A simple procedure for fractionation of transfer RNA

ANALYTICAL

BIOCHEMISTRY

A Simple

33, 120-124 (1970)

Procedure

for Fractionation ENDRE

Department

of Hktology,

Karol&z&a

of Transfer RNA

EGYHAZI Institute,

Received September

104 01 Stockholm,

Sweden

11, 1969

Interest in the biology of transfer RNA (tRNA) has led to the development of a large number of methods for preparative isolation and analytical characterization of different tRNA species such as countercurrent distribution between two phases (1)) partition chromatography (2)) chromatography on columns of hydroxylapatite (3)) methylated albumin-kieselguhr (4)) DEAE-cellulose (5)) and benzoylated DEAEcellulose (6)) as well as chemical procedures involving oxidation of amino acid free tRNA with periodate and separation of the formed dialdehyde tRNA from aminoacyl tRNA (7). In the present communication a simple chromatographic procedure will be described based on the use of methylated albumin coated on nitrocellulose membrane filters (MANM). This technique is capable of resolving individual unacylated tRNA species partially or completely from a crude mixture of Escherich&z coli 4 S RNA species. The procedure eliminates a number of laborious steps involved in the conventional column chromatography such as packing and washing of column and is time-saving due to small volumes of eluting gradient solutions. MATERIALS

AND

METHODS

Escherichia coli W 4 S RNA was obtained from Schwarz BioResearch, Inc., Orangeburg, New York. L- (H3) -Histidine (40 Ci/mmole) , L- (H3) tyrosine (43 Ci/mmole) , L- (H3) -1eucine (22 Ci/mmole) , and L- (H3) methionine (6.9 Ci/mmole) were purchased from the Radiochemical Centre, Amersham, England, and L- (H3)-proline (20 Ci/mmole), DL(HS) -1ysine (16 Ci/mmole) and DL- (H3) -phenylalanine (17 Ci/mmole) from C E A D,Bpartment des Radio8&ments, B. P. No 8 Gif-sur-Yvette, France. The soluble protein solution containing the activating enzymes was prepared from lyophilized cells of strain B (ATCC 11303) of E. coli (Sigma Chemical Company, St. Louis) as described by Kaji (8). Methylation of albumin was carried out according to Mandell and Hershey (9). The tRNA’s were eluted from the MANM filter with a linear 120

FRACTIONATION

OF

TRANSFER

RNA

121

gradient of 0.35-0.75 M NaCl in the presence of a constant concentration of urea (2 M). Methylated albumin-nitrocellulose membrane filters used as chromatographic medium were prepared as follows: 10 nitrocellulose membrane filters, 30 mm in diameter (Sartorius MF 50) were placed in a Pyrex microanalysis filter holder (No. xx 1002500, Millipore Corporation) and 1 ml solution of 1.5% methylated albumin in distilled water was passed through the filters for 5 min. They were then removed from the filter holder and dried for 45 min at 60°C. After this step they were ready for chromatography either immediately or within three weeks after storage at room temperature. For the chromatographic procedure the Pyrex microanalysis filter holder was equipped with a rubber stopper with one inlet for air and one for the gradient solution. It was inserted into the glass funnel 5 mm over the filter, thereby permitting control of the level of gradient solution over the filter. A constant flow rate (0.5 ml/min) of the gradient through the filter was maintained by means of a peristaltic pump (type 10200, LKB-produkter, Stockholm) which delivered the effluent to the fraction collector and the receiving tubes via a recording spectrophotometer (Uvicord II, LKB-produkter, Stockholm) with a flow cell of 3 mm optical path. During the course of the run the gradient level was kept constant over the MANM filter (l-2 mm). One filter fractionates successfully 50 pg of E. coli 4 S RNA. A larger number of MANM filters can hold proportionally more nucleic acid. The filter is equilibrated with the charging buffer (0.05 sodium acetate buffer, pH 5.1, containing 2 M urea) by passing 10 ml of buffer solution through the filter before loading with 4 S RNA. The sample of 4 S RNA dissolved in 1 ml charging buffer was then loaded onto the MANM filter. Contaminating material not sticking to the filter was removed by washing with the charging buffer under control of the recording spectrophotometer. A linear sodium chloride gradient in 2 M urea was used. The total volume of gradient was 50 ml and fraction size was 1 ml. The elution was carried out at room temperature. More than 90% of E. coli RNA could be recovered from the MANM filter. After completion of the elution amino acid acceptor assays were performed for identification of specific tRNA species. The eluted fractions contain NaCl and urea which must be removed from RNA before the assays. This step together with charging reactions was carried out by a modification of the filter disc technique described by Cherayil and Bock (5). 100 ~1 of chromatographic fractions was applied directly to filter paper discs (Munktell’s, 25 mm diameter) which were pierced in the center and threaded on to a glass rod (3 X 400 mm) at 6 mm distances. Rubber tube pieces 6 mm in height inserted between the filter discs kept them separated from

0.2 -ia) ~OOOO(b)

122

ENDRE

EGYHbZI

--*-

LVSINE

+n-

PROLINE

---

METHIONINE

-ON-

PHENVLALANINE

-w-

HISTIDINE

-w-

TVROSINE

5000 -

Fraction

FIG.

