Radioactively labelled amino acids were purchased from Amersham-Buchler (Braunschweig, Germany) and restriction enzymes from New England Biolabs (Beverly, MA). All other chemicals were pro analysi grade and purchased from Merck (Darmstadt, Germany).

Escherichia coli MRE600rif ( 12 ) is a strain which: (i) is adaptable to growth on deuterated media ( 13 ); (ii) contains low levels of ribonuclease I activity ( 14 ); and (iii) tolerates high doses of rifampicin. This strain was used for all cultivations in deuterated media. As a reference strain HB101 ( 15 ) containing the same plasmids as the MRE600rif derivatives was grown in protonated LB medium (see below).

Plasmids

The plasmid pPhe was previously described as pPP15 ( 16 ). It is a pBR322 derivative containing the phe V gene, which codes for tRNA ^Phe , under the control of the natural P2 promoter, which is the second of a tandem promoter pair. Plasmid pMet ( 17 ) was a kind gift of U. RajBhandary. It carries the gene for roman {{t R N A} sub f sup {M e t}} behind the natural promoter. The E.coli roman {{t R N A} sub f sup {M e t}} gene cloned into the plasmid plppMet (previously described as pBStRNAMetfY; 18 ) is under the control of a synthetic lipoprotein promoter lpp ( 19 ) and has several modifications at the level of the 5' maturation sequence allowing maturation by RNase P in vivo . The tRNA transcription region is terminated by the strong terminator of the rrn C operon. This construct allows very high levels of overexpression. Plasmids pBR322 (Boehringer, Mannheim, Germany) and pBluescript (Stratagene, La Jolla, CA) were used for cloning purposes and as control plasmids (minus tRNA gene).

Cloning of tRNA genes

Plasmids pPhe, pMet and plppMet were used directly for transformation of HB101 or MRE600rif respectively by electroporation using a BioRad gene pulser. Plasmid plppMetPhe was constructed by subsequent cloning of the Pst I- Hpa I fragment of pPhe (the fragment contains the tRNA ^Phe gene) and the Xho I- Hin dIII fragment of plppMet (containing the cassette lpp promoter-roman {{t R N A} sub f sup {M e t}} gene- rrn C terminator) into the respective restriction sites within pBluescript. The successful cloning of both tRNA genes were confirmed by sequencing using an automated laser fluorescent (ALF) DNA sequencer (Pharmacia, Uppsala, Sweden).

Growth of cells overexpressing specific tRNAs

Cells were grown in 50 ml batch cultures in Luria-Bertani (LB) medium (1% bactotryptone, 0.5% yeast extract, 0.5% NaCl in H ₂ O) or M3 medium [0.15% NaCl, 0.2% (NH ₄ ) ₂ SO ₄ , 0.01% MgCl ₂ , 0.65% KH ₂ PO ₄ , 1% K ₂ HPO ₄ , pH 7.2] with 0.5% each of protonated acetate and succinate as carbon sources. M3 medium was either made with H ₂ O or D ₂ O. Each medium was supplemented with 200 [mu]g/ml ampicillin (Boehringer) as a selection marker for pMet, plppMet and plppMetPhe or 12 [mu]g/ml tetracyclin (Boehringer) for pPhe respectively. Large-scale fermentation in fully deuterated medium was performed using fed batch cultivation with a computer controlled pregiven growth rate as described previously ( 12 ), except that 200 mg/l ampicillin or 12 mg/l tetracyclin was added at the beginning of cultivation to ensure maintenance of the plasmids. A mixture of 0.67 M succinic and 1.95 M acetic acids served as carbon source. Due to very poor growth the succinic acid was increased to 0.77 M while cultivating the cells carrying plppMetPhe. The critical growth rates ([mu] _crit ) corresponding to the condition of substrate limitation of recombinant strains were significantly reduced ([mu] _crit = 0.05, 0.035 and 0.025/h for the cells carrying pMet, pPhe and plppMetPhe respectively) compared with the host cells of E.coli MRE600rif without a plasmid ([mu] _crit = 0.08/h). The biomass yield coefficient (mol C biomass formed/mol C substrate utilized) was 0.25 for cells carrying pPhe and 0.30 for cells carrying pMet and plppMetPhe and cells without a plasmid.

