Fluorescent labeling of cell-free synthesized proteins with fluorophore-conjugated methionylated tRNA derived from in vitro transcribed tRNA.

A simple and practical method for preparing fluorophore-conjugated methionylated tRNA necessary for tRNA-mediated fluorescent labeling of cell-free synthesized proteins was developed. Without complicated chromatographic purification and subsequent concentration, fluorophore-conjugated methionylated tRNA with higher purity and fluorescence concentration could be synthesized from in vitro transcribed tRNA instead of from a total tRNA mixture, which has been routinely used as a tRNA source. Although fluorophore-conjugated methionylated tRNA derived from in vitro transcribed tRNA was purified by simple phenol extraction following alcohol precipitation, it worked well in tRNA-mediated fluorescent labeling, yielding an improved signal-to-noise ratio and higher fluorescence intensity compared to the conventional total tRNA-based method. Based on its simplicity in the preparation of labeling agent with higher purity and fluorescence concentration, the developed method will accelerate the prevalence of fluorescence-based detection of cell-free synthesized proteins.

Overexpression and purification of mammalian mitochondrial translational initiation factor 2 and initiation factor 3.

Two mammalian mitochondrial initiation factors have been identified. Initiation factor 2 (IF2(mt)) selects the initiator tRNA (fMet-tRNA) and promotes its binding to the ribosome. Initiation factor 3 (IF3(mt)) promotes the dissociation of the 55S mitochondrial ribosome into subunits and may play additional, less-well-understood, roles in initiation complex formation. Native bovine IF2(mt) was purified from liver a number of years ago. The yield of this factor is very low making biochemical studies difficult. The cDNA for bovine IF2(mt) was expressed in Escherichia coli under the control of the T7 polymerase promoter in a vector that provides a His(6)-tag at the C-terminus of the expressed protein. This factor was expressed in E. coli and purified by chromatography on Ni-NTA resins. The expressed protein has a number of degradation products in partially purified preparations and this factor is then further purified by high-performance liquid chromatography or gravity chromatography on anion exchange resins. IF3(mt) has never been purified from any mammalian system. However, the cDNA for this protein can be identified in the expressed sequence tag (EST) libraries. The portion of the sequence encoding the region of human IF3(mt) predicted to be present in the mitochondrially imported form of this factor was cloned and expressed in E. coli using a vector that provides a C-terminal His(6)-tag. The tagged factor is partially purified on Ni-NTA resins. However, a major proteolytic fragment arising from a defined cleavage of this protein is present in these preparations. This contaminant can be removed by a single step of high-performance liquid chromatography on a cation exchange resin. Alternatively, the mature form of IF3(mt) can be purified by two sequential passes through a gravity S-Sepharose column.

In vitro studies of archaeal translational initiation.

Initiation is the step of translation that has incurred the greatest evolutionary divergence. In silico and experimental studies have shown that archaeal translation initiation resembles neither the bacterial nor the eukaryotic paradigm, but shares features with both. The structure of mRNA in archaea is similar to the bacterial one, although the protein factors that assist translational initiation are more numerous than in bacteria and are homologous to eukaryotic proteins. This chapter describes a number of techniques that can be used for in vitro studies of archaeal translation and translational initiation, using as a model system the thermophilic crenarcheon Sulfolobus solfataricus, growing optimally at about 80 degrees in an acidic environment.

Protection-based assays to measure aminoacyl-tRNA binding to translation initiation factors.

To decipher the mechanisms of translation initiation, the stability of the complexes between tRNA and initiation factors has to be evaluated in a routine manner. A convenient method to measure the parameters of binding of an aminoacyl-tRNA to an initiation factor results from the property that, when specifically complexed to a protein, the aminoacyl-tRNA often resists spontaneous deacylation. This chapter describes the preparation of suitable aminoacyl-tRNA ligands and their use in evaluating the stability of their complexes with various initiation factors, such as e/aIF2 and e/aIF5B. The advantages and the limitations of the method are discussed.

Yeast initiator tRNA identity elements cooperate to influence multiple steps of translation initiation.

