Evidence for interaction of an aminoacyl transfer RNA synthetase with a region important for the identity of its cognate transfer RNA.

Recent experiments showed that a single base pair (G3:U70) in the amino acid acceptor helix is a major determinant for the identity of Escherichia coli alanine transfer RNA. Experiments reported here show that bound alanine tRNA synthetase protects (from ribonuclease attack) seven consecutive phosphodiester linkages on the 3'-side of the acceptor-T psi C helix (phosphates 65-71) and a few additional sites that are in scattered locations. There is no evidence for interaction of the enzyme with the anticodon, a sequence which can be varied without effect on recognition by alanine tRNA synthetase.

Recent experiments showed that a single base pair (G3:U7') in the amino acid acceptor helix is a major determinant for the identity of Escherichia coli alanine transfer RNA. Experiments reported here show that bound alanine tRNA synthetase protects (from ribonuclease attack) seven consecutive phosphodiester linkages on the 3'-side of the acceptor-TQC helix (phosphates 65-71) and a few additional sites that are in scattered locations. There is no evidence for interaction of the enzyme with the anticodon, a sequence which can be varied without effect on recognition by alanine tRNA synthetase.
The identity of each transfer RNA (tRNA) is established by interactions with aminoacyl-tRNA synthetases. These interactions determine which triplet nucleotide sequence (anticodon/codon) is assigned to which amino acid. While the three-dimensional structure of transfer RNA is well established, there is at present no high resolution structure of a synthetase-tRNA complex. In early work, a variety of genetic, physical, and chemical experiments suggested regions on the L-shaped tRNA structure which make contact with bound aminoacyl-tRNA synthetase, and this led to a schematic model for the complex (1,2).
Although the basis for the identity of a tRNA had long been obscure, recent evidence shows that a simple structural element can be a major determinant for identity (3). In Escherichia coli tRNAA'*, a single base pair (G3:U7O) in the acceptor helix is a signal for alanine. When this base is introduced into the analogous position of tRNACys (3) tRNAPhe (3, 4), these molecules are aminoacylated with alanine.
E . coli alanine tRNA synthetase is an a4 tetramer of identical 875 amino acid polypeptide chains. Sequences essential for the aminoacyl adenylate synthesis reaction, subunit interactions, and for interaction with tRNAA'" have been defined by the analysis of a set of 18 polypeptide fragments, which were generated by gene deletions (5)(6)(7). Although the physical association of alanine tRNA synthetase and fragments of this enzyme with tRNAA'" have been well studied (7), there have been no investigations of the complex per se. In this paper, we present initial attempts to characterize the synthetase-* This work was supported by Grant GM 15539 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tRNA complex using the complete native protein. These experiments utilize nuclease protection assays to map phosphodiester bonds that are shielded by the bound enzyme. While the nuclease probe is coarse, it nonetheless provides a useful first delineation of physical contacts between the enzyme and nucleic acid. In particular, we wished to determine whether contacts made by the enzyme included sites on the amino acid acceptor helix at the location of the G3:U7' base pair.

EXPERIMENTAL PROCEDURES
Materials-E. coli Ala-tRNA synthetase was purified by Dr. Kelvin Hill in this laboratory according to a method reported elsewhere (8). The concentration of the enzyme was determined by active site titration (9). The was obtained from Subriden RNA (Washington) and further purified by electrophoresis on a 12% polyacrylamide, 7 M urea gel. The [m3'P]ATP and [Y-~'P]ATP were purchased from Du Pont-New England Nuclear. The tRNA-nucleotidyltransferase was kindly provided by Dr. Murray Deutscher (University of Connecticut Health Sciences Center). Cobra venom RNase VI, RNase TI, and RNase CL3 were purchased from Pharmacia LKB Biotechnology Inc. Bovine pancreatic RNase A, calf intestine alkaline phosphatase, and snake venom phosphodiesterase were from Boehringer Mannheim. Polynucleotide kinase was obtained from New England Biolabs. The units of RNase A and RNase VI were taken as those described by the manufacturers.
