Vol. 186, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

July 31, 1992 Pages 956-962

FOOTPRINTING EVIDENCE FOR CLOSE CONTACTS OF THE YEAST tRNAASP ANTICODON REGION WITH ASPARTYL-tRNA SYNTHETASE

Angela GARCIA and Richard GIEGE*

Unite FVopre de Recherche “Structure des Macromolkules Biologiques et Mkanismes de

Reconnaissance”, Institut de Biologie Mok&laire et Cellulaire du Centre National de la

Recherche Scientifique, 15 rue Rent Descartes, 67084 Strasbourg-Cedex, France

Received June 3, 1992

Summary : Chemical footprinting experiments on brewer’s yeast tRNAAsP compl.exed to its cognate aspartyl-tRNA synthetase are reported : they demonstrate that bases of the anticodon loop, including the anticodon itself, are in close proximity with the synthetase. Contacts were determined using dimethylsulfate as the probe for testing reactivity of guanine and cytosine residues in free and complexed tRNA. Results correlate with the decrease in aspartylation activity of yeast tRNAAsp molecules mutated at these contact positions and will be compared with other structural data arising from solution and crystallographic studies on the aspartic acid complex. 0 1992 Academc Press. mc.

One of the crucial step for the fidelity of translation during protein synthesis concerns

the correct aminoacylation of a tRNA species by its cognate aminoacyl-tRNA synthetase.

Because of its biological importance, this process has been intensively studied over the

years, but despite the great knowledge that accumulated, specific recognition of tRNAs by

synthetases is only partly understood (for recent reviews, see ref. l-4). Among

aminoacylation systems, that specific for aspartic acid in yeast has been studied in great

details. In particular, the question of the recognition process of yeast tRNA&P by aspartyl-

tRNA synthetase has been adressed using a variety of different approaches. Functional

studies carried out on tRNA&IJ molecules transcribed in vitro have shown that mutation of

a few specific bases is leading to strong decrease in aspartylation activity (5). The complex

formed between tRNA*W and the synthetase was also analyzed by footprinting methods

using various probes targeting the reactivity of phosphate groups (6-8). Altogether, these

*To whom correspondence should be addressed at Institut de Biologie MolCculaire et Cellulaire du Centre National de la Recherche Scientifique, 15 rue RenC Descartes, F- 67084 Strasbourg Cedex, France. Fax: (33) 88.61.06.80.

Abbreviations. tRNA@, transfer RNA specific for aspartic acid.

Enzymes. Aspartyl-tRNA synthetase (EC 6.1.1.12) ; ribonuclease Tl (EC 3.1.27.3) ;

venom phosphodiesterase (EC 3.1.4.1) ; tRNA nucleotidyl-transferase (EC 2.7.7.21).

0006-291X/92 $4.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproducrion in any form reserved. 956

Vol. 186, No. 2, 1992 tjlOCtiEibl.CAL AND BIG, HYSICXi RESEAHCtI COrL/IMUNICATIONS

experiments have concluded to the same type of tRNA/synthetase interaction : the tRNA

would contact with the synthetase along the variable loop side with interactions in the

region of the anticodon, the D-stem and the 3’-part of the aminoacylation stem. Meanwhile,

the crystallographic structure of the complex has been solved (9). It explicitely shows that

tRNA approaches the synthetase at the above regions in an overall interaction scheme that

was partly foreseen by the footprinting experiments, but without direct contacts with the

variable region. One of the important finding of the X-ray work was the demonstration of

an important conformational change in the ant&don region of complexed tRNA*sP (9) as

compared to the conformation in the free tRNA (10, 11). The tight interaction of the

anticodon as observed in the X-ray structure (9) as well as the mutational analysis showing

that the three bases of the anticodon were identity determinants (5), prompted US to analyze

by chemical footprinting the interaction of the anticodon region with the synthetase.

Fortunately, most of the bases of the ant&don, as already shown by Romby et al. (12) can

be alkylated in the presence of dimethylsulfate (DMS), a probe reacting with the N3 and N7

positions of cytosines and guanines, respectively. Consequently, DMS could be a suitable

tool to determine specific contacts or proximities in solution of the anticodon loop

nucleotides with the synthetase.