All three kingdoms of life employ two methionine tRNAs, one for translation initiation and the other for insertion of methionines at internal positions within growing polypeptide chains. We have used a reconstituted yeast translation initiation system to explore the interactions of the initiator tRNA with the translation initiation machinery. Our data indicate that in addition to its previously characterized role in binding of the initiator tRNA to eukaryotic initiation factor 2 (eIF2), the initiator-specific A1:U72 base pair at the top of the acceptor stem is important for the binding of the eIF2.GTP.Met-tRNA(i) ternary complex to the 40S ribosomal subunit. We have also shown that the initiator-specific G:C base pairs in the anticodon stem of the initiator tRNA are required for the strong thermodynamic coupling between binding of the ternary complex and mRNA to the ribosome. This coupling reflects interactions that occur within the complex upon recognition of the start codon, suggesting that these initiator-specific G:C pairs influence this step. The effect of these anticodon stem identity elements is influenced by bases in the T loop of the tRNA, suggesting that conformational coupling between the D-loop-T-loop substructure and the anticodon stem of the initiator tRNA may occur during AUG codon selection in the ribosomal P-site, similar to the conformational coupling that occurs in A-site tRNAs engaged in mRNA decoding during the elongation phase of protein synthesis.

Preparation and activity of synthetic unmodified mammalian tRNAi(Met) in initiation of translation in vitro.

Translation of eukaryotic mRNA is initiated by a unique amino-acyl tRNA, Met-tRNAi(Met), which passes through a complex series of highly specific interactions with components of the translation apparatus during the initiation process. To facilitate in vitro biochemical and molecular biological analysis of these interactions in fully reconstituted translation initiation reactions, we generated mammalian tRNAi(Met) by in vitro transcription that lacked all eight base modifications present in native tRNAi(Met). Here we report a method for in vitro transcription and aminoacylation of synthetic unmodified initiator tRNAi(Met) that is active in every stage of the initiation process, including aminoacylation by methionyl-tRNA synthetase, binding of Met-tRNAi(Met) to eIF2-GTP to form a ternary complex, binding of the ternary complexes to 40S ribosomal subunits to form 43S complexes, binding of the 43S complex to a native capped eukaryotic mRNA, and scanning on its 5' untranslated region to the correct initiation codon to form a 48S complex, and finally joining with a 60S subunit to assemble an 80S ribosome that is competent to catalyze formation of the first peptide bond using the [35S]methionine residue attached to the acceptor terminus of the tRNAi(Met) transcript.

A subcomplex of three eIF3 subunits binds eIF1 and eIF5 and stimulates ribosome binding of mRNA and tRNA(i)Met.

Yeast translation initiation factor 3 contains five core subunits (known as TIF32, PRT1, NIP1, TIF34 and TIF35) and a less tightly associated component known as HCR1. We found that a stable subcomplex of His8-PRT1, NIP1 and TIF32 (PN2 subcomplex) could be affinity purified from a strain overexpressing these eIF3 subunits. eIF5, eIF1 and HCR1 co-purified with this subcomplex, but not with distinct His8-PRT1- TIF34-TIF35 (P45) or His8-PRT1-TIF32 (P2) sub complexes. His8-PRT1 and NIP1 did not form a stable binary subcomplex. These results provide in vivo evidence that TIF32 bridges PRT1 and NIP1, and that eIFs 1 and 5 bind to NIP1, in native eIF3. Heat-treated prt1-1 extracts are defective for Met-tRNA(i)Met binding to 40S subunits, and we also observed defective 40S binding of mRNA, eIFs 1 and 5 and eIF3 itself in these extracts. We could rescue 40S binding of Met- tRNA(i)Met and mRNA, and translation of luciferase mRNA, in a prt1-1 extract almost as well with purified PN2 subcomplex as with five-subunit eIF3, whereas the P45 subcomplex was nearly inactive. Thus, several key functions of eIF3 can be carried out by the PRT1-TIF32-NIP1 subcomplex.

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.

Crystallization and preliminary X-ray analysis of Escherichia coli methionyl-tRNAMet(f) formyltransferase complexed with formyl-methionyl-tRNAMet(f).

The structure of methionyl-tRNAfMet(f) formyltransferase from E. coli, a monomeric protein of 34 kDa, has previously been determined at 2.0 A resolution. In the present work, this enzyme was crystallized as a complex with its macromolecular product, the initiator formyl-methionyl-tRNAfMet(f) (25 kDa). Polyethylene glycol 5000 monomethylether was used as a precipitating agent. The crystals are orthorhombic and have unit-cell parameters a = 201.7, b = 68.1, c = 86.4 A. They belong to space group P21212 and diffract to 2.8 A resolution. The structure is being solved with the help of a mercury derivative.

