm8-Trm82 heterodimer in complex with tRNA(Phe) has enabled us to propose a low-resolution structure of the ternary complex which defines the tRNA binding mode of Trm8-Trm82 and the structural elements contributing to specificity.

New chromatographic and biochemical strategies for quick preparative isolation of tRNA.

A combination of hydrophobic chromatography on phenyl-Sepharose and reversed phase HPLC was used to purify individual tRNAs with high specific activity. The efficiency of chromatographic separation was enhanced by biochemical manipulations of the tRNA molecule, such as aminoacylation, formylation of the aminoacyl moiety and enzymatic deacylation. Optimal combinations are presented for three different cases. (i) tRNA(Phe) from Escherichia coli. This species was isolated by a combination of low pressure phenyl-Sepharose hydrophobic chromatography with RP-HPLC. (ii) tRNA(Ile) from E. coli: Aminoacylation increases the retention time for this tRNA in RP-HPLC. The recovered acylated intermediate is deacylated by reversion of the aminoacylation reaction and submitted to a second RP-HPLC run, in which deacylated tRNA(Ile) is recovered with high specific activity. (iii) tRNA(i)(Met) from Saccharomyces cerevisiae. The aminoacylated form of this tRNA is unstable. To increase stability, the aminoacylated form was formylated using E.coli: enzymes and, after one RP-HPLC step, the formylated derivative was deacylated using peptidyl-tRNA hydrolase from E.COLI: The tRNA(i)(Met) recovered after a second RP-HPLC run exhibited electrophoretic homogeneity and high specific activity upon aminoacylation. These combinations of chromatographic separation and biochemical modification can be readily adapted to the large-scale isolation of any particular tRNA.

Thermodynamic contribution of nucleoside modifications to yeast tRNA(Phe) anticodon stem loop analogs.

The determination of the structural and functional contributions of natural modified nucleosides to tRNA has been limited by lack of an approach that can systematically incorporate the modified units. We have produced a number of oligonucleotide analogs, of the anticodon of yeast tRNA(Phe) by, combining standard automated synthesis for the major nucleosides with specialty chemistries for the modified nucleosides. In this study, both naturally occurring and unnatural modified nucleotides were placed in native contexts. Each oligonucleotide was purified and the nucleoside composition determined to validate the chemistry. The RNAs were denatured and analyzed to determine the van't Hoff thermodynamic parameters. Here, we report the individual thermodynamic contributions for Cm, Gm, m1G, m5C, psi. In addition m5m6U, m1psi, and m3psi, were introduced to gain additional understanding of the physicochemical contribution of psi and m5C at an atomic level. These oligonucleotides demonstrate that modifications have measurable thermodynamic contributions and that loop modifications have global contributions.

Preparation of active tRNA gene transcripts devoid of 3'-extended products and dimers.

Significant amounts (10-30%) of 3'-extended products with one or two extra nucleotides are synthesized in the course of run-off tRNA gene transcription with T7 RNA polymerase. Denaturing polyacrylamide gel electrophoresis appeared to be insufficient to provide preparative amounts of pure correct-size transcripts. Formation of dimers by tRNA gene transcripts as side products in the course of their activation is also another obstacle in preparation of biologically active transcripts. Here, we have shown that EF-Tu affinity chromatography and/or non-denaturing electrophoresis are simple and efficient tools for isolation of highly active correct-size transcripts. Conditions for transcript activation in vitro should be carefully controlled to prevent dimer formation and obtain reliable data on tRNA transcript structure and function.

A procedure for isolation of pure complexes of acylated and unacylated tRNA(Phe) with ribosomes from Escherichia coli.

