ANALYTICAL
206,363-368
BIOCHEMISTRY
(19%)
Affinity Electrophoretic Detection of Primary Amino Groups in Nucleic Acids: Application to Modified Bases of tRNA and to Aminoacylation Gabor L. Igloi Institut
fLir
Received
May
Biologic
III,
Universitiit
Freiburg,
Schiinzlestrasse
Freiburg,
Germany
14, 1992
Thiolation of primary amino groups in tRNA with the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate gives rise to species which are retarded during electrophoresis in organomercury-containing polyacrylamide gels. Since such amino groups occur, as far as is known, only as part of the modified bases 3-(3-amino-3-carboxypropyl)uridine and N-2-(5amino-5-carboxypentyl)cytidine or as the a-amino group of aminoacylated tRNAs, this extention of the principle of affinity electrophoresis can be used for the detection and analysis of a specific functional group in both single tRNA species and in a mixed population. The strength of the interaction may be quantified and provides information on the chemical environment/ conformation of the derivatized bases. o 1992 Academic Press,
1, D-7800
Inc.
More than 70 different modified bases have been identified in tRNAs to date (1) and new ones are being discovered even before the functions of many of the well-characterized ones have been established. The discovery and structural definition of these bases are largely dependent on data from combined liquid chromatography-mass spectroscopy (LC-MS)’ analyses (2) resulting in the destruction of the sample. We have previously shown (3,4) that the application of certain functional group-specific chemical reagents when combined with the resolution offered by affinity electrophoresis can not only offer an alternative as a method of detec-
1 Abbreviations used: SPDP, IV-succinimidyl 3-(2-pyridyldithio)propionate; acp3U, 3-(3-amino-3-carboxypropylhkline; acp*C (Iysidine), iV-2-(5-amino-5-carboxypentyl)cytidine; LC-MS liquid chromatography-mass spectroscopy; s’U, 4-thiouridine; mnm%*U, 5-methylaminomethyl-2-thiouridine; APM, acryloylaminophenylmercuric acetate; DTT, dithiothreitol. 0003-2697/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
tion but may give valuable information as to the chemical environment, i.e., tRNA conformation, in which a particular modified base may be found (for review see 5). This approach has, so far, been feasible for the detection of free ribose groups (3,6) in, e.g., the Q base, and for the analysis of thiol-containing modifications of 4thiouridine and 5-methylaminomethyl-2-thiouridine (s”U and mnm5s2U) (4,7), where the reactivity of the sulfur is highly dependent on its chemical and physical environment. Although the application to thiol-bearing bases has not as yet been exhaustively examined to include all potentially reactive modifications, it has been of interest to try to extend this type of analysis to other classes of modified bases. Admittedly, affinity electrophoresis may not be the method of choice for detecting important but chemically inert modifications, such as methylations. However, an interaction with the hydrophobic gel matrix which has been postulated to explain the anomalous migration of 5-methylcytidine in DNA oligomers (8) may offer a means of approaching this problem. A number of other rare nucleosides carrying functional groups not present in the standard bases may be further candidates for affinity electrophoretic detection and analysis. In this study, we have considered primary amino groups, as found in acp3U (Scheme 1) (usually at position 47 but sometimes also present in the ~-loop (9)), or in the case of the a-amino group of aminoacylated tRNAs, as targets for affinity electrophoretic analysis. Whereas previously we have tailored the affinity matrix to match the functional group of interest, in this case the alternative of modifying the mobile ligand to a species capable of reacting with an already established matrix has been introduced. Primary amino groups have been derivatized with the bifunctional thiolating reagent N-succinimidyl 3-(2-pyridyldithio)propionate 363
Inc. reserved.
364
GABOR
(SPDP) (shown in Scheme 1) and the products have been shown to interact with the mercurial, acryloylaminophenylmercuric acetate (APM) gel system (4). MATERIALS
AND
METHODS
Bulk Escherichia coli tRNA and those specific for Phe (E. coli, yeast) and Tyr (E. cob) were from Boehringer. tRNALy” (E. coli) was obtained from Subriden RNA (Rolling Bay). Total maize chloroplast tRNA was isolated as described for tobacco (10). Pure E. coli tyrosyltRNA synthetase was a kind gift of Dr. H. Sternbach (Giittingen). APM was synthesized and incorporated at the indicated concentrations into polyacrylamide gels as described previously (4) using O.&mm-thick polyacrylamide gels in the presence of 7 M urea and Trisborate buffer, pH 8.3. Electrophoresis was at about 10 V/cm. 3’-terminal labeling with [32P]pCp was carried out as described (6) and silver staining was according to (11). Thiolation of acp3U with SPDP (Pharmacia) was achieved as follows: tRNA (25 pg or less) was incubated in 10 mM NaH,PO,/Na,HPO, buffer, pH 7.2, 100 mM NaCl at room temperature for 30 min with 20 mM SPDP (diluted from a 40 mM stock solution in ethanol) in a total volume of 100 ~1. This was followed by the addition of either 50 ~1100 mM dithiothreitol (DTT) (to generate the free thiol group) or 50 ~1 water (control). After 20 min, 10 ~1 of 3 M NaOAc was added and the tRNA was recovered by precipitation with 350 ~1ethanol at -20°C. Any residual DTT was removed by purifying the tRNA using a Qiagen column (Diagen). For this, the samples were dissolved in 450 ~1 of 100 mM NaC1/50 mM Mops, pH 7/15% EtOH. They were applied to Qiagen Tip20 columns which were then washed with 1.5 ml of the same buffer. Elution was with 3 X 0.2 ml Qiagen buffer F (1.5 M NaCI, 50 mM Mops, pH 7.5,15% EtOH) and the tRNA was precipitated with 0.75 ml isopropanol at -20°C. In the case of aminoacyl-tRNAs, SPDP treatment followed directly the aminoacylation reaction. Aminoacylation was as described previously (12), except that Tris was replaced by Hepes (to avoid the introduction of additional primary amino groups). After a 30-min incubation at 37”C, the tRNA was obtained by rapid phenol extraction and ethanol precipitation and the tRNA was kept cold for all subsequent operations, apart from the SPDP and DTT reactions, which were as described above. RESULTS
AND
DISCUSSION
The heterobifunctional reagent SPDP has, among other uses, been considered as a thiolating reagent for proteins, in particular (13). This is understandable in view of the limited occurrence of its target functional group, a free primary amino residue, in nucleic acids. It is, however, exactly the specificity of such succinimide derivatives which makes it possible to label selectively