Determination of the cellular tRNA content

For analytical assays, a method for rapid preparation of tRNA ^bulk was adapted from the protocol of Xue et al. ( 20 ). About 0.2 g wet wt E.coli cells were lysed in 1.5 ml 50% phenol in 10 mM Tris-HCl, pH 6.0 (0^oC), 10 mM MgCl ₂ by shaking for 45 min at 4^oC. The aqueous phase was extracted with phenol twice and once with chloroform/isomayl alcohol (24:1). rRNAs were precipitated on ice by adjusting the NaCl concentration to 2 M. The tRNA-containing supernatant was precipitated with ethanol and the pellet was resuspended in a suitable amount of water (~200 [mu]l).

Aminoacylation capacity was checked in an assay system with 50 mM HEPES-KOH, pH 7.8 (0^oC), 10 mM MgCl ₂ , 100 mM KCl, 4 mM [beta]-mercaptoethanol, supplemented with an optimized amount of tRNA-free S-100 post-ribosomal supernatant ( 21 ) as a source of synthetases. An aliquot of 0.2 A ₂₆₀ units tRNA ^bulk preparation, 500 pmol [ ¹⁴ C]Met or [ ¹⁴ C]Phe respectively and 500 pmol [ ³ H]Leu for normalization were incubated for 15 min at 37^oC and afterwards precipitated with ice-cold TCA, filtered through a glassfibre filter (no. 6; Schleicher & Schüll, Dassel, Germany) and counted according to Rheinberger et al . ( 21 ).

Large-scale purification of ^D tRNAs

Escherichia coli cells grown in fully deuterated medium ( 12 ) were taken to prepare tight coupled ribosomes according to Bommer et al . ( 22 ). ^D tRNA ^bulk was isolated from the S-100 fraction by stepwise elution from a DEAE-cellulose 52 (Whatman, Springfield Mill) column ( 23 ). ^D tRNA ^Met was separated from ^D tRNA ^Phe by chromatography of the tRNA on a large-scale DEAE-Sephadex A-50 column (40 * 1200 mm) at 4^oC using a linear gradient (7 l) formed from buffer A [20 mM HEPES-KOH, pH 7.5 (0^oC), 10 mM magnesium chloride, 4 mM [beta]-mercaptoethanol, 250 mM sodium chloride] and buffer B [20 mM HEPES-KOH, pH 7.5 (0^oC), 10 mM magnesium chloride, 4 mM [beta]-mercaptoethanol and 500 mM sodium chloride] ( 24 ). The fractions were checked by an analytical aminoacylation assay for their content of either ^D tRNA ^Phe or ^D tRNA ^Met . Fractions containing one of the two tRNAs were combined and precipitated with ethanol.

The two tRNAs were further purified by HPLC on a preparative Vydac Silica Gel 214 TP-510 column (250 * 10 mm; Vydac). Chromatography was performed as described ( 20 ) except that the buffer system was varied slightly [buffer A, 20 mM ammonium acetate, pH 5.0, 8 mM magnesium chloride, 1 M sodium formiate; buffer B, 20 mM ammonium acetate, pH 5.0, 10% (w/v) methanol].

The ^D tRNA ^Phe fraction was then aminoacylated and N -acetylated ( 21 ). For aminoacylation and formylation of the ^D tRNA ^Met fraction, the tRNA was incubated in 20 mM HEPES-KOH, pH 7.5 (0^oC), 7 mM MgCl ₂ , 150 mM KCl, 3 mM ATP, 4 mM [beta]-mercaptoethanol for 30 min at 37^oC together with a 3-fold excess of [ ¹⁴ C]Met, an optimized amount of tRNA-free S-100 and a 700 molar excess of formyl donor (folinic acid; Serva, Heidelberg, Germany). The reaction was stopped by addition of a 1/10 volume of 3 M sodium acetate, pH 5.0. After two phenol extractions and precipitation with ethanol the remaining formyl donor was separated from the f-[ ¹⁴ C]Met-{roman {{"" sup D} {{t R N A} sub f sup {M e t}}}} by gel filtration using a NAP-25 disposable column (Pharmacia, Uppsala, Sweden).