A simple and rapid procedure for the isolation of pure specific complexes of poly(U).ribosome with acylated and/or unacylated tRNA(Phe) is described. The method is based on the use of conditions that favor the formation of polyribosomes. The polyribosomes are separated from ribosomal subunits and monoribosomes by gel filtration on Sepharose 4B at low Mg2+ concentrations that cause the selective dissociation of tRNA-vacant ribosomes from polyribosomes. The procedure allows the isolation in about one hour of various ribosome.poly(U).tRNA complexes completely free of ribosomal particles unable to bind tRNA. A minor fraction of the purified ribosomal complexes is formed by particles devoid of peptidyltransferase activity. Attempts to eliminate this fraction of inactive ribosomes and to obtain ribosomal complexes fully active in polypeptide synthesis were unsuccessful.

Hamming chromatography.

Selection of molecules with desired properties from random pools of biopolymers has become a powerful tool in biotechnology. On designing an evolution experiment, a certain knowledge of the concomitant fitness landscape is clearly helpful to set up the optimal experimental conditions. The correlation function is a useful means of characterizing a given landscape, since it can be efficiently measured if one has a method of separating a pool of random sequences according to their Hamming distance from a moderately small number of test sequences. In this paper we describe a special type of hybridization chromatography, where a mixture of oligomers (partially) complementary to a given test sequence is hybridized to the test sequence, covalently bound to a matrix. DNA oligomers are eluted in an 'effective temperature gradient' using conditions that minimize the differences of effects of GC versus AT pairs on the melting temperatures. This method should be a means to quickly separate error classes and thus be the crucial step in characterizing fitness landscapes of biopolymers through an experimental approach. It would also be a useful tool to design sequence pools with a bias towards desired mutant spectra.

In vivo deuteration of transfer RNAs: overexpression and large-scale purification of deuterated specific tRNAs.

Structural investigations of tRNA complexes using NMR or neutron scattering often require deuterated specific tRNAs. Those tRNAs are needed in large quantities and in highly purified and biologically active form. Fully deuterated tRNAs can be prepared from cells grown in deuterated minimal medium, but tRNA content under this conditions is low, due to regulation of tRNA biosynthesis in response to the slow growth of cells. Here we describe the large-scale preparation of two deuterated tRNA species, namely D-tRNAPhe and D-tRNAfMet (the method is also applicable for other tRNAs). Using overexpression constructs, the yield of specific deuterated tRNAs is improved by a factor of two to ten, depending on the tRNA and growth condition tested. The tRNAs are purified using a combination of classical chromatography on an anion exchange DEAE column with reversed phase preparative HPLC. Purification yields nearly homogenous deuterated tRNAs with a chargeability of 1400-1500 pmol amino acid/A260 unit. The deuterated tRNAs are of excellent biological activity.

The ribosomal neighbourhood of the central fold of tRNA: cross-links from position 47 of tRNA located at the A, P or E site.

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl)uridine (acp3U) at position 47 of tRNA(Phe) from Escherichia coli was modified with a diazirine derivative and bound to ribosomes in the presence of suitable mRNA analogues under conditions specific for the ribosomal A, P or E sites. After photo-activation at 350 nm the cross-links to ribosomal proteins and RNA were identified by our standard procedures. In the 30S subunit protein S19 (and weakly S9 and S13) was the target of cross-linking from tRNA at the A site, S7, S9 and S13 from the P site and S7 from the E site. Similarly, in the 50S subunit L16 and L27 were cross-linked from the A site, L1, L5, L16, L27 and L33 from the P site and L1 and L33 from the E site. Corresponding cross-links to rRNA were localized by RNase H digestion to the following areas: in 16S rRNA between positions 687 and 727 from the P and E sites, positions 1318 and 1350 (P site) and 1350 and 1387 (E site); in the 23S rRNA between positions 865 and 910 from the A site, 1845 and 1892 (P site), 1892 and 1945 (A site), 2282 and 2358 (P site), 2242 and 2461 (P and E sites), 2461 and 2488 (A site), 2488 and 2539 (all three sites) and 2572 and 2603 (A and P sites). In most (but not all) cases, more precise localizations of the cross-link sites could be made by primer extension analysis.