L. IGLOI
those components in a nucleic acid population which bear primary amino groups (a fact which is utilized in biotin or fluorescent labeling of synthetic oligonucleotides at a Y-terminus-bearing NH, group (14)). In the case of tRNAs, this reaction is, at the present time, therefore, limited to the modified bases acp3U and acp2C (lysidine) (Scheme l), together with the a-amino moiety of aminoacylated tRNAs. The feasibility and specificity of this type of reaction have been demonstrated by coupling affinity probes or biotin to some of these sites via appropriate succinimide esters (15). Analogously, reaction with SPDP leads to thiolation and thus makes the reaction products amenable to characterization by affinity electrophoresis on a mercurial derivative of acrylamide. acp3U Figure 1B shows the electrophoretic pattern obtained when SPDP-derivatized E. coli tRNALy” (acp3U,,) was subjected to analysis on a mercurial affinity matrix. A retarded band whose appearance is dependent on cleavage of the S-S bond within the coupled reagent, by DTT, is observed. Incubation of the disulfide with water, instead of DTT, does not liberate a species amenable to interaction with the affinity electrophoretic matrix. The absence of a discernable SPDP reaction of yeast tRNAPhe (Fig. lA), which exemplifies tRNA species lacking an acp3U, confirms the fact that the common bases are not modified by this type of reagent (15). The strong retardation in the case of tRNALW seen at an immobilized ligand concentration of 0.1 pg/ml signifies an affinity comparable to that of the monophosphorothioate ester interaction observed previously (4) (see also below). The heterogeneous state of modification indicated by an only partial conversion of the tRNA to a retardable state reflects either the true in uivo state of the tRNA (undermodification, either naturally or by nature of the bacterial growth conditions) or an incomplete reaction of potentially available modified bases with SPDP. Undermodification has previously been observed with all tRNAs subjected to affinity electrophoretie analysis but the source of this heterogeneity has not been investigated in each case. Certainly, in the case of s*U, growth conditions have been shown to affect the state of modification (Emilsson and Kurland, personal communication) while commercial preparations of E. coli tRNAPhe (acp3U4,) h ave failed to react with SPDP (not shown). On the other hand, it cannot be denied that this indirect approach to the detection of specific modified bases, unlike the analyses relying on the interaction of an intrinsic partner with the immobilized ligand, e.g., s*U, must depend on the coupling yield between the free amino group and the succinimide ester. This has been determined in a number of instances to be between 60 and 90% (15) but a quantitative analysis of modified
AFFINITY
A
CH=CH
ELECTROPHORESIS
OF
365
tRNA
2
Lo I NH
0
0 - S-S-CH#$-O-N
3 OS
HgOAc
OH
OH
bH
Structures of the main of tRNAs containing
base content of a particular into account.
bH
acp%J
a mw SCHEME 1. retie fractionation
O*
components of the analytical system described in the text. (A) Reagents used during the modified bases (B), whose reactive amino groups are marked by asterisks.
tRNA
must take this factor
Other Base Modifications Treatment of bulk tRNA, in this case species isolated from higher plant chloroplasts, gives rise to a series of retarded bands (Fig. 1C). The individual moieties responsible for this pattern have not yet been fully characterized but, according to our present knowledge of modified bases in chloroplast tRNAs, one may hazard some observations as to the modifications involved. acp3U has been identified in a number of chloroplast tRNAs (9) at position 47. Furthermore, the only other primary amino-bearing base known to date, acp2C (lysidine), whose presence has been confirmed in tRNA”“(CAU) in E. coli (16) and plant mitochondria (17), is expected to play a similar crucial role in the chloroplast tRNA’“(CAU) by preventing the incorporation of Ile at Met codons. The multiple bands resulting from the SPDP reaction with bulk chloroplast tRNAs may then be correlated with acp3U at positions of differing reactivities/conformational flexibilities (see below), or within tRNAs of different sizes, or with a mixture of tRNA species with differing modifications, e.g., acp3U or acp2C. Aminoacyl-tRNAs The ester linkage between the 3’-terminal ribose and the carboxyl group of an amino acid is labile (see, e.g.,
affinity
electropho-
18) and, under standard conditions used for polyacrylamide gel electrophoresis, survives intact to only a limited extent. Fractionation in acid gels has recently been introduced to overcome this problem (19), while modification of the a-amino group has been practiced for some time in an effort to stabilize this ester bond (e.g., 20). Conversion of the -NH, to the thiol needed for affinity electrophoretic purposes simultaneously leads to a significant stabilization of the aminoacyl-tRNA at pH 8.3 under electrophoretic buffer conditions (Fig. 2). Above pH 9 the protection against hydrolysis is no longer observed (not shown). In order to monitor the migration of the aminoacyl moiety of the aminoacylated tRNA, rather than the tRNA itself, bulk E. coli tRNA was aminoacylated with a mixture of 16 14C-labeled amino acids, in the presence of an E. co& SlOO extract and the product treated with SPDP, as described under Materials and Methods. Subsequent affinity electrophoresis and fluorography (Fig. 3A) revealed a strongly retarded radioactive fraction which, in fact at an APM concentration of 0.5 pg/ml (1.25 PM), barely enters the gel, cf. the migration of the reaction product with acp3U. The control lane in which the [14C]aminoacyl-tRNA had not been treated with SPDP gives rise to two very faint nonretarded bands (due to species having escaped deacylation during electrophoresis) at a position equivalent to the tRNA marker. This indicates that retardation is due to the thiol group, now linked to the a-amino residue at the 3’ terminus, interacting with the affinity ma-
366
GABOR
L. IGLOI
B
FIG. 1. Affinity electrophoretic fractionation of SPDP-treated tRNAs. Yeast tRNAPh” (A), E. coli tRNA”” (B), and maize chloroplast total tRNA (C) were reacted with SPDP as described under Materials and Methods, or left untreated (control). In each case, onehalf of the product was reduced with DTT while the other half was incubated with water, as indicated. The tRNAs (approx. 50 rig/lane) were fractionated on a 5% polyacrylamide gel containing 7 M urea and 0.25 PM (0.1 pglml) APM. The bands were visualized by silver staining (11).
