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Crystallization of RNA-protein complexes
METHODS: A Companion to Methods in Enzymology Vol. 1, No. 1, August, pp. 7 5 - 8 2 , 1990
Crystallization of RNA-Protein Complexes J o h n J. P e r o n a Department of Pharmaceutical Chemistry, 926 Medical Sciences Building, University of California, San Francisco, California 94143
The widespread occurrence of RNA-protein interactions in critical cellular processes makes an understanding of the structural basis for recognition and sequence discrimination of paramount importance. Until recently, however, the difficulty in obtaining large quantities of homogeneous RNA had made structural investigations of many interesting species by biophysical methods impossible. Summarized here are the recent applications of several in vivo expression systems to the overproduction of a number of RNA species. The potential usefulness in crystallization of RNA synthesized in vitro by T7 RNA polymerase is also discussed. Together with advancements in separation methods by HPLC, these two methods make a much wider range of systems accessible to structural analysis. A comparison of the successful cocrystallizations of two complexes of arninoacyl-tRNA synthetases with their cognate tRNA substrates is presented. © 1990 Academic Press, Inc.
RNA plays a central and increasingly recognized role in many aspects of cellular structure and function. RNAs possess both functional and structural roles in the transmission of genetic information from DNA to prot e i n - - a s messenger molecules, as components of the splicing apparatus and of the ribosome, and as small adaptor molecules (tRNA) which provide the critical link between nucleotide and amino acid sequence information (1). Retroviral genomes are composed entirely of RNA, demonstrating the ability of this molecule to serve also as an organism's storehouse of genetic information. Certain RNAs also function enzymatically in self-splicing and tRNA maturation reactions (2, 3). The crystal structures of several tRNAs have been determined at atomic resolution (4-6; R. Basavappa and P. B. Sigler, personal communication). From these structures it is clear t h a t large RNAs are in general likely to possess compact, globular conformations containing singlestranded loop regions as well as helical sections. In tRNA, the tertiary fold is maintained in part via hydrogen bonding and hydrophobic stacking interactions between nucleotide bases in parts of the polynucleotide chain t h a t are distant from each other in primary se1046-2023/90 $3.00
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
quence. Additionally, the sugar-phosphate backbone possesses considerable flexibility, which is likely to play a critical role in the interactions of the RNA with proteins. The stereochemical rules governing these proteinnucleic acid interactions are at present largely unknown and constitute a major area of biophysical investigation (7). X-ray crystallographic methods currently offer the only well-established means for obtaining atomic-resolution views of RNA-protein interaction. However, the preparation of large quantities of homogeneous RNA and its subsequent cocrystallization with protein have proven to be very challenging steps t h a t have thus far limited investigation to a small number of systems. Crystals of RNA t h a t diffract to medium resolution or better have thus far been reported only for a variety of transfer RNA species (8-11, 43), for a fragment of 5 S RNA (12), and recently for a 14-bp helical RNA duplex for which the structure has been determined at high resolution (13). In most of these cases, the RNA was obtained either by purification directly from large quantities of bacterial or yeast cells (8-11, 43) or by chemical synthesis in solution (13). The limitations inherent in these methods (requirements for high intracellular abundance and difficulties in reproducibility for the former; size limitations for the latter) do much to explain why the current database of available RNA structures is so limited. Described here are several bacterial expression systems, originally developed for the overproduction of proteins, that have been applied to the in vivo synthesis of multimilligram quantities of a number of RNA species. The usefulness of in vitro synthesized RNA in the preparation of high-resolution diffracting crystals is also discussed. Together, these systems hold the promise of significantly enlarging the number of RNA-protein complexes t h a t may become amenable to crystallographic study. The in vivo overproduction of glutaminespecific tRNA via an engineered bacterial strain was instrumental in the recent growth of crystals of the Escherichia coli glutaminyl-tRNA synthetase:tRNAa.n complex (14). A comparative analysis of the crystallization conditions used for this complex with those utilized 75
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JOHN J. PERONA
in the crystallization of the yeast aspartyl-tRNA synthetase:tRNA Asp complex (15) is presented. Although due regard for the structural properties and solution behavior of RNA is warranted, in general the approaches t hat have been used for successful crystallization of RNA and of RNA-protein complexes have been quite similar to those used in many laboratories over the past generation for the crystallization of proteins.