GTP consumption of elongation factor Tu during translation of heteropolymeric mRNAs.

The stoichiometry of elongation factor Tu (EF-Tu) and GTP in the complex with aminoacyl-tRNA and the consumption of GTP during peptide bond formation on the ribosome were studied in the Escherichia coli system. The ribosomes were programmed either with two different heteropolymeric mRNAs coding for Met-Phe-Thr-Ile ... (mMFTI) or Met-Phe-Phe-Gly ... (mMFFG) or with poly(U). The composition of the complex of EF-Tu, GTP, and Phe-tRNA(Phe) was studied by gel chromatography. With equimolar amounts of factor and Phe-tRNA(Phe), a pentameric complex, (EF-Tu.GTP)2.Phe-tRNA(Phe), was observed, whereas the classical ternary complex, EF-Tu.GTP.Phe-tRNA(Phe), was found only when Phe-tRNA(Phe) was in excess. Upon binding of the purified pentameric complex to ribosomes carrying fMet-tRNA(fMet) in the peptidyl site and exposing a Phe codon in the aminoacyl site, only one out of two GTPs of the pentameric complex was hydrolyzed per Phe-tRNA bound and peptide bond formed, regardless of the mRNA used. In the presence of EF-G, the stoichiometry of one GTP hydrolyzed per peptide bond formed was found on mMFTI when one or two elongation cycles were completed. In contrast, on mMFFG, which contains two contiguous Phe codons, UUU-UUC, two GTP molecules of the pentameric complex were hydrolyzed per Phe incorporated into dipeptide, whereas the incorporation of the second Phe to form tripeptide consumed only one GTP. Thus, generally one GTP is hydrolyzed by EF-Tu per aminoacyl-tRNA bound and peptide bond formed, and more than one GTP is hydrolyzed only when a particular mRNA sequence, such as a homopolymeric stretch, is translated. The role of the additional GTP hydrolysis is not known; it may be related to frameshifting of peptidyl-tRNA during translocation.

Purification and crystallization of the ternary complex of elongation factor Tu:GTP and Phe-tRNA(Phe).

Elongation factor Tu (EF-Tu) is the most abundant protein in prokaryotic cells. Its general function in protein biosynthesis is well established. It is a member of the large family of G-proteins, all of which bind guanosine phosphates (GDP or GTP) as cofactors. In its active GTP bound state EF-Tu binds aminoacylated tRNA (aa-tRNA) forming the ternary complex EF-Tu:GTP:aa-tRNA. The ternary complex interacts with the ribosome where the anticodon on tRNA recognises a codon on mRNA, GTPase activity is induced and inactive EF-Tu:GDP is released. Here we report the successful crystallization of a ternary complex of Thermus aquaticus EF-Tu:GDPNP and yeast Phe-tRNA(Phe) after its purification by HPLC.

Fractionation and identification of cytoplasmic tRNAS and structural characterization of a phenylalanine and a leucine tRNA from cucumber hypocotyls.

Total cytoplasmic tRNAs from cucumber hypocotyls were fractionated by two-dimensional polyacrylamide gel electrophoresis into about 56 species. Of these, 32 tRNA species were identified by aminoacylation as tRNA specific for 15 amino acids. Furthermore, cytoplasmic tRNA(Phe) and tRNA(Leu(NAA)) were purified to homogeneity by the combination of RPC-5 column chromatography and two-dimensional polyacrylamide gel electrophoresis. The primary structure of tRNA(Leu(NAA)) and the modified nucleotides of tRNA(Phe) have been determined. The sequence of cucumber cytoplasmic tRNA(Leu(NAA)) is identical to that of bean cytoplasmic tRNA(Leu(NAA)) except for three base differences in the variable loop.

Phenylalanyl-tRNA synthetase from Thermus thermophilus can attach two molecules of phenylalanine to tRNA(Phe).