trix and confirms the stabilizing effect of the SPDP modification; only a fraction of the attached label survives the electrophoretic conditions in the unmodified lane. In the case of the SPDP-modified product, the amount of nonretarded material just visible on longer exposure indicates that the reaction is virtually complete.
100 -o-o90 +SPDP 80
\
‘0
- SPDP
\ =O I 0
I 20
FIG. 3. Detection of aminoacylated tRNAs by affinity electrophoresis. (A) Bulk E. coli tRNA was aminoacylated with a *‘C-labeled amino acid mixture using an SlOO extract. One-half of the product, after isolation as described under Materials and Methods, was left unthiolated while the other was thiolated with SPDP/DTT, as indicated. Affinity electrophoresis was carried out under the conditions specified in the legend to Fig. 1, except that the APM concentration was 1.25 pM (0.5 pg/ml) and the bands were detected by fluorography. As a marker, “P-labeled total chloroplast tRNA was run alongside the samples (control). The position of the bottom of the sample well is indicated by an arrow. (B) E. coli tRNA% was aminoacylated with nonradioactive tyrosine in the presence of pure tyrosyl-tRNA synthetase (Tyr + TRS) or an SlOO extract (Tyr + SlOO), as indicated. Controls were carried out in the absence of Tyr, lanes (TRS), (SlOO), or after deacylation. Affinity electrophoretic analysis was as described in the legend to Fig. 1 using an APM concentration of 0.1 pg/ml and the bands were detected by silver staining (11). The retarded band corresponding to aminoacylated tRNA is marked with an arrowhead.
I 40
1 60
80
I 100
, 120 1 (mln)
FIG. 2. Stabilization of the aminoacyl-tRNA ester bond by reaction with SPDP. E. coli tRNA was aminoacylated with a “C-labeled amino acid mix and then treated with SPDP (+) or used as a control (-), as described under Materials and Methods. The stability of the labeled tRNA at room temperature at pH 8.3 in TBE buffer was monitored by removing aliquots at the times shown and determining the number of TCA-precipitable counts remaining.
Having demonstrated the affinity electrophoretic retardation of tRNA species known to be aminoacylated (by radioactive assay) this method could now be used to test for aminoacylation per se, in the example of the tyrosine system. Commercial E. coli tRNA% was incubated with pure E. coli tyrosyl-tRNA synthetase and tyrosine, as described under Materials and Methods. SPDP modification was followed by affinity electrophoresis and silver staining (Fig. 3B). A substantial proportion of the tRNA is retarded by the interaction between the thiol and the mercury, while the rest migrates as unmodified tRNA. (Note that the concentration of APM in the gel (0.1 pg/ml, 0.25 PM) is far below that
AFFINITY
ELECTROPHORESIS
required to observe an interaction between tRNA% s~IJ,,~ and the matrix (10 pg/ml) (4)). Two control lanes demonstrate that the retardation is a consequence of aminoacylation (followed by thiolation). First, in the absence of the amino acid, no slow-migrating product is observed. Second, treatment of the product of aminoacylation with NaOAc, pH 9, for 30 min at 37°C (effectively, hydrolyzing any ester bond between the amino acid and the tRNA) prior to SPDP modification also abolishes the affinity interaction. Using an E. coli SlOO, tyrosyl-tRNA synthetase activity could be detected according to this protocol, although the situation is complicated by the presence in the extract of some high-molecular-mass nucleic acids which are detected by staining. Furthermore, proteolytic degradation in the extract liberates free tyrosine, which then gives rise to an aminoacylation-specific signal in the control reaction lacking added amino acid. Quantification Interaction
and Comparison
of the Binding
The relative extent of retardation of tRNAs which have been thiolated with SPDP at different sites is noteworthy. The rate of electrophoretic migration reflects the strength of the affinity between the interacting partners (21) and may, indeed, be used to quantify the binding. The binding constant between this matrix and a number of thiol derivatives has been determined rigorously by measuring the APM concentration-dependent variation of the electrophoretic mobility, according to the simplified affinity electrophoretic equation (for a more extensive discussion of quantitative aspects, see 22,23). Assuming that the complex once formed has zero mobility, the dependence of the mobility on the apparent binding constant K is given by l/r
,
= l/R,[l
+ c/K],
where r is the migration distance of one interacting partner in the presence of the immobilized ligand with a concentration c, while RO is the mobility at c = 0. For comparative purposes, one may use the previously determined affinity if one considers the concentration of APM at which the mobility of a component of unknown K, is identical to the relative mobility of a species of known KI . The equation is then approximated by K,/c, = K2/c2, where c1 and c2 are the APM concentrations, where species 1 and 2 have identical relative mobilities -a value which may be obtained from the known plot of { 1 - r/R,}-’ vs c-l for, say, s4U-containing tRNA. Table 1 gives the values for K which have been deduced in this manner for the SPDP derivative of acp3U and of aminoacyl-tRNA and summarizes the binding affinities which have been determined for this affinity matrix, to date.