Labeling of tRNAs-tRNA was labeled at either the 5'-or 3'-end with 3zP according to standard procedures (lo), except for a few modifications. The end-labeled tRNA was resolved by electrophoresis on a 12% polyacrylamide, 7 M urea sequencing gel. Typically, more than 2 X lo6 cpm of labeled tRNA was obtained from 0.5 pg of tRNA.
The end-labeled tRNA was excised from the gel and purified according to Wurst et al. (11). RNase A Protection Assays-For assays of protection of the 3'-end of the tRNA, the digestion conditions were adjusted to obtain a limited number of cleavages. The buffer used for RNase A partial hydrolysis was 30 mM sodium cacodylate (pH 5.5), 10 mM MgC12, and 2 mM dithiothreitol, and contained 400 nM [3'-32P]tRNAAla in a reaction volume of 100 pl. (Assays were also done at pH 7.0 in 50 mM HEPES' buffer). Upon addition of 0.05 unit of RNase A, the digestion was performed at 0 "C in the absence and in the presence of Ala-tRNA synthetase. The concentrations of the synthetase are given in the figure legends. Aliquots (20 pl) were withdrawn to a Whatman (grade 3) filter pad at various times. The reaction was stopped immediately by placing the filter in 5% trichloroacetic acid. The filter was washed twice with 5% trichloroacetic acid, twice with ethanol, once with ether, and then dried. The radioactivity on the filter was determined by scintillation counting.
In order to identify the positions of tRNAMa digested by RNase A, the [5'-32P]tRNA (0.4 p~) was digested at pH 5.5 by 0.05 unit of RNase A in the absence and in the presence of the synthetase (2.8

p~) .
The reactions were stopped by adding an equal volume of a pheno1:chloroform solution. After re-extracting the organic phase, the combined aqueous solutions containing tRNA fragments were precipitated, dried, and then dissolved in a 10 M urea, 10 mM EDTA solution. A defined amount of radioactivity was subjected to electrophoresis on a 12% polyacrylamide, 7 M sequencing gel.
RNase VI Protection Assay-The cobra venom RNase VI protection assay was done in 10 mM potassium phosphate (pH 7.0), 10 mM MgC12, and 2 mM dithiothreitol at 0 "C (12) with a final reaction volume of 30 p1 that contained 6.5 p~ tRNAA'" and 60 p~ Ala-tRNA synthetase. The ratio between RNase VI and tRNA was 0.2 unit/pg of tRNA. The digestion products were resolved by electrophoresis on 15 and 20% polyacrylamide sequencing gels.
cleavage gives a mixture of 2'-and 3'-phosphates. As a result, with 5"labeled tRNA, oligonucleotides (up to dodecamer) produced by RNase TI and RNase A, and by hydroxide ion cleavage, migrate more rapidly than the corresponding oligonucleotides produced by cobra venom RNase VI cleavage. With 3'4abeled tRNA, the converse is true.
Sequence Identification of tRNAA"-The sequence of tRNAALa was taken from Sprinzl et al. (13) who list two isoacceptor tRNAALa sequences from E. coli with a UGC anticodon. The differences between these two sequences are in the acceptor stem. We determined the sequence of the 5'-side of the acceptor stem using RNase TI (Gspecific) and RNase CL3 (C-specific). The sequence of tRNAA'a'UGC that matched with our result is shown in Fig. 1 where phosphates p have been numbered consecutively from the 5'-end.