METHODS (a) I n Vivo O v e r p r o d u c t i o n of R N A
Overproduction of RNA in vivo can offer the advantage of obtaining a product t hat corresponds to the natural, biologically active molecule. Work in this area has thus far emphasized the use of bacterial expression systems, originally developed for the overproduction of proteins, to obtain multimilligram quantities of t R N A and 5 S RNA species. Both constitutive and inducible expression vectors have been employed; Table 1 lists a n u m b e r of these systems with an indication of the yields th at have thus far been obtained. Methods of cell growth and induction, preparation of crude RNA fractions, and subsequent separations on high-resolution chromatographic resins vary among the different systems, and the reader is referred to the references for a detailed description of individual protocols. H e r e the emphasis is on a comparative assessment and a description of common issues facing the biochemist who may wish to use a similar method for expression of a novel RNA. One general problem t h a t must be confronted when expressing RNA i n vivo is t h a t the overproduced product may be detrimental to cellular function. For example, expression levels of tRNAs cloned as operons onto mul-
ticopy plasmids have often not been high. T h e reason for this may be t h a t there is a negative control exerted on t RN A gene expression or, alternatively, th a t transformants containing such plasmids are unstable. Inducible systems provide a means to circumvent this problem by allowing cell growth to proceed to the mid-logarithmic stage before expression of the RNA is turned on. After induction, it is often the case t hat the cells no longer grow or divide at normal rates; nonetheless, large quantities of RNA are still produced. Considerable overproduction of RNA using inducible systems has been achieved in two cases (Table 1). Overexpression ofE. coli t RN A Ginwas achieved on expression vector PLc28, which utilizes the k leftward operator and promoter sequences to stimulate transcription of two tandem t RN A Gin genes in a temperature-inducible fashion (14, 19). T h e host strain used for expression harbors a defective X prophage carrying the ci857 temperaturelabile repressor gene, which represses the operon at 28°C but not at 42°C. T h e expression of tRNA~ in was carried out by temperature induction, but it should be noted t h a t the system allows also for induction via addition of naladixic acid: r e c A - s t i m u l a t e d protease activity is thereby induced, leading to cleavage of the c I repressor (47). Extremely high levels of expression of tR N A GIn have been obtained with this system; up to 70% of the small RNA in the crude RNA extract corresponds to this isoacceptor, an overproduction of 80- to 100-fold (Table 1). T he high levels obtained are undoubtedly due partly to the presence of two copies of the gene, which derive originally from the isolation of a transducing phage carrying a segment of the E. coli chromosome containing a total of seven t RN A genes (48). Importantly, no evidence for the existence of higher molecular weight transcripts was found when extracts from cells carrying the plasmid were analyzed. This indicates th a t the cellular enzymes responsible for correct processing of the pre-
TABLE
1
I n Vivo Expression Systems for Overproduction of RNA
Expressionvector
Inducibility
PL promoter in PLc28
Temperature
E. coli tRNA~in
IPTG
5 S RNA fragment
In vivo T7 RNA
Species expressed
polymerasesystem E. coli sup F tRNAw~ lac or tac promoter in
pBR322-derived vectors lpp promoter in pEMBL vectors rrnB ribosomalcistron in pBR322-derivedvector
IPTG
E. coli tRNAL~u(UAG)and
Yields
Ref.