Phenylalanyl-tRNA synthetase from the extreme thermophilic bacterium Thermus thermophilus can incorporate more than one molecule of phenylalanine into the tRNA(Phe). It is shown that the 'hyperaminoacylated' tRNA(Phe) is the bis-2',3'-O-phenylalanyl-tRNA(Phe), and its formation is typical for the thermophilic enzyme but does not occur for E. coli phenylalanyl-tRNA synthetase under the same conditions.

Construction and processing of transfer RNA precursor models.

Several "dimeric" tRNA molecules were constructed as potential substrates for ribonuclease P (RNase P) and for M1 RNA, the catalytic subunit of RNase P. Construction was affected by the T4 RNA ligase-mediated coupling of a mature Escherichia coli tRNA (acceptor substrate) and nucleotides 1-36 of yeast tRNAPhe (donor substrate), followed by annealing of the 3'-half of yeast tRNAPhe (nucleotides 38-76). E. coli RNase P and M1 RNA were both found to cleave the dimeric tRNA precursor model constructed from E. coli tRNAPhe (5'-tRNA) and yeast tRNAPhe (3'-tRNA) in a reaction that was dependent on the presence of the annealed 3'-half molecule derived from yeast tRNAPhe, or on some conformation imposed by the presence of this species; the product had the same mobility as authentic E. coli tRNAPhe on a polyacrylamide gel. By utilizing tRNA precursor models radiolabeled at phosphodiesters immediately preceding or following the putative site of processing, cleavage of the substrate by both M1 RNA and the holoenzyme was demonstrated to occur at the expected phosphate ester linkage. The results obtained here suggest that the endonucleolytic separation of two tRNAs by RNase P is dependent on one or more structural features in the 3'-half of the 3'-tRNA, and thus are consistent with the report of McClain et al. (McClain, W. H., Guerrier-Takada, C., and Altman, S. (1987) Science 238, 527-530) that identifies the T stem and loop as a possible recognition site.

Characterization and preliminary crystallographic studies on large ribosomal subunits from Thermus thermophilus.

Diffracting crystals, suitable for X-ray crystallographic analysis, have been obtained from large (50 S) ribosomal subunits from Thermus thermophilus. These crystals, with P4(1)2(1)2 symmetry and a unit cell of 495 A x 495 A x 196 A, reach typically a size of 0.15 mm x 0.25 mm x 0.35 mm. Using synchrotron radiation at cryo-temperature, these crystals diffract X-rays to better than 9 A resolution, and do not show any measurable decay after a few days of irradiation. They complete a series of crystals, grown by us, from ribosomal particles of the same source, including a 30 S subunits, 70 S ribosomes and complexes of the latter with: (1) an oligomer of 35 uridine residues and (2) the same oligonucleotide together with approximately two Phe-tRNA(Phe) molecules. Crystallographic analysis of the various members of this series should provide information for investigating the conformational changes that take place upon the association of ribosomes from their subunits as well as upon binding of non-ribosomal components that participate in protein biosynthesis.

[An approach to isolating individual tRNA from the human placenta by affinity chromatography. Isolation of highly purified Phe-tRNA Phe]. / Podkhod k polucheniiu individual'nykh tRNK iz platsenty cheloveka, osnovannyi na metode affinnoi khromatografii. Vydelenie vysokoobogashchennogo preparata Phe-tRNK Phe.

A new approach to isolation of individual tRNAs from eukaryotes based on affinity chromatography is suggested. At first, using a sorbent with oligonucleotide pTGGT attached, the total tRNA with native CCA-ends was obtained. Then by means of a sorbent with oligonucleotide pTTCAG immobilized, which is complementary to a part of the tRNA(Phe) anticodon loop, tRNA(Phe) with the acceptor activity greater than 1000 pmole/unit was isolated.

Presence of the hypermodified nucleotide N6-(delta 2-isopentenyl)-2-methylthioadenosine prevents codon misreading by Escherichia coli phenylalanyl-transfer RNA.