RESULTS
RNase A Protection-Bovine pancreatic RNase A cleaves specifically after pyrimidines with a preference for singlestranded regions of tRNA (14). It is also known that the C-C-A sequence of the 3' terminus of tRNA is especially susceptible to RNase A attack (15). The effect of binding of Ala-tRNA synthetase to the 3' terminus of tRNAAL" was quantitatively tested by exposing [3'-32P]tRNAALa to RNase A. Plots of the fraction of acid-precipitable radioactivity remaining on the filter pad uersus time of exposure to RNase A are shown in Fig. 2. Oligonucleotides greater than 6 residues are retained on the filter during trichloroacetic acid precipitation, but smaller oligomers may not be precipitated and are then washed away from the filter (16). Thus, the method used in Fig. 2 gives a measure of detecting the RNase A attack on the 3'-end of tRNA. The data show that the synthetase shields the 3' terminus of tRNAAL" and that increasing amounts of Ala-tRNA synthetase afford increased protection. The concentration of the synthetase required for full protection at pH 5.5 was much less than that required at pH 7.0. This result is consistent with the observation that the interaction between tRNA and aminoacyl-tRNA synthetases is much weaker at higher pH values (2). The use of bovine serum albumin instead of Ala-tRNA synthetase did not protect tRNAAL" from RNase A attack. Protection of the 3' terminus from RNase A attack has also been observed for the interaction of E. coli Ile-tRNA synthetase with tRNAILe (17). Fig. 3 shows the pattern of partial digestion by RNase A and the protection by Ala-tRNA synthetase. In summary, positions p9, p17, p18, p33-p35, p37 and p75-p76 of the free tRNA are digested by RNase A (shown by arrows in Fig. 1). There are additional digestions in the helical segments of tRNAA'", but this is probably due to secondary cleavages resulting from the loss of the integrity of the three-dimensional structure of tRNA after the primary cleavages have occurred (not shown in Fig. 3). We did not give serious consideration to these secondary digestions. Position 15 of tRNAA'" is G, and, therefore, the cleavage by RNase A at p16 is not expected. This cleavage may be due to a contaminating RNase.
In the presence of Ala-tRNA synthetase, p9, p17, p18, and the 3' terminus (p75 and p76) of tRNAAL" were protected from RNase A attack (shown by the subset of arrows that are slashed in Fig. 1). In these experiments, we define protection by observations made at 1-and at 2-min time points, where multiple cleavages are minimized. The different intensities of the bands for specific fragments in the presence and in the absence of the synthetase were clear and reproducible, even if the protections at some positions were not as strong as at others. According to the gel pattern in Fig. 3, the anticodon loop of free tRNAA'" is one of the main targets of RNase A digestion. However, the nucleotides in the anticodon loop were not protected by Ala-tRNA synthetase. This result con- trasts with the protection of the anticodon loop of tRNA1Ie by Ile-tRNA synthetase (17).
RNase Vl Protection-Ribonuclease from cobra venom Naja oxiana preferentially cuts the double-stranded regions of RNA molecules without any base specificity (18). Although it also digests single-stranded polynucleotides, its affinity for double-stranded regions is much higher (19). In the protection assays, the reaction conditions were again defined to make limited cuts in the tRNA.
In the absence of Ala-tRNA synthetase, the prominent positions digested by RNase V1 are p4-p8, p15, p28-p30, p42, p49, and p63-p70 (Figs. 4 and 5). Most of these cleavages are in the anticodon and acceptor stems. (The minor cleavages of [3'-32P]tRNA at the anticodon loop may be the products of either a secondary digestion during the reaction and/or to tRNA molecules that were nicked during sample preparation). Some of the band intensities are rather weak compared to others (e.g. p71 and p72 in Fig. 5A). Because small oligonucleotides are not efficiently precipitated, they are difficult to detect on the gel. The digestion at p15 of the D-loop is possibly due to structure formed by the tertiary interaction between Us and AI4 as indicated in the three-dimensional structure of yeast tRNAPhe (20). These two residues are strongly conserved and make reversed Hoogsteen hydrogen bonds. Additionally, it is possible that C4' makes a reversed Watson-Crick type base pair with G15, as shown in the structure of tRNAPhe (20). When [3'-32P]tRNA was used, digestions at p40 and p41 were observed, and these