1 mg pure RNA per gram cells (60-70% in extract) 4 mg ptJre RNA per gram cells 35-40% of small RNA in crude extract
(14)
Not characterized
P. Moore,personal communication
D. SoU,personal communication (16)
tRNAT M mutants Constitutive
E. coli synthetic
Not characterized
(17)
Constitutive
suppressor tRNAs 5 S RNA
Up to 7.5 mg pure
(18)
..RNAper gram cells
CRYSTALLIZATION OF RNA-PROTEIN COMPLEXES cursor tRNA to its mature form were able to cope with the increased rates of synthesis. However, all tRNAs and many other RNAs of interest also contain a variety of modified bases that are produced via post-transcriptional reactions carried out by highly specific modifying enzymes (33). Overexpression of these RNAs then raises the question of whether the material produced is in fact fully modified, because it seems quite possible that the modification enzymes might not be able to keep up with the increased rates of RNA synthesis. This issue has not yet been addressed in a rigorous manner. However, trial purifications of tRNA2atn on reversed-phase chromatographic resins showed that closely spaced peaks of glutamine-accepting activity, of roughly equivalent size, were eluted. The most likely explanation for this phenomenon is that the peaks represented differently modified forms of this tRNA Species (14). In the case of tRNA~ in the base modifications are all small in size, and their underrepresentation, if present, might in any case be unlikely to prove refractory to crystal growth (see below). In general, however, correct processing and proper base modification are issues that are likely to be import a n t in the in vivo expression of many RNAs. They may prove to be particularly troublesome in the heterologous expression in E. coli of RNAs from other, higher organisms. The second inducible system that has been utilized for the in vivo overproduction of RNA is that originally developed by Studier et al. (20), which employs T7 RNA polymerase to direct the expression of cloned genes. In this system, genes for the RNAs of interest are cloned behind the T7 promoter sequences in vectors derived originally from pBR322. The expression strain carries bacteriophage DE3, a derivative of phage },, as a lysogen. This phage carries a DNA fragment containing the lacU V 5 promoter followed by the gene for T7 RNA polymerase (20). Induction is achieved by addition of IPTG to growing cultures, which stimulates transcription of the polymerase from the lysogenic sequences. The expressed polymerase then transcribes the RNA gene of interest from the plasmid vector. An additional feature of this system is that a further level of control is possible through the use of T7 lysozyme, a natural inhibitor of T7 RNA polymerase (21). The T7 lysozyme is expressed from a second plasmid which is compatible with that carrying the RNA gene. This system has been used recently for the expression of several RNAs in high yield (Table 1) and is the system of choice when the toxicity of the gene product demands carefully regulated expression. Using this method the level of expression in crude extracts of the suppressor tRNA wyr(Table 1) is observed to be about half that obtained for tRNA G1~1overproduction from the PLc28 vector. This difference might be due to a number of factors: for example, decreased processing rates of the precursor form of the tRNA or an increased cell toxicity associated with overexpression of the suppressor tRNA species.
7-1
Preparation of a crude RNA extract may be carried out by any of the well-established means for carrying out bacterial cell lysis. However, a crude extract containing a small RNA of interest may be obtained as well by a direct phenol extraction of cells (14). In this method, actual lysis does not occur; rather, the action of the phenol on the cell walls of the bacteria is sufficient to liberate low-molecular-weight macromolecules, including small RNAs. The resulting extract contains little high-molecular-weight nucleic acid or protein and is therefore less likely to contain potentially deleterious nuclease activity. Subsequent purifications of tRNA from such an extract are further optimized by a short incubation at high pH to induce chemical deacylation, eliminating the possibility of altered retention of acylated and deacylated tRNAs on the same chromatographic matrix. Incubation of the crude extract at 4°C in the presence of 1 M NaC1 further serves to precipitate any higher molecular weight RNA that was obtained in the phenol extraction. Prior to application on expensive