New chromatographic and biochemical strategies for quick preparative isolation of tRNA

Abstract

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.

Figure 1

Isolation of tRNA^Phe from tRNA^bulk of E.coli. (A) RP-HPLC. Aliquots of 20 A₂₆₀ units of tRNA^bulk dissolved in 50 µl of water were applied to a Delta Pak C4 15 µ 300 Å 3.9 × 300 mm column (Waters) equilibrated in buffer A (20 mM ammonium acetate pH 5.25, 10 mM magnesium acetate, 400 mM NaCl). Retained material was eluted with the indicated gradient (dotted line) of buffers A and B (60% methanol in buffer A). The shaded region under the chromatographic profile at 260 nm (line) indicates the position where Phe acceptor activity elutes. (B) Hydrophobic chromatography. Aliquots of 6000 A₂₆₀ units of E.coli tRNA^bulk were applied to phenyl-Sepharose (2.3 × 60 cm) equilibrated in 50 mM ammonium acetate pH 5.3, 10 mM magnesium acetate, 1.5 M ammonium sulfate. Adsorbed material was eluted with a negative ammonium sulfate gradient from 1.4 to 0.9 M (dotted line). The shaded region under the chromatographic profile at 280 nm (line) indicates the location of Phe acceptor activity. (C) RP-HPLC chromatography [as described in (A)] of pooled material (250 A₂₆₀ units) from the shaded region shown in (B). (D) Rechromatography of material recovered from the shaded peak in (C) (75 A₂₆₀ units). Phenylalanine acceptor activity was determined as described in Materials and Methods. The insert in (B) shows the electrophoretic pattern of the pool of fractions from hydrophobic chromatography containing tRNA^Phe acceptor activity (shaded region) and in (C) the insert shows the electrophoretic analysis of purified tRNA^Phe.

Figure 2

Isolation of tRNA^Ile from tRNA^bulk of E.coli. (A) Aliquots of 20 A₂₆₀ units of tRNA^bulk from E.coli were submitted to RP-HPLC as described in Figure 1A using an optimized gradient of buffer B (dotted line) for separation of Ile acceptor activity. The shaded region under the chromatographic profile at 260 nm (line) indicates the position where isoleucine acceptor activity elutes. (B) RP-HPLC Separation of tRNA^bulk after preparative aminoacylation with [¹⁴C]isoleucine (diluted to 9 c.p.m./pmol with non-labeled isoleucine) as indicated in Materials and Methods. Samples (5 µl) were analyzed for the presence of [¹⁴C]Ile-tRNA^Ile via liquid scintillation counting (circle). The fractions comprising the radioactivity peak were pooled and analyzed by denaturing PAGE (see insert). (C) Recovery of tRNA^Ile after deacylation via RP-HPLC. The isoleucine acceptor activity of pooled material (shaded region) was determined by analytical aminoacylation using [¹⁴C]isoleucine (730 c.p.m./pmol) and submitted to electrophoretic analysis (insert).

Figure 3

Isolation of tRNA_i^Met from S.cerevisiae. (A) Analysis of methionine acceptor activity in a RP-HPLC separation of tRNA^bulk components. Aliquots of 10 A₂₆₀ units of tRNA^bulk were applied to a Nucleosil C4 7 µ 300 Å 2.4 × 250 mm column (Macherey-Nagel) equilibrated with buffer A and eluted with the indicated gradient of buffer B (dotted line) at a flow rate of 0.5 ml/min. The tRNA content of the fractions (500 µl each) was precipitated with ethanol and redissolved in 300 µl of water. The methionine acceptor activity of 20 µl samples from every fraction was determined performing aminoacylation tests under standard conditions for heterologous initiator tRNA (see preparation of fMet-tRNA_i^Met in Materials and Methods) using 1 nmol [¹⁴C]methionine (550 c.p.m./pmol) and 10 µg crude synthetases from E.coli. Line, absorbance at 260 nm; circle, c.p.m. of [¹⁴C]methionine incorporated in aminoacylation tests. The shaded region indicates the elution position of [¹⁴C]Met-tRNA_i^Met when aminoacylation was performed before the HPLC separation. (B) Similar to (A), but with aminoacylation and formylation prior to RP-HPLC separation. Excess formyl donor was removed from the sample by gel filtration on Sephadex G-25. The radioactivity profile (circle) indicates the position of f[¹⁴C]Met-tRNA_i^Met and the insert contains the denaturing PAGE pattern of pooled radioactive fractions. (C) RP-HPLC purification of deacylated tRNA_i^Met. Aliquots of 6000 pmol f[¹⁴C]Met-tRNA_i^Met (550 c.p.m./pmol, 809 pmol/A₂₆₀ unit) were incubated with 1 mg of purified PTH in 6 ml of deacylation mix for 20 min at 30°C (an analytical cold TCA precipitation made at the end of the incubation indicated 97.9% deacylation). After phenol extraction and ethanol precipitation the material was submitted to RP-HPLC as described in (A). The tRNA content of the fractions was precipitated with ethanol and redissolved in 300 µl of water and the [¹⁴C]methionine acceptor activity determined (circle). The material eluting from 19 to 24 min corresponded to tRNA_i^Met with an average acceptor activity of 1700 pmol/A₂₆₀ and showed a unique homogeneous band upon denaturing PAGE analysis (insert).

Figure 4

Isolation and activity test of the PTH from E.coli. (A) SDS–PAGE analysis of the fractions obtained during the isolation procedure described in Materials and Methods and Table 1. Lane 1, molecular weight markers; lane 2, S-100 fraction; lane 3, 70% saturated pellet after dialysis; lane 4, unbound fraction on CM cellulose; lane 5, 50 mM NaCl wash; lanes 6 and 7, final product from two different isolations. (B) Samples of 0.1 A₂₆₀ units of f[¹⁴C]Met-tRNA_i^Met before (lane 1) and after (lane 2) 15 min incubation under deacylation conditions were applied to a denaturing 12.5% polyacrylamide gel. The RNA species were visualized via toluidine blue staining (left). Fluorography (right) was of the same gel after destaining and treatment with Amplify (Amersham). A second sample of deacylated tRNA (corresponding to 40 pmol of the original fMet-tRNA_i^Met) was incubated under methionylation conditions for 15 min at 37°C and re-aminoacylation was estimated to be 97.4% by cold TCA precipitation.