A second HPLC chromatography on the Vydac Silica Gel column using the same conditions as described above but on the aminoacyl-tRNAs (Ac-[ ¹⁴ C]Phe- ^D tRNA ^Phe and f-[ ¹⁴ C]Met-{roman {{"" sup D} {{t R N A} sub f sup {M e t}}}}) yielded essentially pure tRNAs (>1300 pmol/A ₂₆₀ unit).

For isolation of highly purified deacylated tRNAs the N -aminoacyl-tRNAs can be deacylated using purified RNase-free peptidyl-tRNA hydrolase (PTH, see next section). The enzyme works quite efficiently on Ac-Phe-tRNA in 20 mM HEPES-KOH, pH 7.6 (0^oC), 10 mM MgCl ₂ , 4 mM [beta]-mercaptoethanol within 10 min at 30^oC, whereas the deacylation of f-Met-tRNA needs a basic milieu (pH 8.0) and a longer incubation time (20 min, 30^oC) to achieve quantitative deacylation ( 25 ). The optimal ratio of enzyme to tRNA depends on its activity and has to be optimized for each preparation (it is in the region of 150 ng enzyme/pmol tRNA).

Purification of peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase (PTH, EC 3.1.1.29) was purified from E.coli . A sample of 100 ml S-100 fraction from E.coli was fractionated by stepwise precipitation with 32 and 72.5% (w/v) (NH ₄ ) ₂ SO ₄ . The second pellet (72.5%) was dissolved in 10 mM KH ₂ PO ₄ , pH 6.4, and dialysed three times each for 5 h against 100 volumes of the same buffer. The protein solution (~150 ml) was then applied to a CM-cellulose column (60 ml) equilibrated with the same buffer at a flow rate of 0.5 ml/min. The unbound and weakly bound material was washed out with 250 ml 10 mM KH ₂ PO ₄ , pH 6.4, 50 mM NaCl. The enzyme activity was eluted with 500 mM NaCl, concentrated and desalted by ultrafiltration using Centricon^tm 10 microconcentrators (Amicon) and stored in 10 mM KH ₂ PO ₄ , pH 6.4, with 5% (w/v) glycerol at -80^oC.

Gel electrophoresis of tRNA

Approximately 0.2 A ₂₆₀ units of RNA were analysed on a 15% denaturing polyacrylamide gel (acrylamide:bis-acrylamide 38:2% w/v, 7 M urea). The gel was stained with 0.1% (w/v) toluidine blue.

Ribosome binding assay

Preparation of E.coli 70S ribosomes and site-specific binding of tRNAs to 70S particles followed the protocols described ( 22 ). The ribosomes were programmed with a 5-fold excess of a 46 nt mRNA analogue containing unique codons for Met and Phe in the middle (MF-mRNA; 26 ) and protonated or deuterated Ac-[ ¹⁴ C]Phe-tRNA (up to a 5-fold molar excess over ribosomes) was added either directly for P site binding or after pre-incubation with a 2-fold molar excess of {roman {{"" sup D} {{t R N A} sub f sup {M e t}}}} for A site binding of Ac-[ ¹⁴ C]Phe-tRNA. The extent of binding was determined by nitrocellulose filtration and location to the A or P site was distinguished by puromycin reactivity ( 22 ).

RESULTS AND DISCUSSION

For the structural investigation of functional ribosomal complexes with neutron scattering methods it was necessary to develop an efficient method to obtain specific highly purified 100% deuterated tRNAs from cell extracts. To optimize the tRNA yield from deuterated cells MRE600rif was transformed with tRNA overexpression constructs and grown in batch or fed batch cultivation ( 12 ). The tRNAs from these cells were purified in a multistep protocol and the quality of the resulting specific tRNAs was determined.