The overall structure of transfer RNA is optimized for its various functions by a series of unique post-transcriptional nucleotide modifications. Since many of these modifications are conserved from prokaryotes through higher eukaryotes, it has been proposed that most modified nucleotides serve to optimize the ability of the tRNA to accurately interact with other components of the protein synthesizing machinery. When a cloned synthetic Escherichia coli tRNAPhe gene was transfected into a bacterial host that carried a defective phenylalanine tRNA-synthetase gene, tRNAPhe was overexpressed by 11-fold. As a result of this overexpression, an undermodified tRNAPhe species was produced that lacked only N6-(delta 2-isopentenyl)-2-methylthioadenosine (ms2i6A), a hypermodified nucleotide found immediately 3' to the anticodon of all major E. coli tRNAs that read UNN codons. To investigate the role of ms2i6A in E. coli tRNA, we compared the aminoacylation kinetics and in vitro codon-reading properties of the ms2i6A-lacking and normal fully modified tRNAPhe species. The results of these experiments indicate that while ms2i6A is not required for normal aminoacylation of tRNAPhe, its presence stabilizes codon-anticodon interaction and thereby prevents misreading of the genetic code.

Mischarging Escherichia coli tRNAPhe with L-4'-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine, a photoactivatable analogue of phenylalanine.

The Boc-protected derivative of a photoactivatable, carbene-generating analogue of phenylalanine, L-4'-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine [(Tmd)Phe], was used to acylate 5'-O-phosphorylcytidylyl(3'-5')adenosine (pCpA). A diacyl species was isolated which upon successive treatments with trifluoroacetic acid and 0.01 M HCl yielded a 1:1 mixture of 2'(3')-O-(Tmd)phenylalanyl-pCpA and of its 2'-5'-phosphodiester isomeric form. Adapting a procedure introduced by Hecht's group [Heckler, T.G., Chang, L.H., Zama, Y., Naka, T., Chorghade, M.S., & Hecht, S.M. (1984) Biochemistry 23, 1468-1473], brief incubation of a 15 molar excess of this material with Escherichia coli tRNAPhe, missing at the acceptor stem the last two nucleotides (pCpA), in the presence of T4 RNA ligase and ATP afforded "chemically misaminoacylated" tRNAPhe in approximately 50% yield. Following chromatographic purification on DEAE-Sephadex A-25, benzoylated DEAE-cellulose, and Bio-Gel P-6, the misaminoacylated tRNAPhe was characterized by (i) urea-polyacrylamide gel electrophoresis, (ii) enzymatic reaminoacylation under homologous conditions following chemical deacylation, and (iii) its ability to stimulate protein synthesis in an in vitro translation system which, through the addition of the phenylalanyl-tRNA synthetase inhibitor phenylalaninyl-AMP, was unable to charge its endogenous tRNAPhe. The data demonstrate that we have prepared a biologically active misaminoacylated tRNAPhe.

Bovine mitochondrial tRNAPhe, tRNASer (AGY) and tRNASer (UCN): preparation using a new detection method and their properties in aminoacylation.

Bovine mitochondrial tRNAPhe, tRNASer (AGY), and tRNASer (UCN) possessing unusual structures were purified using a new hybridization assay system and their properties in aminoacylation were examined. Bovine mitochondrial phenyl-alanyl- and seryl-tRNA synthetases could aminoacylate the same amino acid-specific tRNAs obtained not only from the mitochondria but also from other sources such as E. coli, Thermus thermophilus, bovine and yeast cytosols and archaebacteria, Sulfolobus acidocaldarius. On the contrary, none of both bacterial and cytosolic synthetases could aminoacylate the same amino acid specific tRNAs from the heterologous sources with some exceptions. We consider that the bovine mitochondrial aminoacyl-tRNA synthetases have considerably simple recognition mechanism toward the substrate tRNAs compared with the non-mitochondrial ones. This mechanism may be correlated with the occurrence of structural varieties of the mitochondrial tRNA species with unusual structures.