OF
367
tRNA TABLE
1
Interaction Constants (PM) between Thiol-Containing Macromolecules and APM, as Determined by Affinity Electrophoresis in Polyacrylamide Gels Containing ‘7 M Urea tRNAMEt(s’U) (E. coli) tRNAG’“(mnm6s2U) (E. coli) tRNALyB(acp3UISPDP) (E. coli) Aminoacyl-tRNA/SPDP (a-amino) 5’-Phosphorothioatemonoester RNA monophosphorothioate RNA triphosphoro-y-thioate a From
8.0” 90.0” 0.1 0.02 0.3” t0.1”
(4).
This difference in affinity, however, cannot be seen as a simple intrinsic property of the molecules involved in complex formation since in both cases identical thiol groups become bound to identical mercury derivatives. It must then reflect some aspect of the residual molecules (tRNAs) and, since, as far as the chemical composition is concerned, even these are very similar, one is left with the interpretation that conformational differences may be the basic properties governing the strength of complex formation. This conclusion is not entirely unexpected since our previous studies have shown that a change in the overall conformation of a tRNA, as induced by denaturing agents, can lead to significant changes in the affinity electrophoretic migration in the case of s4U/APM or mnm5s2U/APM (4). These changes have been interpreted in terms of a structural flexibility or opening permitting a more favorable, stronger interaction. In the present case, this argument would again be applicable. The 3’ terminus, bearing the amino acid, is known from X-ray data of several tRNAs (24,25) to be in an exposed position and a thiol group here would be ideally situated, even in a native conformation, for the interaction considered here. On the other hand, acp3U4,, at a position in the molecule which is susceptible to chemical attack (24), is nevertheless flanked by bases which are involved in tertiary base pair interactions and must, therefore, be part of a more rigid architecture which, as has been shown previously (4), is only incompletely destroyed by 7 M urea. An interaction with the matrix is apparently sterically hindered, leading to a relatively weaker interaction. It should be mentioned that, based on the observation that multiple interacting sites within one molecule lead to a higher affinity (4,26), were one to aminoacylate a tRNA which simultaneously also contains an acp3U moiety, SPDP treatment of this species would result in a further retardation during affinity electrophoresis. In a comparison between the behavior of different sulfur-containing compounds in this affinity electrophoretie system, one must also take into account the chemical environment/reactivity of the sulfur. This is re-
368
GABOR
fleeted in the order-of-magnitude differences in the “dissociation constants” on going from sulfur atoms which form part of an aromatic nucleus, as in s4U or s2U derivatives, to the strongly nucleophilic phosphorothioate monoesters (Table 1) and the aliphatic thiols described here. The actual numerical values attributed to these latter affinities toward the organomercurial may, in view of problems involved in quantifying such tight, quasicovalent binding, only be approximate. Nevertheless, the fact that one can, on a gel, clearly distinguish the tRNA thiolated at position 47 from SPDP-modified aminoacyl-tRNA is a compelling demonstration of the sensitivity of affinity electrophoresis to the electronic and conformational intricacies of biomolecules (5). ACKNOWLEDGMENTS I thank Dr. H. K&se1 for fruitful discussions. I am grateful to Mrs. E. Schiefermayr for dedicated technical assistance and to Mrs. S. Krien for photographic work. This investigation has been supported, in part, by the Deutsche Forschungsgemeinschaft (Grant SFB 206).
1. Bj8rk, G. R., Ericson, J. U., Gustafsson, C. E. D., Hagervall, T. G., Jonason, Y. H., and Wikstrbm, P. M. (1987) Annu. Reu. Biochem. 66.263-287. 2. Edmonds, C. G., Cram, P. F., Gupta, R., Hashizume, T., Hocart, C. H., Kowalak, J. A., Pomerantz, S. C., Stetter, K. O., and McCloskey, J. A. (1991) J. Bacterial. 173, 31383148. 3. Igloi, G. L., and KBssel, H. (1985) Nucleic Acids Res. 13, 6881-
6898. 4. Igloi, G. L. (1988) Biochemistry 27,3842-3849. 5. Igloi, G. L. (1992) in Molecular Interactions in Bioseparation (Ngo, T. T., Ed.), Plenum, New York, in press. G. L., and Kossel, H. (1987) R., Ed.), Vol. 155, pp. 433-448,
7. Bezerra, R., and Favre, A. (1990) Biochem. Biophys. mun. 166,29-37. 8. Fritzsche, E., Hayatsu, H., Igloi, G. L., Iida, S., and (1987) Nucleic Acids Res. 15,5517-5528. 9. Sprinzl, M., Dank, N., Neck, S., and Acids Res. Suppl. 19, 2127-2171.
in Methods Academic
in Enzymology Press, San Diego,
Schon,
Res. ComKiissel,
A. (1991)
H.
Nucleic
10. Steinmetz, A., and Weil, J.-H. (1986) in Methods in Enzymology (Weissbach, A., and Weissbach, H., Eds.), Vol. 118, pp. 212-231, Academic Press, San Diego, CA. 11. Igloi, G. L. (1983) Anal. Biochem. 134, 184-188. 12. Igloi, G. L., von der Haar, F., and Cramer, F. (1979) in Methods in Enzymology (Moldave, K., and Grossman, L., Eds.), Vol. 59, pp. 282-291, Academic Press, San Diego, CA. 13. Carlsson,
J., Drevin,
H., and AxBn,
R. (1978)
Biochem
J.
173,
723-737. 14. Chollet, A., and Kawashima, 1529-1541.
E. H. (1985)
Nucleic
Acids
Res.
13,
15. Ofengand, J., Denman, R., Nurse, K., Liebman, A., Malarek, D., Focella, A., and Zenchoff, G. (1988) in Methods in Enzymology (Noller, H. F., and Moldave, K., Eds.), Vol. 164, pp. 372-397, Academic Press, San Diego, CA. 16. Muramatsu, T., Nishikawa, K., Nemoto, F., Kuchino, Y., Nishimura, S., Miyazawa, T., and Yokoyama, S. (1988) Nature 336, 179-181. 17. Weber, (1990)
REFERENCES
6. Igloi, (Wu, CA.
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18. Igloi,
F., Dietrich, Nucleic Acids
A., Weil, Res. 18,
G. L., von der Haar,
J.-H.,
and Marechal-Drouard,
L.