Here we describe the large-scale preparation for two deuterated tRNAs, tRNA ^Phe and roman {{t R N A} sub f sup {M e t}}. Those two deuterated tRNAs were required in large amounts to isolate ribosomal elongation complexes for neutron scattering analysis. However, the protocol can be easily adapted for preparation of other tRNA species.

Evaluation of different constructs for overexpression of tRNAs

Three prerequisites have to be taken into account in the design of overexpression constructs for large-scale preparation of fully deuterated tRNAs. (i) Since growing cells in fully deuterated media is very expensive, it is important that ribosomes and tRNAs can be prepared from a single batch. Because in vitro ribosome activity drops when protein synthesis is hampered in vivo , the level of tRNA overexpression should not interfere with cellular protein synthesis. (ii) Proton incorporation into tRNAs by metabolism-inducing agents like IPTG should be avoided, as well as changes in growth conditions due to a temperature shift. Therefore, overexpression should be constitutive. Consequently, the use of strong induction systems ( 27 ) or runaway replication plasmids ( 28 ), in which overproduction of a single tRNA kills the cell, are not suitable for overproducing deuterated tRNAs, although the yield of specific tRNAs is very high in these systems (up to 70% of total tRNA). (iii) A strain capable of growing in deuterated medium is needed. The most suitable strain for this purpose is MRE600, which can be grown in fully deuterated medium to very high densities ( 12 ).

In this study we tested four different constructs (Fig. 1 ) for their effect on tRNA content in deuterated cells. One construct contains the gene for tRNA ^Phe under the control of its natural promoter P2 (pPhe), two constructs harbour genes for roman {{t R N A} sub f sup {M e t}}, either with the natural promoter (pMet) or an artificial constitutive promoter of the lipoprotein Lpp (plppMet), and one construct contains both tRNA genes (plppMetPhe). The last plasmid was constructed by cloning the tRNA ^Phe gene (the Pst I- Hpa I fragment of pPhe) into the respective restriction sites of the high copy number plasmid pBluescript. Subsequently the expression cassette for roman {{t R N A} sub f sup {M e t}} from plasmid plppMet (a Xho I- Hin dIII fragment comprising the lpp promoter, the gene for roman {{t R N A} sub f sup {M e t}} and the rrn C terminator) was inserted, yielding plasmid plppMetPhe. The presence of both tRNA genes in the plasmid construct was shown by sequencing the region of the plasmid using an automated DNA sequencer (data not shown).

Figure 1 . Characterization of the tRNA-overexpressing plasmids used. ( A ) The tRNA gene-containing regions. ( B ) The plasmids: 1, pPhe; 2, pMet; 3, plppMet; 4, plppMetPhe. The tRNA ^Met gene codes for initiator tRNA _f ^Met in all cases.

The overexpression level of the different plasmid constructs was analysed for different strains (MRE600rif versus HB101) in minimal and rich medium (M3 versus LB) with or without deuteration (M3/D ₂ O versus M3/H ₂ O). To compare data from different tRNA preparations the cellular levels of chargeable tRNA ^Phe or tRNA ^Met were normalized with respect to chargeable tRNA ^Leu . The level of tRNA ^Leu is assumed to be largely independent of the overexpression constructs. Representative results for batch cultivations are shown in Figure 2 .

Figure 2 . Expression of tRNA ^Met ( A ) or tRNA ^Phe ( B ) in different strains and under different growth conditions. The respective chargeable tRNA content is normalized to the chargeable cellular content of tRNA ^Leu .

In general the overexpression level of tRNAs tested in protonated rich medium was higher in HB101 (white columns) compared with MRE600rif (hatched columns), but it was still significant in MRE600rif, the strain used for growth in deuterated minimal medium. The level of tRNA ^Met overproduction (Fig. 2 A) from its natural promoter (pMet) was not very high (3- to 4-fold) under the different conditions tested. For this reason {roman {{"" sup D} {{t R N A} sub f sup {M e t}}}} expression was tested with the gene under the control of the strong lpp promoter (plppMet and plppMetPhe respectively). These constructs worked quite efficiently in HB101 (~15-fold overexpression), but were less active in MRE600rif, where overexpression was only ~5-fold.