Following these lines, the present paper is a complement to the crystallographic

studies on the tRNA*sP/aspartyl-tRNA synthetase complex. It presents novel footprinting

data in solution indicating protections of anticodon loop residues of tRNAAsP by the

synthetase. It completes footprinting investigations already done on this complex with

phosphate specific reagents, using for the fist time a chemical probe, DMS, specific for the

bases. Results show that the 3’-part of the anticodon loop is in proximity or even in contact

with the synthetase, with in particular the positions N7 of G34 and N3 of C36 clearly

protected against alkylation. Because these two bases, G34 and C36, have been identified as

identity determinants for aspartylation (5), it can be concluded that anticodon residues

participate specifically in this process by direct and specific contacts with the synthetase.

MATERIALS AND METHODS

tRNA, enzymes and chemicals. Yeast tRNAASP was prepared from total brewer’s yeast Saccharomyces cerevisiae (Boebringer-Mannheim France SA, Meylan) by counter- current distribution (13) followed by column cbromatographies on BD-cellulose and Sepharose 4B matrices (14). Dimeric a2 aspartyl-tRNA syntbetase from baker’s yeast was purified to homogeneity according to a standard procedure (15). Snake venom phosphodiesterase was from Worthington (Freehold, USA) ; tRNA nucleotidyl-transferase was prepared from commercial baker’s yeast (FALA, Strasbourg, France) according to Rether er al. (16). Ribonuclease Tl was from Sankyo (Tokyo, Japan). [a-szP]ATP at 4 10 Ci/mol and L-[3H] aspartic acid at 32 Ci/mol were from Amersham France SA, Les Ulis. DMS was purchased from Aldrich-Chimie (Strasbourg, France).

End-labeling of tRNA, To label tRNA at its 3’-end, molecules were first deprived of their CCA-end sequence by limited snake venom phosphodiesterase hydrolysis and reconstituted in the presence of cold CTP, [a-32P]ATP and tRNA nucleotidyl-transferase

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(16). Labeled tRNAs were purified by high voltage gel electrophoresis on 15 % (w/v) polyacrylamide/8M urea gels.

Alkylation of bases with dimethylsulfate. Chemical modification experiments were conducted on 3’-end labeled tRNA@’ molecules. Alkylation of N7 positions in guanine residues and N3 positions in cytosine residues with DMS and analysis of the results on polyacrylamide gels, were done according to well established procedures (12, 17). Alkylation of bases was done under native conditions (12 mn, 37 “C, 10 mM MgClz), semi-denaturing conditions (12 mn, 37 “C, 1 mM EDTA) and denaturing conditions (1 mn, 90 ‘C, 1 mM EDTA). The reaction mixtures were buffered at pH 7.2 with 50 mM sodium cacodylate. Labeled tRNAhP (2 PM, 100 000 cpm) was mixed with 0.25 % (v/v) DMS. The reaction mixtures were supplemented with 4 uM aspartyl-tRNA synthetase in footprinting samples. This enzyme concentration is IOO-fold above the K,,, for tRNA*sp and ensures a almost complete complexation of the tRNA. After modification, synthetase was removed by phenol extraction. Modified guanine bases were split in 1 M Tris-HCl at pH 8.2 containing 0.2 M sodium borohydride ; modified cytosine residues were split in hydrazine 10 %I (v/v). Incubations were made at 0 “C during 5 mn. Chain scission was induced by aniline at 60 “C. Control experiments were performed under native conditions without DMS, but with the same borohydride and aniline treatments. Fragments of tRNA resulting from DMS modification and subsequent splitting were analyzed by polyacrylamide gel electrophoresis followed by autoradiography of the gels.

Enzymatic activity measurements of aspartyl-tRNA synthetase. Enzymatic activity of the synthetases (DMS treated or not) was measured at limiting enzyme concentration at 30 “C in the following incubation mixture : 100 mM Hepes-KOH buffer at pH 7.2 containing 30 mM KCl, 5 mM ATF, 10 mM MgC12,2.5 mM glutathione, 50 pM L-[WI aspartic acid and an excess of tRNA ; after incubation samples were treated in the conventional manner (18).