preparative-scale H P L C columns, binding of the crude tRNA fraction at 0.2 M NaC1 to a DEAE-cellulose resin, followed by batch elution at 1 M NaC1, serves to further clarify the extract and to remove potential contaminants that could damage the H P L C resin (14). The polyanionic character of the sugar-phosphate backbone of RNA and the presence of the hydrophobic bases suggest that both anion-exchange and reversedphase resins will be useful in obtaining separations. This was borne out in early separations of bulk E. coli tRNAs, which utilized matrices such as DEAE-Sephadex, RPC5, and BD-cellulose (22). More recently, a number of H P L C resins that produce considerably higher-resolution separations in a shorter time have been developed (23). In the case of the expression of tRNA2aln (Table 1, Ref. (14)), the levels of expression were such that a single H P L C anion-exchange column (DEAE 5PW, Waters) was sufficient to increase the purity of the material to 90-95%. Recently, excellent separations of crude tRNA enriched for the selenocysteine-inserting tRNA set species (32) have been obtained on a C4 reversed-phase H P L C support (Phenomenex W-Porex 5-C4) (J. Perona and D. Soll, unpublished results). The use of Sephacryl S-200 as well as various Sephadex size-exclusion columns has provided useful large-scale separations of 5 S RNA and of enzymatically prepared fragments of this molecule (18). The central role of tRNA in ensuring the faithful transmission of genetic information has sometimes been invoked as a rationale for suggesting that its overexpression is likely to be deleterious to the host cell. This is because of the complex equilibrium that exists between the approximately 60 different tRNA species and the 20 aminoacyl-tRNA synthetase enzymes responsible for their correct acylation. The existence of these parallel synthetase-tRNA systems in vivo is likely to aid in suppressing deleterious noncognate interactions (25). Over-
78
JOHN J. PERONA
expression of a single species can therefore upset this delicate balance (24), possibly leading to misincorporation of amino acids into cellular proteins and ensuing deleterious effects on cell viability. Similarly, because the individual nucleotides that specify the identity of tRNAs are often shared among several species (39), the expression of tRNA mutants may also result in misrecognition events. Mutant species may also suffer from incorrect processing of 5' and 3' flanking sequences, as well as potentially decreased thermodynamic stability and resistance to nucleases. Despite these potential difficulties, the stable expression of a number of synthetic suppressors in vivo has been achieved (Table 1), although the levels of their expression have not been rigorously characterized. It is noteworthy that the yields of both intact 5 S RNA and a 5 S RNA fragment are considerably higher than those Obtained for tRNA, by both inducible and constitutive overexpression (Table 1). Although careful regulation of the levels of ribosomal RNA in E. coli occurs as an integral part of ribosome assembly, one reason for the increased levels of expression relative to tRNA may be that overproduction of one component of the particle does not cause deleterious effects on the overall assembly pathway. It is possible that heterologous expression in E. coli of RNAs performing functions not present in bacteria (for example, small nuclear RNAs) will be favored by the absence of a normal cellular process with which they could interfere. In general, many of the considerations applicable to the expression of proteins in E. coli will also be relevant to the expression of RNA. Among these considerations are the strength and inducibility of the promoter, the copy number of the plasmid, and the presence of an efficient terminator following the gene (47). These features will be of equal relevance whether the RNA gene is derived synthetically or is obtained via cloning from a natural source. The expression of RNA, of course, does not benefit or suffer from any of the features governing translational regulation of expression. Thus far, the problem of intracellular aggregation of RNA leading to the formation of inclusion bodies, a major problem in the expression of some proteins, has not been reported. Crystals of overexpressed RNA have been grown of a fragment of the constitutively overproduced intact 5 S RNA (12), as well as of the complex of tRNA Ginwith the cognate glutaminyl-tRNA synthetase (14). Methods for growth of RNA-protein crystals are discussed below.