Importance of structural features for tRNA(Met) identity.

We showed previously that the tRNA tertiary structure makes an important contribution to the identity of yeast tRNA(Met) (Senger B, Aphasizhev R, Walter P, Fasiolo F, 1995, J Mol Biol 249:45-58). To learn more about the role played by the tRNA framework, we analyzed the effect of some phosphodiester cleavages and 2'OH groups in tRNA binding and aminoacylation. The tRNA is inactivated provided the break occurs in the central core region responsible for the tertiary fold or in the anticodon stem/loop region. We also show that, for tRNA(Met) to bind, the anticodon loop, but not the anticodon stem, requires a ribosephosphate backbone. A tertiary mutant of yeast tRNA(Met) involving interactions from the D- and T-loop unique to the initiator species fails to be aminoacylated, but still binds to yeast methionyl-tRNA synthetase. In the presence of 10 mM MgCl2, the mutant transcript has a 3D fold significantly stabilized by about 30 degrees C over a wild-type transcript as deduced from the measure of their T(m) values. The k(cat) defect of the tRNA(Met) mutant may arise from a failure to overcome an increase of the free energetic cost of distorting the more stable tRNA structure and/or a tRNA based MetRS conformational change required for formation of transition state of aminoacylation.

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.

Topography of the Escherichia coli initiation factor 2/fMet-tRNA(f)(Met) complex as studied by cross-linking.

trans-Diamminedichloroplatinum(II) was used to induce reversible cross-links between Escherichia coli initiation factor 2 (IF-2) and fMet-tRNA(f)(Met). Two distinct cross-links between IF-2 and the initiator tRNA were produced. Analysis of the cross-linking regions on both RNA and protein moieties reveals that the T arm of the tRNA is in the proximity of a region of the C-terminal domain of IF-2 (residues Asn611-Arg645). This cross-link is well-correlated with the fact that the C-domain of IF-2 contains the fMet-tRNA binding site and that the cross-linked RNA fragment precisely maps in a region which is protected by IF-2 from chemical modification and enzymatic digestion. Rather unexpectedly, a second cross-link was characterized which involves the anticodon arm of fMet-tRNA(f)(Met) and the N-terminal part of IF-2 (residues Trp215-Arg237).

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.

Primary and higher order structures of nematode (Ascaris suum) mitochondrial tRNAs lacking either the T or D stem.

By fractionation using polyacrylamide gel electrophoresis and/or a preparative hybrid selection method employing solid-phase DNA probes, we prepared and characterized mitochondrial tRNAs from the body wall muscle of Ascaris suum, all of which are thought to lack either the T stem or the D stem from their gene sequences (Okimoto, R., and Wolstenholme, D. R. (1990) EMBO J. 10, 3405-3411). Some of the partially purified tRNAs were appreciably aminoacylated with an extract of A. suum mitochondria. The three species sequenced had CCA sequence at their 3'-ends, and tRNA(Met) had 5-formylcytidine at the anticodon first position, a new modified nucleoside found at the same position of bovine mitochondrial tRNA(Met) (Moriya, J., Yokogawa, T., Wakita, K., Ueda, T., Nishikawa, K., Crain, P. F., Hashizume, T., Pomerantz, S. C., McCloskey, J. A., Kawai, G., Hayashi, N., Yokoyama, S., and Watanabe, K. (1994) Biochemistry 33, 2234-2239). Enzymatic probing of these tRNAs supported the secondary structural model proposed by Okimoto and Wolstenholme in the reference cited above. Chemical probing of tRNA(Phe) demonstrated the existence of tertiary interactions between the (T arm-variable loop)-replacement loop and the D arm. The results suggest that these tertiary interactions enable the bizarre tRNAs of nematode mitochondria to maintain an L-shape-like structure in order to function in the nematode mitochondrial translation system.

A novel modified nucleoside found at the first position of the anticodon of methionine tRNA from bovine liver mitochondria.