were reproducible (Fig. 5). However, these cleavages were not observed when [5'-32P]tRNA was used (Fig. 4). Because p42 is the prime target of RNase V1 attack, the cleavages at p40 and p41 are more difficult to detect with 5'-labeled tRNAA1'. In the presence of Ala-tRNA synthetase, p14, p40-p42, and p65-p71 were protected from RNase V1 attack. With this ribonuclease, protection was defined by observations at 5and at 10-min time points. Even if the band intensity for p71 is weak, the protection from RNase V1 attack at this position is clear and reproducible. Similarly, the protections at p40 and p41, which are adjacent to the strong protection at p42, are evident even if the intensities at these positions are weak (Fig. 5B). The protection by a bound cognate synthetase at position 41 was also found for E. coli and yeast tRNAPhe, and for yeast tRNAVa1 (12,19). An enhancement of cleavage is observed at position p28. This may result from a conforma- There is protection by bound enzyme of a run of consecutive phosphates (p65-p71) on the 3'-side of the acceptor helix. However, protection of the 5'-side of the helix was not observed. With the experimental conditions used in Fig. 4A, there was evidence of some protection of p4 and p5 at the 5min reaction time, but we did not consider this protection as significant because, as the digestion progressed further, the effect became less obvious. Fig. 1 summarizes the positions of tRNA"" cleaved by RNase A and RNase VI. It also shows those positions protected from RNase attack by Ala-tRNA synthetase. The protections at some positions are much weaker than at others, and the consistent results are listed in Fig. 1.

DISCUSSION
The use of cobra venom RNase V1 provides valuable information for probing at the acceptor and anticodon stems. The pattern of limited digestion by RNase V1 can be compared with that of digestion of other tRNAs. In the case of yeast tRNAPhe and yeast tRNAArg, no digestions were observed on the 5'-side of the acceptor stem, while yeast tRNAV"' showed extensive digestions on both sides of the acceptor stem (12,19). The common features of RNase V1 digestion for all of these tRNAs (including E. coli tRNAA'") are that the acceptor stem and the anticodon stem are the primary targets of RNase V1 attack. The variations shown at the other positions may reflect subtle differences in the structures of these tRNA molecules.
The results show the significance of the role of the acceptor stem in the interaction between tRNAA'" and Ala-tRNA synthetase. The G3:U7O base pair of tRNANa is known to be a recognition site by Ala-tRNA synthetase. Neither the A3:U7' nor the G3:C7' mutant of the G3:U7' base pair is aminoacylated with alanine by the Ala-tRNA synthetase in uivo or in vitro, whereas mutations at many other positions do not prevent aminoacylation by the synthetase (3). The results of the ribonuclease protection experiments suggest that the enzyme contacts a series of sites in the acceptor stem that include ribose-phosphate backbone flanking the G3:U70 base pair. Because the 3'-end of tRNAA'" is also protected from RNase A attack (Fig. I), it appears that the synthetase binds along the 3'-side of the acceptor stem and into the single-stranded regions of the 3'-end of tRNAA'". While the data on ribonuclease protection of the acceptor stem correlate with the importance of this region as determined in genetic studies, the protection of parts of the anticodon stem contrasts with the lack of an effect (on recognition in uiuo) of mutations in this part of the molecule (3). Possibly Ala-tRNA synthetase binds to phosphates and makes no contacts with specific bases in the anticodon stem or possibly it is sufficiently close to this part of tRNAA'" to afford protection from nuclease attack without making any binding interactions.
When the protected regions in Fig. 1 are viewed on the three-dimensional structure of tRNA, the pattern of protection of consecutive phosphates in the 3"region suggests that the enzyme wraps around the acceptor helix. The protected position p9 on the 5'-side is proximal to the lower parts of the acceptor helix and, if the enzyme wraps along the backbone of the 3'-side of the acceptor helix, then a contact between p9 of tRNA and the protein seems inevitable. The contact of the enzyme on the 3"side of the acceptor stem could also extend to parts of the D-loop to accommodate the protections shown at p15, p17, and p18 of tRNAA'". The enzyme must be extended in shape, however, in order for another part (or domain) to shield a portion of the anticodon stem.