(b) Crystallization of RNA-Protein Complexes (i) General considerations. Once a sufficient quantity of purified RNA has been obtained, its cocrystallization with protein can be attempted. For an initial scanning of potential growth conditions, the quantity of RNA available need not be large; as little as 5 mg can suffice for these trials, as a considerable proportion of
the molecular mass of the complex will be formed by the protein. The method of choice, for its adaptability to rapid scanning of a wide variety of conditions, is that of vapor diffusion using hanging drops (34). When a novel crystallization is being attempted, it is probably best at first not to demand absolute homogeneity of the sample. In general, a purity of about 90% is sufficient for initial crystallization trials. The need for a high degree of homogeneity may be less stringent in the case of cocrystallization with a protein that binds the RNA specifically. For example, glutaminyl-tRNA synthetase will specifically bind and cocrystallize with tRNA Gl" despite the fact that between 5 and 10% of the tRNAs present in the crystallization drop are noncognate species (14). However, if a reasonable initial scanning of conditions does not produce suitable crystals, then improvement of the homogeneity of the samples can be one of the most important steps in the continuation of the project. The crystallization of the yeast aspartyl-tRNA synthetase: tRNA Asp complex provides a case study in the importance of sample purity, as discussed below (35). It is convenient to store the RNA in a way that will simplify its subsequent use in crystallization trials. In the case of tRNA Gin, the procedure is to store the final purified material (approximately 30-40 mg from a 10liter cell growth) as an ethanol precipitate in aliquots of approximately 5 mg. When needed, the RNA in each aliquot is recovered, resuspended in distilled water at high concentration, and lyophilized as separate pellets of approximately 0.5 rag. Each pellet then provides material sufficient for scanning 20-30 separate conditions. If proper precautions to eliminate nuclease contamination are followed, the RNA may be stored for many years in this manner without harm. Determination of RNA concentration from uv absorption is not straightforward, because the absorbance of an RNA in its native state is hypochromic relative to that when it is in a disordered, random coil conformation. Large hypochromic effects are thus indicative of a structure containing large amounts of base pairing and hydrophobic stacking; e.g., tRNAs exhibit an effect of approximately 30% (40). The importance of the RNA:protein stoichiometry as a variable in crystallization (41; also see below) emphasizes the importance of an accurate determination. In the case of tRNA, differences due to length and nucleotide sequence are small, and standard values of the extinction coefficients and hypochromicity that are likely to be accurate within 10% are available (40). For other RNAs, the extinction coefficient may be determined (for a known sequence) by complete hydrolysis of the molecule and measurement of the absorption of the nucleotide mixture or, alternatively, may be calculated based on the assumption that base interactions occur only between neighboring nucleotides (42). For large RNAs the experimental measurement will probably give a more accurate determination. In light of the potential
CRYSTALLIZATION OF RNA-PROTEIN COMPLEXES importance of the RNA:protein ratio to crystallization and the difficulty in quantitating RNA concentration, the best approach to exploring this parameter in a novel system is to test a range of stoichiometries larger than that otherwise considered necessary. High-quality crystals of RNA have proven to be quite difficult to obtain, largely because the nature of the material gives rise to sample heterogeneities which can be quite difficult to detect (35). Sequence heterogeneities within an RNA sample may be due to its susceptibility to nucleases or to nicking of the backbone as a consequence of alkaline hydrolysis or metal ion-catalyzed cleavage. Proper precautions in the preparation of samples can minimize these effects. However, heterogeneity can also occur at the conformational level due to the intrinsic flexibility of the polynucleotide backbone. It is also important to be aware that the conformation of RNA will vary as a consequence of differing conditions of salt and ionic strength (38). In the case of tRNA, which is by far the most intensively studied species, various classes of unfolded structure occur at low ionic strength in the absence of magnesium ions. Some tRNAs are even known to adopt alternative stable structures. To obtain a more homogeneous population of species prior to crystallization attempts, protocols such as heating of the material to 60°C in the presence of 10-20 mM magnesium ions, followed by slow cooling, may be carried out. However, the early observation that PheRS can promote a conformational change in tRNA Phe from an inactive to an active form suggests t h a t protein binding may well serve to stabilize a single conformation of the RNA (36). This provides reason for optimism that the crystallization of RNA-protein complexes may suffer less from the problem of RNA conformational variability. (ii) Crystallization of in vitro synthesized