1. Methylated

albumin-nitrocellulose

no

membrane

(MANM)

filter

chromatog

FRACTIONATION

OF

TRANSFER

RNA

123

each other. The array of discs was then dried in air until the blank sheen has disappeared and subsequently placed in a cylinder-shaped graduated glass (50 x 350 mm) containing 70% ethanol/O.03 M KC1 for 20 min at 4°C to remove excess of salts. This washing procedure was repeated once, after which the array of discs was dried in air and then subjected to amino acid acceptor assay. The incubation mixture which was added to the filter paper discs contained (per ml) : 40 pmole TrisHCl buffer, pH 7.65, 4 pmole MgCl,, 0.5 pmole EDTA, 1 pmole ATP, 20 pmole KCl, 2 pmole ,&mercaptoethanol, 10 $Zi (H3)-amino acid (743 CiJmmole) , and 20 ~1 E. coli soluble protein (100-200 Fg) containing amino acyl tRNA synthetases; 100 ~1 of this mixture was added to each filter paper disc with a micropipet at 4°C and the set of filter papers was kept in a humidified cylinder-shaped glass container, of the kind described above, for 40 min at room temperature. After this time period, no further increase in the amounts of RNA-attached amino acid could be obtained. For removal of unreacted radioactive amino acid the paper discs were then dried in air and treated with the following solvents at 4°C: 10% TCA for 20 min, 5% for 15 min, and 70% ethanol/O.1 M NaCl for 15 min. All wash solutions contained 5 mg unlabeled amino acid per liter. Finally, the array of discs was washed in an ethanol/ether mixture (3:1), dried, and transferred to scintillation vials. 10 ml of a toluene solution containing 5 gm PPO, 0.5 gm POPOP, and 50 ml NCS (Nuclear Chicago) per liter was added to each vial. The samples were then counted in a Packard (model 3380) liquid scintillation spectrometer with an efficiency around 30%. RESULTS

Results of fractionating E. coli 4 S RNA are demonstrated in Figure 1; optical density pattern are seen in (a), distribution of acceptor activities for 7 amino acids in (b-e). Every tube was assayed for acceptor activity. Since the activating enzymes are present at different concentrations during the assays, the height of the individual peaks does not reflect the true quantity of each tRNA species measured. A distinct separation of lysine tRNA from proline tRNA can be seen in Figure lb. An almost complete separation could be obtained between methionine raphy of E. co& 4 S RNA. 109 pg stripped 4 S RNA (30 min buffer, pH 8.9) was loaded onto a double MANM filter in (0.05 M sodium acetate buffer, pH 5.1, containing 2 M urea). out with a gradient of NaCl dissolved in the charging buffer 50 ml at a rate of 0.5 ml/min. Assays for amino acid acceptor formed as described in the text. (a) Optical density profile, for lysine and proline, (c) for methionine and phenylalanine, tyrosine, (e) for leucine.

at 37°C in Tris-HCl 1 ml charging buffer Elution was carried in a total volume of activity were per(b) acceptor activity (d) for histidine and

124

ENDRE

EGYHAZI

and phenylalanine tRNAs (Fig. lc), likewise between histidine and tyrosine tRNA’s (Fig. Id). The resolution of tyrosine tRNA into two peaks can also be seen in this case. The leucine tRNA was broadly distributed and was resolved into multiple peaks (Fig. le). The salt concentrations at which various tRNA’s were eluted showed only slight variation in different experiments. Summing up, a chromatographic procedure based on elution of tRNA from methylated albumin coated nitrocellulose membrane filters to separate individual tRNA species from a crude E. coli 4 S preparation has bseen developed. This technique is capable of giving a high resolution display of the profiles of several acceptor activities in a single run. The technique should, in view of its simplicity and rapidity, be valuable for analytical purposes such as establishing variation in tRNA composition due to developmental, environmental, or nutrional influence (lO12) # ACKNOWLEDGMENTS This work was supported by grant 68:47 from the Swedish Cancer Society and grant GM 14896 from the National Institutes of Health, U. S. Public Health Service. Miss Agneta Askendal provided skillful technical assistance. REFERENCES 1. APGAR, J., HOLLEY, R. W., AND MERRILL, S. H., J. Biol. Chem. 237, 796 (1962). 2. MUENCH, K., AND BERG, P., Biochemistry 5, 970 (1966). 3. MUENCH, K., AND BERG, P., Biochemistry 5, 982 (1966). 4. SUEOKA, N., AND YAMANA, T., Proc. Natl. Acad. Sci. U. S. 48, 1454 (1962). 5. CHEFLAYIL, J. D., AND BOCK, R. M., Biochemistry 4, 1174 (1965). 6. GILLAM, I., MILLWARD, S., BLEW, D., TIGERSTROM, M., WIMMEB, E., AND TENER, G. M., Biochemistry 6, 3043 (1967). 7. STEPHENSON, M. L., AND ZAMECNIK, P. L., Biochem. Biophys. Res. Commun. 7, 91 (1962). 8. KAJI, A., in “Methods in Enzymology” (Grossman, L., and Moldave, K., eds.), Vol. 12, Part B. Sect. XI. p. 692. Academic Press, New York, 196% 9. MANDELL, J. D., AND HERSHEY, A. D., Anal. Biochem. 1, 66 (1960). 10. DOI, R. H., KANEKO, I., AND IGARASHI, R., J. Biol. Chem. 243, 945 (1968). 11. YEGIAN, C. D., AND STENT, G. S., J. Mel: Biol. 39, 45 (1969). 12. DELLWEG, H., GERNER, R., AND WACKER, A., J. Neurochem. 15, 1109 (1966).