Methionine tRNA was purified from bovine liver mitochondria, and its nucleotide sequence was determined. The tRNA possesses only three posttranscriptionally modified nucleosides, two pseudouridines in the anticodon and T stems and a previously unknown nucleoside specified by the gene sequence as cytidine, in the first position of the anticodon. Structure analysis of the anticodon nucleoside by mass spectrometry revealed a molecular mass 28 Da greater than that of cytidine, and unmodified ribose, with substitution at C-5 implied by hydrogen-deuterium exchange experiments. Proton NMR of the intact tRNA showed presence of a formyl moiety, thus leading to the candidate structure 5-formylcytidine (f5C), not a previously known compound. The structure assignment was confirmed by chemical synthesis and comparison of data from combined HPLC/mass spectrometry and proton NMR for the natural and synthetic nucleosides. The potential function of f5C in the tRNA(Met) anticodon is discussed with regard to codon-anticodon interactions.

Separation of formyl-methionyl transfer RNA, methionyl transfer RNA, and transfer RNAfmet using mixed-mode high-performance liquid chromatography on C6-modified aminopropylsilyl-hypersil.

Preparative amounts of formyl-methionyl-tRNAfmet, methionyl-tRNAfmet, and tRNAfmet were separated from each other with baseline resolution in 30 min using mixed-mode HPLC on hexanoic anhydride-modified aminopropylsilyl-Hypersil 2. Pure tRNAfmet was aminoacylated with [35S]methionine in the presence or absence of a formyl donor and was immediately fractionated on the column. Two isoacceptors, tRNA1fmet and tRNA2fmet, as well as aminoacyl-tRNA synthetases were clearly separated from each other. The purified f[35S]-methionyl-tRNA was biologically active in that as much as 98% could be bound to ribosomes in response to AUGUAA in vitro. Formyl-methionine was released from this complex by the action of termination factor and greater than 92% of bound formyl-methionine was released by puromycin.

Effect of 3-deazaadenosine on methionine metabolism in isolated perfused livers.

The effect of the purine analog 3-deazaadenosine (dzAdo) on the metabolism of sulfur-containing compounds was examined in hepatocytes. The uptake of exogenous methionine by the liver was not affected by the addition of dzAdo to the perfusate, while the intracellular concentrations of S-adenosyl-L-methionine (AdoMet) and S-adenosyl-L-homocysteine (AdoHcy) continued to increase as long as exogenous methionine was available. In addition, large amounts of 3-deazaadenosyl-L-homocysteine (dzAdoHcy) accumulated in the cell. The specific radioactivity of the carbon chain of dzAdoHcy was the same as that of AdoMet and AdoHcy. Consequently, an equivalent amount of homocysteine (Hcy) must have been generated via hydrolysis of AdoHcy. Free Hcy could not be detected either in the tissue or perfusate when dzAdo was present, while Hcy was excreted into the perfusate by control livers. Consequently, the AdoHcy and DzAdoHcy that accumulate in the cell not only function as inhibitors of methylation reactions, but serve as a trap for Hcy. This could result in methionine starvation and hence, inhibition of protein synthesis.

Total chemical synthesis of a 77-nucleotide-long RNA sequence having methionine-acceptance activity.

Chemical synthesis is described of a 77-nucleotide-long RNA molecule that has the sequence of an Escherichia coli Ado-47-containing tRNA(fMet) species in which the modified nucleosides have been substituted by their unmodified parent nucleosides. The sequence was assembled on a solid-phase, controlled-pore glass support in a stepwise manner with an automated DNA synthesizer. The ribonucleotide building blocks used were fully protected 5'-monomethoxytrityl-2'-silyl-3'-N,N-diisopropylaminophosphoram idites. p-Nitro-phenylethyl groups were used to protect the O6 of guanine residues. The fully deprotected tRNA analogue was characterized by polyacrylamide gel electrophoresis (sizing), terminal nucleotide analysis, sequencing, and total enzyme degradation, all of which indicated that the sequence was correct and contained only 3-5 linkages. The 77-mer was then assayed for amino acid acceptor activity by using E. coli methionyl-tRNA synthetase. The results indicated that the synthetic product, lacking modified bases, is a substrate for the enzyme and has an amino acid acceptance 11% of that of the major native species, tRNA(fMet) containing 7-methylguanosine at position 47.