RNA. A second means of obtaining multimilligram quantities of RNA utilizes the technology of in vitro transcription of RNA genes by T7 RNA polymerase, as developed by Uhlenbeck (26, 27). The methodologies involved have been well described, and here the potential usefulness of this material for the preparation of high-resolution diffracting crystals is discussed. A number of technical difficulties associated with the synthesis of RNA by this method bear directly on the issue of its applicability for this purpose. It appears that RNA synthesized in vitro by T7 RNA polymerase will take on a native, biologically active conformation. The transcript most fully analyzed, that of yeast tRNA Phe, has been examined by aminoacylation activity and thermal melting profile (28), precise lead cleavage at a defined site in the D-loop (29), and proton N M R studies (30). All of these investigations support the conclusion that the molecule adopts a fully native conformation, despite the complete absence of modified nucleotides. Thus, it appears that the accuracy of incor-
79
poration of ribonucleotides by the enzyme under the conditions of large-scale in vitro transcription is quite high. Nonetheless, the fidelity of the enzyme has been a source of concern, based upon the observation that NMR linewidths of 5 S RNA molecules seemed to be unduly broad (31). This suggested the possibility of a high rate of misincorporation leading to a heterogeneous population of species. Recently, however, this issue has been reexamined (49). The conclusion is that RNA synthesized by T7 RNA polymerase from identical DNA templates either in vitro or in vivo gives NMR spectra of comparable quality. Broadened linewidths obtained from the original preparations have been traced in many cases to aggregation of the material as a consequence of the presence of 5' overhanging guanine residues. Therefore, transcripts containing such residues should probably be avoided in crystallization attempts unless conditions under which aggregation does not occur are found. Of potentially serious concern is the fact that the T7 RNA potymerase often produces transcripts that are lengthened at their 3' termini by one or occasionally two or more nontemplate encoded nucleotides. This observation, coupled with the fact that large amounts of abortive transcripts ranging in length from two to six nucleotides are always synthesized, emphasizes strongly the need for purification of the desired transcript prior to its use in crystallization attempts. For preparative-scale quantities of material, anion-exchange and reversed-phase H P L C are the methods of choice. Additionally, it will be important to characterize the full-length products obtained via gel electrophoresis, using a separation length appropriate for distinguishing transcripts differing in size by a single nucleotide. Apparently, the extent of 3'terminal heterogeneity cannot be predicted in advance for any given transcript, and reaction conditions for minimization of this effect have thus far not been found (27). Growth of high-quality crystals of RNA produced in this manner may thus in some cases depend upon the adequate separation of transcripts of lengths differing by only a single nucleotide, in sufficient yield for crystallization trials. In the case of tRNAs, aminoacylation of the mixture of full-length and extended transcripts will result in the charging of only properly terminated species. The acylated tRNAs will then be separated more readily from the uncharged extended molecules on HPLC, where the resin and conditions can be chosen to exploit the interaction of the amino acid moiety with the column. The lability of the aminoacyl linkage at high pH should be borne in mind when such a separation is attempted. A final consideration relates to the requirement of T7 RNA polymerase for a specific promoter sequence, rich in G residues at positions +1 to +6, for efficient initiation of transcription (27). It may therefore be difficult to obtain multimilligram quantities of RNAs possessing unfavorable 5'-terminal sequences. This problem can be
80
JOHN J. PERONA
circumvented in the case of t R N A transcripts by synthesizing a precursor molecule instead of the mature species. The precursor contains an extended 5' flank of 1015 nucleotides possessing the consensus T7 promoter sequence. The correct 5' terminus can then be obtained via in vitro enzymatic processing using the catalytic M1 RNA of RNase P, which can itself be produced by T7 RNA polymerase transcription in vitro (37). It is important to be aware that the synthesis of some mutant tRNAs by this method may not be possible, since the rate and accuracy of RNase P cleavage are affected by the structure of the mature species (46). A number of mutant t R N A Gin species have recently been synthesized and characterized successfully using this methodology (M. Jahn and D. Soll, personal communication). High-resolution diffracting crystals containing unmodified R N A produced by in vitro transcription have recently been grown of several mutants of E. coli, tRNA2aln, complexed with glutaminyl-tRNA synthetase (J. Arnez and T. A. Steitz, personal communication). The high similarity in structure demonstrated between native and synthesized RNAs provides reason for optimism that this technique will continue to bear fruit in the near future.