5027-5030.
F., and Cramer,
F. (1977)
Biochemistry
16,1696-1702. 19. Varshney, U., Lee, C.-P., Chem. 266.24712-24718.
and
RajBhandary,
U. (1991)
J. Biol.
20. Herve, G., and Chapeville, F. (1965) J. Mol. Biol. 13, 757-766. 21. Horejsi, V. (1981) Anal. Biochem. 112, l-8. 22. Beg-Hansen, T. C., and Takeo, K. (1980) Electrophoresis 1,6771. 23. Horejsi, V., and Ticha, M. (1986) J. Chromatogr. 376,49-67. 24. Kim, S-H. (1978) in tRNA (Altman, S., Ed.), pp. 248-293, MIT Press, Cambridge, MA. 25. Woo, N. H., Roe, B. A., and Rich, A. (1980) Nature 286,346-351. 26. Dubreuil, cl&Acids
Y. L., Expert-Besancon, Res. 19,3653-3660.
A., and Favre,
A. (1991)
Nu-
206,363-368
BIOCHEMISTRY
(19%)
Affinity Electrophoretic Detection of Primary Amino Groups in Nucleic Acids: Application to Modified Bases of tRNA and to Aminoacylation Gabor L. Igloi Institut
fLir
Received
May
Biologic
III,
Universitiit
Freiburg,
Schiinzlestrasse
Freiburg,
Germany
14, 1992
Thiolation of primary amino groups in tRNA with the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate gives rise to species which are retarded during electrophoresis in organomercury-containing polyacrylamide gels. Since such amino groups occur, as far as is known, only as part of the modified bases 3-(3-amino-3-carboxypropyl)uridine and N-2-(5amino-5-carboxypentyl)cytidine or as the a-amino group of aminoacylated tRNAs, this extention of the principle of affinity electrophoresis can be used for the detection and analysis of a specific functional group in both single tRNA species and in a mixed population. The strength of the interaction may be quantified and provides information on the chemical environment/ conformation of the derivatized bases. o 1992 Academic Press,
1, D-7800
Inc.
More than 70 different modified bases have been identified in tRNAs to date (1) and new ones are being discovered even before the functions of many of the well-characterized ones have been established. The discovery and structural definition of these bases are largely dependent on data from combined liquid chromatography-mass spectroscopy (LC-MS)’ analyses (2) resulting in the destruction of the sample. We have previously shown (3,4) that the application of certain functional group-specific chemical reagents when combined with the resolution offered by affinity electrophoresis can not only offer an alternative as a method of detec-
1 Abbreviations used: SPDP, IV-succinimidyl 3-(2-pyridyldithio)propionate; acp3U, 3-(3-amino-3-carboxypropylhkline; acp*C (Iysidine), iV-2-(5-amino-5-carboxypentyl)cytidine; LC-MS liquid chromatography-mass spectroscopy; s’U, 4-thiouridine; mnm%*U, 5-methylaminomethyl-2-thiouridine; APM, acryloylaminophenylmercuric acetate; DTT, dithiothreitol. 0003-2697/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
tion but may give valuable information as to the chemical environment, i.e., tRNA conformation, in which a particular modified base may be found (for review see 5). This approach has, so far, been feasible for the detection of free ribose groups (3,6) in, e.g., the Q base, and for the analysis of thiol-containing modifications of 4thiouridine and 5-methylaminomethyl-2-thiouridine (s”U and mnm5s2U) (4,7), where the reactivity of the sulfur is highly dependent on its chemical and physical environment. Although the application to thiol-bearing bases has not as yet been exhaustively examined to include all potentially reactive modifications, it has been of interest to try to extend this type of analysis to other classes of modified bases. Admittedly, affinity electrophoresis may not be the method of choice for detecting important but chemically inert modifications, such as methylations. However, an interaction with the hydrophobic gel matrix which has been postulated to explain the anomalous migration of 5-methylcytidine in DNA oligomers (8) may offer a means of approaching this problem. A number of other rare nucleosides carrying functional groups not present in the standard bases may be further candidates for affinity electrophoretic detection and analysis. In this study, we have considered primary amino groups, as found in acp3U (Scheme 1) (usually at position 47 but sometimes also present in the ~-loop (9)), or in the case of the a-amino group of aminoacylated tRNAs, as targets for affinity electrophoretic analysis. Whereas previously we have tailored the affinity matrix to match the functional group of interest, in this case the alternative of modifying the mobile ligand to a species capable of reacting with an already established matrix has been introduced. Primary amino groups have been derivatized with the bifunctional thiolating reagent N-succinimidyl 3-(2-pyridyldithio)propionate 363
Inc. reserved.