RESULTS AND DISCUSSION

In this work, it was firstly intended to study the possible interaction of the anticodon

region of tRNA*sP with aspartyl-tRNA synthetase. As the anticodon loop is rich in

cytosine and guanine residues, we choose to probe this region with DMS (17). The reactive

positions towards this probe are usually located either in single-stranded regions at N3C

and N7G positions or in helices at N7G positions. In that case, reactivities occur only when

the G residues are surrounded by at least one pyrimidine residue or when they are present

in non-regular helices. The above conclusions were derived from comparative studies on

tRNAhP and tRNAPhe (12). For tRNAAsP, experiments showed that all G and C residues

of the anticodon loop were fully reactive, together with conserved G18 and Gl9 located in

the D-loop. Some potential positions embedded in double-helices were slightly alkylated

but became fully reactive under semi-denaturationg conditions (e.g. without magnesium

ions at 37 “C) or under denaturating conditions (at 90” C).

Since DMS alkylates proteins (19), it was necessary as a prerequisite to the

footprinting experiments on complexed tRNA*sP to verify the effect of the alkylating

reagent on aspartyl-tRNA synthetase activity. Alkylation of free enzyme under conditions

of chemical mapping of the tRNA/synthetase complex provoked partial enzyme

inactivation. However, the remaining specific activity of aspartyl-tRNA synthetase was still

21 % that of the non-treated protein. Interestingly enough, the inactivation is significantly

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Vol. 186, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

N7G

[RNA complex tRNA complex

NS,D CR,C N&DC R,C H Tl C,

-65

-68

Figure 1. Alkylation of N7G and N3C positions in tRNA*sp. (N), (SD) and (D) tRNA treated with DMS in native, semi-denaturating or denaturating conditions, respectively ; (RS) tRNA complexed to the synthetase and treated with DMS ; (C) control treatment of free tRNA or comP!exed tRNA in the absence of DMS ; (H) formamide ladder ; (lY) Partial digestion of the tRNA by ribonuclease Tl ; (Co) labeled tRNA without any treatment. The numbering of bands corresponds to that of phosphate groups and follows the nomenclature of the tFWA data bank. Full and empty arrows indicate the protection of N7G and N3C Positions respectively in the complexed tRNA.

reduced in the presence of tRNA. Here, only 50 % of the catalytic activity of aspartyl-tRNA

synthetase is lost. This indicates that aspartyl-tRNA synthetase is rather unsensitive to

alkylation by DMS, especially when complexed with tRNA4. Similar observations were

reported on other aminoacylation systems where DMS was the probe to determine tRNA

contacts with their cognate synthetases, like on beef tRNATrP (20) and E. coli tRNAThr

(21). Altogether these data justify the use of DMS as a tool to monitor contacts of the

anticodon region of tRNAAV with aspartyl-tRNA synthetase.

Figure 1 displays the autoradiogram of a typical polyacrylamide gel of a DMS

alkylation experiment on N7G and N3C positions of free or complexed yeast tRNA&P. As

expected from former studies on free tRNA (12), the anticodon stem and loop residues

G30, G34, C36, mlG37 and C38 are all readily alkylated under native conditions. In the

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Vol. 186, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A c c

G U-A

C-G

C-G

G-C

U-G

G-C

D AA

A- U 10 u

G YUuGA

70

60

“A GCCCC I I I I I A

II II m5C G G G G G GAAU

D c A

US0 G A

TY’

20 G-C A G

G- C

C- G

30 0-u 40 c- G

Y El

U mlG

o,Fl

Figure 2. Cloverleaf structure of yeast tRNA*sP (27) with residues protected agamst DMS alkylation in the complex with cognate aspartyl-tRNA synthetase. Protected guanine and cytosine residues are circled or boxed. Regions that could not be tested are indicated by lines.

complex, the NT position of G30 in the anticodon stem and of G34 in the first position of

the anticodon are clearly protected against alkylation by the interacting aspartyl-tRNA

synthetase (see full arrows on the autoradiogram). Similarly, the N3 positions of C36 and

C38 become unreactive in the complexed tRNA (see open arrows on the autoradiogram).