Changing to minimal medium (solid black columns) slightly increased the overexpression level for roman {{t R N A} sub f sup {M e t}}, whereas tRNA ^Phe overexpression from pPhe or plppMetPhe was increased by a factor of two to three upon a shift to minimal medium (Fig. 2 B). In contrast, the switch from H ₂ O to D ₂ O in minimal medium had almost no effect on overexpression (data not shown).

Under the conditions of fed batch cultivation, which allows growth of cells to very high densities, overexpression was clearly reduced (Fig. 3 ; note the different scales on the y -axis in Figs 2 and 3 ). Nevertheless, tRNA ^Phe content of the cells was 3-fold higher in the presence of pPhe or 7-fold higher when expressed from plppMetPhe. Thus the overexpression plasmids had clear effects, since the higher ratio of tRNA ^Phe to other tRNAs increased the yield and facilitated purification of deuterated tRNA ^Phe . In the case of {roman {{"" sup D} {{t R N A} sub f sup {M e t}}}} little overexpression was observed (up to 2-fold).

Figure 3 . Expression of tRNA ^Phe and tRNA ^Met in MRE600rif grown in fully deuterated minimal medium using the fed batch cultivation technique. The tRNA content is normalized to the cellular content of tRNA ^Leu .

Purification of deuterated tRNAs

The large-scale tRNA purification method described here combines well-known classical chromatographic techniques with the powerful tool of HPLC.

First, tRNA ^bulk has to be isolated from the S-100 fraction, which contains more or less all cellular proteins and small nucleic acid compounds. Both tRNAs and proteins bind tightly to a DEAE-cellulose 52 matrix at low salt concentration (~150 mM NaCl). In a batch-like operation tRNA ^bulk separates from the cellular protein fraction. While the proteins elute at salt concentrations <250 mM NaCl, the tRNAs remain attached to the matrix until 300 mM NaCl. The tRNA ^bulk preparation contains in addition some other small ribonucleic acid components, such as 5S rRNA. The preparation of 100 g E.coli cells grown in fully deuterated minimal medium ( 12 ) yields ~2000 A ₂₆₀ units ^D tRNA ^bulk .

During the late sixties several groups tried to isolate specific tRNAs in large amounts by chromatographic methods. Ion exchange chromatography followed by a hydrophobic column, such as BD-cellulose ( 29 , 30 ), was found to be a useful combination. In principal we followed this isolation procedure, using in the first step a classical DEAE-Sephadex A50 ion exchanger ( 24 ), afterwards switching to a reversed phase HPLC system.

After the DEAE-Sephadex A50 ion exchange column analytical aminoacylation shows a nearly complete separation of ^D tRNA ^Met and ^D tRNA ^Phe in the wide ^D tRNA ^bulk signal (Fig. 4 ). A sharp selection of the fractions to be combined yields a 2- to 6-fold enrichment of ^D tRNA ^Met and ^D tRNA ^Phe (Table 1 ).

Figure 4 . Absorption profile at 260 nm of ^D tRNA ^bulk applied to a DEAE-Sephadex A50 column and distribution of tRNA ^Met and tRNA ^Phe in the linear NaCl gradient.