364
GABOR
(SPDP) (shown in Scheme 1) and the products have been shown to interact with the mercurial, acryloylaminophenylmercuric acetate (APM) gel system (4). MATERIALS
AND
METHODS
Bulk Escherichia coli tRNA and those specific for Phe (E. coli, yeast) and Tyr (E. cob) were from Boehringer. tRNALy” (E. coli) was obtained from Subriden RNA (Rolling Bay). Total maize chloroplast tRNA was isolated as described for tobacco (10). Pure E. coli tyrosyltRNA synthetase was a kind gift of Dr. H. Sternbach (Giittingen). APM was synthesized and incorporated at the indicated concentrations into polyacrylamide gels as described previously (4) using O.&mm-thick polyacrylamide gels in the presence of 7 M urea and Trisborate buffer, pH 8.3. Electrophoresis was at about 10 V/cm. 3’-terminal labeling with [32P]pCp was carried out as described (6) and silver staining was according to (11). Thiolation of acp3U with SPDP (Pharmacia) was achieved as follows: tRNA (25 pg or less) was incubated in 10 mM NaH,PO,/Na,HPO, buffer, pH 7.2, 100 mM NaCl at room temperature for 30 min with 20 mM SPDP (diluted from a 40 mM stock solution in ethanol) in a total volume of 100 ~1. This was followed by the addition of either 50 ~1100 mM dithiothreitol (DTT) (to generate the free thiol group) or 50 ~1 water (control). After 20 min, 10 ~1 of 3 M NaOAc was added and the tRNA was recovered by precipitation with 350 ~1ethanol at -20°C. Any residual DTT was removed by purifying the tRNA using a Qiagen column (Diagen). For this, the samples were dissolved in 450 ~1 of 100 mM NaC1/50 mM Mops, pH 7/15% EtOH. They were applied to Qiagen Tip20 columns which were then washed with 1.5 ml of the same buffer. Elution was with 3 X 0.2 ml Qiagen buffer F (1.5 M NaCI, 50 mM Mops, pH 7.5,15% EtOH) and the tRNA was precipitated with 0.75 ml isopropanol at -20°C. In the case of aminoacyl-tRNAs, SPDP treatment followed directly the aminoacylation reaction. Aminoacylation was as described previously (12), except that Tris was replaced by Hepes (to avoid the introduction of additional primary amino groups). After a 30-min incubation at 37”C, the tRNA was obtained by rapid phenol extraction and ethanol precipitation and the tRNA was kept cold for all subsequent operations, apart from the SPDP and DTT reactions, which were as described above. RESULTS
AND
DISCUSSION
The heterobifunctional reagent SPDP has, among other uses, been considered as a thiolating reagent for proteins, in particular (13). This is understandable in view of the limited occurrence of its target functional group, a free primary amino residue, in nucleic acids. It is, however, exactly the specificity of such succinimide derivatives which makes it possible to label selectively
L. IGLOI
those components in a nucleic acid population which bear primary amino groups (a fact which is utilized in biotin or fluorescent labeling of synthetic oligonucleotides at a Y-terminus-bearing NH, group (14)). In the case of tRNAs, this reaction is, at the present time, therefore, limited to the modified bases acp3U and acp2C (lysidine) (Scheme l), together with the a-amino moiety of aminoacylated tRNAs. The feasibility and specificity of this type of reaction have been demonstrated by coupling affinity probes or biotin to some of these sites via appropriate succinimide esters (15). Analogously, reaction with SPDP leads to thiolation and thus makes the reaction products amenable to characterization by affinity electrophoresis on a mercurial derivative of acrylamide. acp3U Figure 1B shows the electrophoretic pattern obtained when SPDP-derivatized E. coli tRNALy” (acp3U,,) was subjected to analysis on a mercurial affinity matrix. A retarded band whose appearance is dependent on cleavage of the S-S bond within the coupled reagent, by DTT, is observed. Incubation of the disulfide with water, instead of DTT, does not liberate a species amenable to interaction with the affinity electrophoretic matrix. The absence of a discernable SPDP reaction of yeast tRNAPhe (Fig. lA), which exemplifies tRNA species lacking an acp3U, confirms the fact that the common bases are not modified by this type of reagent (15). The strong retardation in the case of tRNALW seen at an immobilized ligand concentration of 0.1 pg/ml signifies an affinity comparable to that of the monophosphorothioate ester interaction observed previously (4) (see also below). The heterogeneous state of modification indicated by an only partial conversion of the tRNA to a retardable state reflects either the true in uivo state of the tRNA (undermodification, either naturally or by nature of the bacterial growth conditions) or an incomplete reaction of potentially available modified bases with SPDP. Undermodification has previously been observed with all tRNAs subjected to affinity electrophoretie analysis but the source of this heterogeneity has not been investigated in each case. Certainly, in the case of s*U, growth conditions have been shown to affect the state of modification (Emilsson and Kurland, personal communication) while commercial preparations of E. coli tRNAPhe (acp3U4,) h ave failed to react with SPDP (not shown). On the other hand, it cannot be denied that this indirect approach to the detection of specific modified bases, unlike the analyses relying on the interaction of an intrinsic partner with the immobilized ligand, e.g., s*U, must depend on the coupling yield between the free amino group and the succinimide ester. This has been determined in a number of instances to be between 60 and 90% (15) but a quantitative analysis of modified
AFFINITY
A
CH=CH
ELECTROPHORESIS
OF
365
tRNA
2
Lo I NH
0
0 - S-S-CH#$-O-N
3 OS
HgOAc
OH
OH
bH
Structures of the main of tRNAs containing
base content of a particular into account.
bH
acp%J
a mw SCHEME 1. retie fractionation
O*
components of the analytical system described in the text. (A) Reagents used during the modified bases (B), whose reactive amino groups are marked by asterisks.
