The purification and aminoacylation of yeast tRNALys were performed a described elsewhere (Johnson et al., 1976; Krieg et al., 1989). As before, the amount of Lys tRNAs were expressed as pmol [14C] Lys/A260U (Krieg et al., 1989; Crowley et al., 1993) where a specific activity of 1600 pmol [14C] Lys/A260U constituted pure Lys-tRNA. I normally used Lys-tRNAs that have specific activities > 1000pmol [14C] Lys/A260 U to perform the chemical modifications. The chemical modification of the Lys epsilon-amino group was performed using the N-hydroxysuccinimide ester of 5-azide-2-nitrobenzoic acid (ANB-NOS; Pierce Chemicals) in a dark room illuminated only by red safety lights. Prior to labeling the Lys-tRNA, the ANB-NOS reagent was brought to room temperature, and 10mg was dissolved in 1.75mL of dimethylsulfoxide (DMSO; Gold Label, Aldrich Chemicals) per labeling reaction.
The reaction was performed in a 3-dram glass vial with constant stirring, and immersed in an ice-water bath. For each reaction, 5100pmol of [14C] Lys-tRNA was added to the glass vial that contained ddH2O and 50mM potassium phosphate (pH 7.0) so that the volume was 750uL and then stirring speed was set. 1.75mL of the ANB-NOS solution was then added to the vial and the stirring speed was adjusted to attain a vigorous vortex. Subsequently, 15uL of freshly prepared 4N KOH was added to the reaction to deprotonate the epsilon-amino group of lysine and initiate the modification process. After exactly 20 seconds, 15uL 4N HOAc was added to the mix to neutralize the base and stop the reaction. The modified Lys-tRNAs were then prepared for ethanol precipitation by adding 2.5mL of 2M KoAc (pH 5.0), mixed thoroughly using a pasteur pipet and transferred to a SW-28 polyallomer centrifuge tube (Beckman Instruments). The reaction was repeated, and this second labeling reaction was pooled together with the first reaction in the SW-28 tube. The tRNAs were precipitated with 27mL 100% ethanol, mixed using a 25mL glass pipet, covered with parafilm, and then covered with aluminum foil to prevent exposure to light. The Lys-tRNAs were then precipitated overnight at -20°C. The labeled product was spun at 28, 000 rpm using a SW-28 rotor (Beckman Instruments) for 2.5 hours at 4°C. The resultant tRNA pellets were dried at room temperature, and nitrogen gas before being resuspended in buffer A [1mM KOAc (pH 5.0), 5mM MgCl 2 ]. The modified tRNAs were dialyzed three times against buffer A for 2 hours to remove salts and excess dye. The ANB-labeled Lys-tRNA (see Fig. 2 (DWA)) was then aliquoted, covered with aluminum foil and stored at -95°C.
Characterization of epsilon-ANB-Lys-tRNA
epsilon-ANB-Lys-tRNA was normally characterized prior to its use in photocrosslinking experiments. Typically, 2uL of the dialyzed ANB-Lys-tRNA was aliquoted into each of four duplicate tubes, then diluted to 400uL with buffer A. Two tubes were used to measure the absorbance at 260 nm to obtain the tRNA concentration, while the remaining two tubes were used in scintillation counting to determine the amount of [14C]Lys-tRNA. Typically, my ANB-Lys-tRNAs preparations had specific activities that ranged from 500-1100 pmol [14C]-Lys/A260 U. The decrease in the specific activity after modification was presumed to be caused by the absorption of the ultraviolet radiation by the photoreactive probe and increase the apparent absorption at 260nm.
The chemical purity of ANB-Lys-tRNA (~15000 dpm) was assayed by paper electrophoresis as described earlier (Johnson et al., 1976). Briefly, epsilon-ANB-Lys-tRNA (~15000 dpm) was hydrolyzed in 50mM triethylamine (final concentration) at 37°C for 1.5 hours or overnight and dried under nitrogen gas(or 25000 cpm in 2uL ; add 2uL of 100mM (14uL/mL), 98% triethylamine, freshly made, at 37°C for 1.5 hours or overnight and dried under nitrogen gas). The sample was resuspended in water and spotted onto electrophoresis paper (Whatmann) at the origin (20 cm from the anode, 80cm from the cathode). Free 0.1M lysine, Nalpha-acetyl-Lys(which was not used) and 2mg/mL of Nepsilon-acetyl-Lys standards were also spotted onto the same electrophoresis paper at different lanes and all samples were separated in buffer B [10% (v/v) glacial acetic acid/1% (v/v) pyridine] at 25 V/cm for 3 hours. After drying overnight, the sample lane was cut into one hundred 1 cm strips and then counted in a liquid scintillation counter, while the standards were stained with ninhydrin to determined the respective position of the unlabeled, alpha-labeled and epsilon-labeled lysine. Typically, greater than 82% of the Lys was labeled ANB at the epsilon-amino group of Lys, while <3% was labeled at the alpha-amino group, and <12% remained unmodified. A typical profile of an ANB-Lys-tRNA sample that was separated by paper electrophoresis is shown in Fig. 3 (see DWA).