To obtain further purification the tRNAs are loaded on an HPLC reversed phase system. A silica gel-based C ₄ column (Vydac) in combination with a reversed salt gradient ( 20 , with minor changes) gives the best resolution for a population of different tRNAs. The elution pattern of a ^D tRNA ^Phe -rich fraction and a ^D tRNA ^Met -rich fraction differ dramatically. Whereas ^D tRNA ^Phe elutes at a higher concentration of buffer B with only a little contamination by other tRNAs (see Fig. 5 A), ^D tRNA ^Met elutes at the beginning of the gradient within the major peak of the tRNAs (Fig. 5 B). In the middle of the ^D tRNA ^Phe signal a maximal purity of 1350 pmol/A ₂₆₀ unit could be obtained by restrictive selection of fractions. This means enrichment by a factor of 15 compared with the ^D tRNA ^Phe fraction after the DEAE-Sephadex A50 column (or 50-fold relative to ^D tRNA ^bulk ; if tRNA ^Phe is overexpressed these factors decrease due to the higher starting value) and for many purposes this ^D tRNA ^Phe can be used directly as deacylated tRNA. If the ^D tRNA ^Phe is to be aminoacylated a second HPLC step follows after aminoacylation (see next paragraph) and thus the yield of deacyl- ^D tRNA ^Phe may be increased at the cost of purity by a less restrictive selection in this step (Table 1 ). In contrast, enrichment of ^D tRNA ^Met in that single step by a factor of two to three is rather low. Nevertheless, the chromatographic step is important for ^D tRNA ^Met purification, since it eliminates all components that elute at a higher concentration of buffer B. This is a prerequisite for further purification.

Figure 5 . Absorption profile at 260 nm of an HPLC preparation of deacylated ^D tRNA ^Phe ( A ) and deacylated ^D tRNA ^Met ( B ) and of the tRNAs after charging and acetylation or formylation resulting in Ac[ ¹⁴ C]Phe- ^D tRNA ^Phe ( C ) or formyl-Met- ^D tRNA _f Met ( D ) respectively. The acceptor activity of each fraction was determined before the fractions were pooled, as indicated by the shadowed area. The gradient is indicated by a dashed line.

Aminoacylation and N -acetylation of a tRNA changes its behaviour on a reversed phase column drastically. The small substituent gives the tRNA a more hydrophobic character, resulting in a later elution in the same gradient compared with deacylated tRNA. This phenomenon is used for further purification of the ^D tRNAs.

Charging and acetylation efficiency is ~70% in the case of ^D tRNA ^Phe , which means that 70% of the deacylated ^D tRNA ^Phe isolated so far can be transformed to Ac-Phe- ^D tRNA ^Phe , whereas aminoacylation and formylation of {roman {{"" sup D} {{t R N A} sub f sup {M e t}}}} is more or less quantitative (>90% of the input material can be obtained as f-Met-{roman {{"" sup D} {{t R N A} sub f sup {M e t}}}} ⁾ .

The charged and N -blocked tRNAs (Ac-Phe- ^D tRNA ^Phe and f-Met-{roman {{"" sup D} {{t R N A} sub f sup {M e t}}}} ⁾ elute at a higher buffer B concentration in a second reversed phase HPLC step, away from all contaminants (Fig. 5 C and D). The quality of the purified tRNAs was satisfactory; 1400-1600 pmol/A ₂₆₀ unit for Ac-Phe- ^D tRNA ^Phe and 1300-1400 pmol/A ₂₆₀ unit for f-Met-{roman {{"" sup D} {{t R N A} sub f sup {M e t}}}} (Table 1 ), corresponding to a 40- to 50-fold enrichment relative to ^D tRNA ^bulk.

Quality of the purified deuterated tRNAs

The deuterated tRNAs obtained using the overexpression strains and the purification protocol described here are of excellent quality, as checked by three different sets of experiments. First, homogeneity of the isolate was controlled either by PAGE or by analytical HPLC analysis, where a single peak was observed (not shown). For biological activity interaction with synthetase was tested in a charging assay. Finally, interaction with the ribosome was analysed in a site-specific binding assay.