tRNA
must take this factor
Other Base Modifications Treatment of bulk tRNA, in this case species isolated from higher plant chloroplasts, gives rise to a series of retarded bands (Fig. 1C). The individual moieties responsible for this pattern have not yet been fully characterized but, according to our present knowledge of modified bases in chloroplast tRNAs, one may hazard some observations as to the modifications involved. acp3U has been identified in a number of chloroplast tRNAs (9) at position 47. Furthermore, the only other primary amino-bearing base known to date, acp2C (lysidine), whose presence has been confirmed in tRNA”“(CAU) in E. coli (16) and plant mitochondria (17), is expected to play a similar crucial role in the chloroplast tRNA’“(CAU) by preventing the incorporation of Ile at Met codons. The multiple bands resulting from the SPDP reaction with bulk chloroplast tRNAs may then be correlated with acp3U at positions of differing reactivities/conformational flexibilities (see below), or within tRNAs of different sizes, or with a mixture of tRNA species with differing modifications, e.g., acp3U or acp2C. Aminoacyl-tRNAs The ester linkage between the 3’-terminal ribose and the carboxyl group of an amino acid is labile (see, e.g.,
affinity
electropho-
18) and, under standard conditions used for polyacrylamide gel electrophoresis, survives intact to only a limited extent. Fractionation in acid gels has recently been introduced to overcome this problem (19), while modification of the a-amino group has been practiced for some time in an effort to stabilize this ester bond (e.g., 20). Conversion of the -NH, to the thiol needed for affinity electrophoretic purposes simultaneously leads to a significant stabilization of the aminoacyl-tRNA at pH 8.3 under electrophoretic buffer conditions (Fig. 2). Above pH 9 the protection against hydrolysis is no longer observed (not shown). In order to monitor the migration of the aminoacyl moiety of the aminoacylated tRNA, rather than the tRNA itself, bulk E. coli tRNA was aminoacylated with a mixture of 16 14C-labeled amino acids, in the presence of an E. co& SlOO extract and the product treated with SPDP, as described under Materials and Methods. Subsequent affinity electrophoresis and fluorography (Fig. 3A) revealed a strongly retarded radioactive fraction which, in fact at an APM concentration of 0.5 pg/ml (1.25 PM), barely enters the gel, cf. the migration of the reaction product with acp3U. The control lane in which the [14C]aminoacyl-tRNA had not been treated with SPDP gives rise to two very faint nonretarded bands (due to species having escaped deacylation during electrophoresis) at a position equivalent to the tRNA marker. This indicates that retardation is due to the thiol group, now linked to the a-amino residue at the 3’ terminus, interacting with the affinity ma-
366
GABOR
L. IGLOI
B
FIG. 1. Affinity electrophoretic fractionation of SPDP-treated tRNAs. Yeast tRNAPh” (A), E. coli tRNA”” (B), and maize chloroplast total tRNA (C) were reacted with SPDP as described under Materials and Methods, or left untreated (control). In each case, onehalf of the product was reduced with DTT while the other half was incubated with water, as indicated. The tRNAs (approx. 50 rig/lane) were fractionated on a 5% polyacrylamide gel containing 7 M urea and 0.25 PM (0.1 pglml) APM. The bands were visualized by silver staining (11).
trix and confirms the stabilizing effect of the SPDP modification; only a fraction of the attached label survives the electrophoretic conditions in the unmodified lane. In the case of the SPDP-modified product, the amount of nonretarded material just visible on longer exposure indicates that the reaction is virtually complete.
100 -o-o90 +SPDP 80
\
‘0
- SPDP
\ =O I 0
I 20
FIG. 3. Detection of aminoacylated tRNAs by affinity electrophoresis. (A) Bulk E. coli tRNA was aminoacylated with a *‘C-labeled amino acid mixture using an SlOO extract. One-half of the product, after isolation as described under Materials and Methods, was left unthiolated while the other was thiolated with SPDP/DTT, as indicated. Affinity electrophoresis was carried out under the conditions specified in the legend to Fig. 1, except that the APM concentration was 1.25 pM (0.5 pg/ml) and the bands were detected by fluorography. As a marker, “P-labeled total chloroplast tRNA was run alongside the samples (control). The position of the bottom of the sample well is indicated by an arrow. (B) E. coli tRNA% was aminoacylated with nonradioactive tyrosine in the presence of pure tyrosyl-tRNA synthetase (Tyr + TRS) or an SlOO extract (Tyr + SlOO), as indicated. Controls were carried out in the absence of Tyr, lanes (TRS), (SlOO), or after deacylation. Affinity electrophoretic analysis was as described in the legend to Fig. 1 using an APM concentration of 0.1 pg/ml and the bands were detected by silver staining (11). The retarded band corresponding to aminoacylated tRNA is marked with an arrowhead.
I 40
1 60
80
I 100
, 120 1 (mln)
FIG. 2. Stabilization of the aminoacyl-tRNA ester bond by reaction with SPDP. E. coli tRNA was aminoacylated with a “C-labeled amino acid mix and then treated with SPDP (+) or used as a control (-), as described under Materials and Methods. The stability of the labeled tRNA at room temperature at pH 8.3 in TBE buffer was monitored by removing aliquots at the times shown and determining the number of TCA-precipitable counts remaining.
Having demonstrated the affinity electrophoretic retardation of tRNA species known to be aminoacylated (by radioactive assay) this method could now be used to test for aminoacylation per se, in the example of the tyrosine system. Commercial E. coli tRNA% was incubated with pure E. coli tyrosyl-tRNA synthetase and tyrosine, as described under Materials and Methods. SPDP modification was followed by affinity electrophoresis and silver staining (Fig. 3B). A substantial proportion of the tRNA is retarded by the interaction between the thiol and the mercury, while the rest migrates as unmodified tRNA. (Note that the concentration of APM in the gel (0.1 pg/ml, 0.25 PM) is far below that
AFFINITY
ELECTROPHORESIS
required to observe an interaction between tRNA% s~IJ,,~ and the matrix (10 pg/ml) (4)). Two control lanes demonstrate that the retardation is a consequence of aminoacylation (followed by thiolation). First, in the absence of the amino acid, no slow-migrating product is observed. Second, treatment of the product of aminoacylation with NaOAc, pH 9, for 30 min at 37°C (effectively, hydrolyzing any ester bond between the amino acid and the tRNA) prior to SPDP modification also abolishes the affinity interaction. Using an E. coli SlOO, tyrosyl-tRNA synthetase activity could be detected according to this protocol, although the situation is complicated by the presence in the extract of some high-molecular-mass nucleic acids which are detected by staining. Furthermore, proteolytic degradation in the extract liberates free tyrosine, which then gives rise to an aminoacylation-specific signal in the control reaction lacking added amino acid. Quantification Interaction
and Comparison
of the Binding
The relative extent of retardation of tRNAs which have been thiolated with SPDP at different sites is noteworthy. The rate of electrophoretic migration reflects the strength of the affinity between the interacting partners (21) and may, indeed, be used to quantify the binding. The binding constant between this matrix and a number of thiol derivatives has been determined rigorously by measuring the APM concentration-dependent variation of the electrophoretic mobility, according to the simplified affinity electrophoretic equation (for a more extensive discussion of quantitative aspects, see 22,23). Assuming that the complex once formed has zero mobility, the dependence of the mobility on the apparent binding constant K is given by l/r
,
= l/R,[l
+ c/K],
where r is the migration distance of one interacting partner in the presence of the immobilized ligand with a concentration c, while RO is the mobility at c = 0. For comparative purposes, one may use the previously determined affinity if one considers the concentration of APM at which the mobility of a component of unknown K, is identical to the relative mobility of a species of known KI . The equation is then approximated by K,/c, = K2/c2, where c1 and c2 are the APM concentrations, where species 1 and 2 have identical relative mobilities -a value which may be obtained from the known plot of { 1 - r/R,}-’ vs c-l for, say, s4U-containing tRNA. Table 1 gives the values for K which have been deduced in this manner for the SPDP derivative of acp3U and of aminoacyl-tRNA and summarizes the binding affinities which have been determined for this affinity matrix, to date.