All the residues becoming non reactive in the complex are reported on a tRNA cloverleaf

structure (Figure 2). On the contrary, the N7 atom of mlG37 and G39 remain reactive in the

complex. Also worth mentioning, no G residue located in stem regions of tRNA*sP and

unreactive in the free tRNA, becomes reactive in the complex. This means that no structural

perturbation occurs in stem regions of the complexed tRNA.

How do these results correlate with other footprinting data concerning the anticodon

region of yeast tRNAksp ? Experiments performed with phosphate specific reagents yielded

similar protections patterns, indicating the existence in the anticodon arm of an extended

stretch of the tRNA backbone in contact with the synthetase. With the photoactivatable

aryldiazonium reagent, the majority of phosphates in the anticodon loop (except P38 to P39

that were split unspecifically) were unreactive upon complexation with the synthetase (7).

With ethylnitrosourea, Romby et al. (6) found P37 and P38 protected (degradations induced

by reaction conditions were occuring in the 5’-part of the loop and hid possible protections

in this region). More recently, a method based on cleavages of phosphorothioate containing

tRNA transcripts by iodine (22) was applied on the aspartic acid system and identified non

reactive phosphates at ant&don positions 34 and 35 in complexed tRNAASP (8). In the

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Vol. 186, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

antic&on stem, protected phosphates were detected on both RNA strands depending on the

probe used. Probably some of the backbone contacts involve H-bonds or salt-bridges

between phosphates and amino acids counterparts that contribute to the binding energy of

the tRNA to the enzyme, but in any case these contacts can mediate the specificity of

tRNA*sP aminoacylation. However, some of the phosphates identified in the above

footprinting experiments are probably located in the neighborhood of nucleotide bases that

contribute to specific binding and thus to identity of tRNAAsP.

The present DMS footprinting experiments suggest that specific contacts with

aspartyl-tRNA synthetase occur in the ant&don loop. Protection of G34 and C36 against

alkylation, together with the results of the mutational analysis of tRNA*sP (5), prove that

these residues participate directly in the aspartic acid identity of the tRNA. The base

mlG37, on the other hand, remains reactive and although being very close to the synthetase

(as deduced from the protection of surrounding c38 and C36 bases) does not participate in

tRNA*sP identity (5). The protected residue c38 may also be involved in specific

recognition, in a similar way as was seen in the crystallographic structure of complexed E.

coli tRNAG*n (23, 24). Finally, protection of the N7 position of G30 indicates that the

synthetase approaches the ant&don stem by the major groove side, in agreement with the

recent crystallographic data on the aspartic acid complex (9).

Interestingly, interaction points of yeast tRNAAsP and E. coli tRNAGln anticodon

loops with their respective synthetases are similar, although they belong to different

synthetase families (25). The tRNA Gin bases from C34 to y38 in the crystallographic

structure are interacting directly with E. co/i glutaminyl-tRNA synthetase (24). According

to the present study, bases 34, 36 and 38 of tRNA&P are in contact with aspartyl-tRNA

synthetase. In both X-ray structures of the glutamine and aspartic acid complexes,

important conformational changes within anticodon loops were reported leading to

unstacking of anticodon residues (9,23,24). This facilitates recognition of the anticodons

by the synthetases. Such conformational changes may occur in other tRNAs known to

interact with synthetases through antic&on base contacts, as found in the methionine (26),

threonine (21) and tryptophan (20) systems after DMS footprinting, and conformational

rearrangements in the anticodon loop and stem were indeed proposed in the case of E. cofi

tRNAfMe* complexed to methionyl tRNA synthetase (26). Thus, unstacking of anticodon

may be a general process allowing synthetases to read anticodon sequences, and

consequently footprinting experiments probing bases could help to define whether

anticodon loops are subjected to tight interactions with synthetases that lead to such

conformational changes.

ACKNOWLEDGMENTS

This work was partly supported by grants from the Centre National de la Recherche Scientifique (CNRS), the Minis&e de la Recherche et 1’Enseignement Sup&ieur (MRES), and UniversitC Louis Pasteur (Strasbourg, France).

Vol. 186, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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