Table 1 . Characterization of fractions in a ^D tRNA purification protocol with overexpressed ^D tRNA ^{Phe D} tRNA ^Phe fraction -

Purification step	Fraction	D tRNA Met fraction
		Yield	Chargeable	Enrichment	Yield	Yield	Chargeable	Enrichment	Yield
		with Met	factor			with Phe	factor
		(A 260 units)	(pmol/A 260 unit)	(relative to D tRNA bulk )	(%)	(A 260 units)	(pmol/A 260 unit)	(relative to D tRNA bulk )	(%)
Control	H tRNA bulk	-	83	-	-	-	39
DEAE-cellulose	D tRNA bulk	(4500)	24	1	100	(4500)	67	1	100
52 batch column			(35)				(27)
DEAE-Sephadex A50 column	Enriched D tRNA bulk fractions	640	120 (207)	5	71	1580	150 (94)	2.2	79
First reversed	Deacylated D tRNA	145	312	13	42	52	1092	16	25
phase HPLC			(427)				(564)
Charging and	N-blocked	17	1320	55	21	13	1540	23	13
second HPLC	aminoacyl- D tRNA		(1360)				(1430)
PTH treatment/	Deacylated D tRNA		1010	42	14		Not done
deacylation			(965)

The table shows a typical example using a ^D tRNA ^bulk fraction obtained from cells with the plasmid pPhe. The starting material of 4500 A ₂₆₀ units ^D tRNA ^bulk was taken as 100%. At this stage ^D tRNA ^Met and the overexpressed ^D tRNA ^Phe species were not separated. (For comparison the respective charging values for a ^D tRNA preparation from cells without the overexpression plasmid are given in parentheses.)

(i) Deacylated deuterated tRNAs are fully active in interaction with their corresponding aminoacyl synthetases: ^D tRNA ^Phe could be charged to ~1350 pmol radioactive [ ¹⁴ C]Phe/A ₂₆₀ unit when the middle fraction of the corresponding preparative HPLC peak (see Fig. 5 A) was taken. In contrast, purification of highly purified deacylated {roman {{"" sup D} {{t R N A} sub f sup {M e t}}}} ^D required charging and deacylation procedures, since deacylated {roman {{"" sup D} {{t R N A} sub f sup {M e t}}}} could not be separated in a single HPLC step. {roman {{"" sup D} {{t R N A} sub f sup {M e t}}}} could be recharged to ~1000 pmol/A ₂₆₀ unit after deacylation using PTH treatment (see Table 1 ). In both cases charged tRNA could be separated from uncharged material, yielding an even better quality.

(ii) A prerequisite for the structural investigation of ribosomal complexes is a purity of the ^D tRNAs of >1000 pmol/A ₂₆₀ unit chargeable with its cognate amino acid. A purity of >= 1000 pmol/A ₂₆₀ unit is sufficient for preparation of specific ribosomal complexes, since the in vitro accuracy of the decoding process ensures specificity of tRNA binding. The purity obtained for deuterated tRNAs were higher than this minimal value. Deuterated tRNAs showed normal behaviour in binding to ribosomes. In 70S ribosomes programmed with heteropolymeric MF-mRNA deacylated {roman {{"" sup D} {{t R N A} sub f sup {M e t}}}} blocked the ribosomal P site quantitatively. More than 80% of Ac-Phe-tRNA bound in the second binding step was directed to the ribosomal A site. Deuterated Ac-[ ¹⁴ C]Phe-tRNA was as active as its protonated counterpart (Fig. 6 ) in binding to the ribosomal P site (binding to ribosomes programmed with MF-mRNA saturates at 0.6 molecules/ribosome, which means that 60% of the 70S ribosomes carried an Ac-[ ¹⁴ C]Phe-tRNA in the P site). For A site binding the activity of deuterated Ac-[ ¹⁴ C]Phe-tRNA was ~90% compared with that of protonated material (binding of 0.45 molecules/ribosome in comparison with 0.5 for the protonated counterpart).

Figure 6 . Binding of protonated or deuterated Ac[ ¹⁴ C]Phe-tRNA ^Phe to the ribosomal P and A sites respectively.

ACKNOWLEDGEMENTS

We thank Dr M. Springer for help and discussion. We are grateful to Dr U. L. RajBhandary for providing us with the roman {{t R N A} sub f sup {M e t}} plasmid and for advice and suggestions on the manuscript. Work in his laboratory is supported by grant GM17151 from the National Institutes of Health. This work was supported by grant CIPA-CT93-0158(DG12HSMU) from the European Commission.

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