OF
367
tRNA TABLE
1
Interaction Constants (PM) between Thiol-Containing Macromolecules and APM, as Determined by Affinity Electrophoresis in Polyacrylamide Gels Containing ‘7 M Urea tRNAMEt(s’U) (E. coli) tRNAG’“(mnm6s2U) (E. coli) tRNALyB(acp3UISPDP) (E. coli) Aminoacyl-tRNA/SPDP (a-amino) 5’-Phosphorothioatemonoester RNA monophosphorothioate RNA triphosphoro-y-thioate a From
8.0” 90.0” 0.1 0.02 0.3” t0.1”
(4).
This difference in affinity, however, cannot be seen as a simple intrinsic property of the molecules involved in complex formation since in both cases identical thiol groups become bound to identical mercury derivatives. It must then reflect some aspect of the residual molecules (tRNAs) and, since, as far as the chemical composition is concerned, even these are very similar, one is left with the interpretation that conformational differences may be the basic properties governing the strength of complex formation. This conclusion is not entirely unexpected since our previous studies have shown that a change in the overall conformation of a tRNA, as induced by denaturing agents, can lead to significant changes in the affinity electrophoretic migration in the case of s4U/APM or mnm5s2U/APM (4). These changes have been interpreted in terms of a structural flexibility or opening permitting a more favorable, stronger interaction. In the present case, this argument would again be applicable. The 3’ terminus, bearing the amino acid, is known from X-ray data of several tRNAs (24,25) to be in an exposed position and a thiol group here would be ideally situated, even in a native conformation, for the interaction considered here. On the other hand, acp3U4,, at a position in the molecule which is susceptible to chemical attack (24), is nevertheless flanked by bases which are involved in tertiary base pair interactions and must, therefore, be part of a more rigid architecture which, as has been shown previously (4), is only incompletely destroyed by 7 M urea. An interaction with the matrix is apparently sterically hindered, leading to a relatively weaker interaction. It should be mentioned that, based on the observation that multiple interacting sites within one molecule lead to a higher affinity (4,26), were one to aminoacylate a tRNA which simultaneously also contains an acp3U moiety, SPDP treatment of this species would result in a further retardation during affinity electrophoresis. In a comparison between the behavior of different sulfur-containing compounds in this affinity electrophoretie system, one must also take into account the chemical environment/reactivity of the sulfur. This is re-
368
GABOR
fleeted in the order-of-magnitude differences in the “dissociation constants” on going from sulfur atoms which form part of an aromatic nucleus, as in s4U or s2U derivatives, to the strongly nucleophilic phosphorothioate monoesters (Table 1) and the aliphatic thiols described here. The actual numerical values attributed to these latter affinities toward the organomercurial may, in view of problems involved in quantifying such tight, quasicovalent binding, only be approximate. Nevertheless, the fact that one can, on a gel, clearly distinguish the tRNA thiolated at position 47 from SPDP-modified aminoacyl-tRNA is a compelling demonstration of the sensitivity of affinity electrophoresis to the electronic and conformational intricacies of biomolecules (5). ACKNOWLEDGMENTS I thank Dr. H. K&se1 for fruitful discussions. I am grateful to Mrs. E. Schiefermayr for dedicated technical assistance and to Mrs. S. Krien for photographic work. This investigation has been supported, in part, by the Deutsche Forschungsgemeinschaft (Grant SFB 206).
1. Bj8rk, G. R., Ericson, J. U., Gustafsson, C. E. D., Hagervall, T. G., Jonason, Y. H., and Wikstrbm, P. M. (1987) Annu. Reu. Biochem. 66.263-287. 2. Edmonds, C. G., Cram, P. F., Gupta, R., Hashizume, T., Hocart, C. H., Kowalak, J. A., Pomerantz, S. C., Stetter, K. O., and McCloskey, J. A. (1991) J. Bacterial. 173, 31383148. 3. Igloi, G. L., and KBssel, H. (1985) Nucleic Acids Res. 13, 6881-
6898. 4. Igloi, G. L. (1988) Biochemistry 27,3842-3849. 5. Igloi, G. L. (1992) in Molecular Interactions in Bioseparation (Ngo, T. T., Ed.), Plenum, New York, in press. G. L., and Kossel, H. (1987) R., Ed.), Vol. 155, pp. 433-448,
7. Bezerra, R., and Favre, A. (1990) Biochem. Biophys. mun. 166,29-37. 8. Fritzsche, E., Hayatsu, H., Igloi, G. L., Iida, S., and (1987) Nucleic Acids Res. 15,5517-5528. 9. Sprinzl, M., Dank, N., Neck, S., and Acids Res. Suppl. 19, 2127-2171.
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