(iii) Crystallizations of aminoacyl-tRNA synthetase: tRNA complexes. Table 2 summarizes the crystallization conditions used in the preparation of crystals of two aminoacyl-tRNA synthetase:tRNA complexes, the only high-resolution diffracting crystals of RNA-protein complexes thus far reported (14, 15). Examination of the conditions used reveals both similarities and differences and highlights specific issues that are relevant to the preparation of crystals of other RNA-protein complexes. A major consideration relates to the means by which large quantities of material were made available for the crystallization attempts. Both GlnRS and tRNA Gin have been overexpressed using engineered bacterial strains, leading to the facile production of multimilligram quantities of both macromolecules (14). The high levels of expression facilitated the development of rapid purification methods, and crystals have been readily obtained and reproduced without difficulty. By contrast, AspRS and t R N A Asp were each obtained via very large-scale purifications from large quantities of yeast cells (8, 15, 35). Gradual improvement in the procedures used, necessitated as a consequence of difficulties with yields and reproducibility, has resulted in a succession of crystal forms of increasing quality grown
TABLE 2
Crystallization of Two Aminoacyl-tRNA Synthetase:tRNA Complexes Conditions
Y e a s t A s p R S - t R N A Asp
E. coli G l n R S - t R N A GI~
RNA:protein stoichiometry Precipitating agent Starting concentration Final concentration M a c r o m o l e c u l e c o n c e n t r a t i o n s (initial) Protein RNA Temperature M g 2+ ions Buffer Other substrates
2.4:1 AmSO4 25% 62%
1:1 AmSO4 24%
10 m g / m l 4.8 m g / m l 4 °C 5 mM MgCle 40 mM T r i s - m a l e a t e / N a O H None
6.7-10.0 m g / m l 2.6-3.9 m g / m l 17°C 20 mM MgSO4 80 mM P i p e s 4 mM A T P 4 mM A M P + 60 mM g l u t a m i n e 4 mM A T P + 60 mM g l u t a m i n e 7.4
pH
7.5
48%
C r y s t a l properties
Diffraction limit Space group Cell d i m e n s i o n s
Solvent c o n t e n t No. c o m p l e x e s / a s y m m e t r i c u n i t
2.7 .~ p21212 a = 210.4 A b = 145.3 h c = 86.0 A 69% 1
2.5 .~ C2221 a = 242.8 b = 93.5 h c = 115.7/k 70% 1
Note. C o n d i t i o n s for t h e y e a s t A s p R S t R N A A~pc o m p l e x are generally as reported (15), with s m a l l modifications (M. Ruff, p e r s o n a l c o m m u n i cation). T h e original crystal f o r m of t h e G l n R S : t R N A GInc o m p l e x was grown f r o m solutions c o n t a i n i n g s o d i u m citrate as t h e p r e c i p i t a t i n g a g e n t (14); t h e form reported here was u s e d for t h e s t r u c t u r e d e t e r m i n a t i o n (44).
CRYSTALLIZATION OF RNA-PROTEIN COMPLEXES under similar conditions (15, 35, 41), culminating in an orthorhombic crystal t hat diffracts to 2.7 .~ resolution (15). Th e message is clear: while good expression systems do not guarantee the growth of adequate crystals, they reduce the need for such heroic efforts, which in the long run will result in a much greater number of crystallization attempts and, presumably, successes. Both complexes crystallize in the presence of magnesium ions, and this cation has also been present for many of the RNAs crystallized to date (8-13, 43). Detailed structural analysis of yeast t R N A phe has demonstrated a role for magnesium in stabilizing the structure, and binding sites were determined for a number of the ions in hydrated form, as well as for two spermine molecules (45). In fact, alternative crystal forms of tRNA Phe were grown from solutions t hat differed only slightly in the stoichiometric ratio of magnesium ions to spermine molecules (9). Clearly, the role of these cations in ensuring structural stability, thereby favoring crystallization, justifies extensive exploration of growth conditions in their presence. A variation in magnesium ion concentration of 5 mM or less can cause significant effects on crystal growth and properties. Other multiply charged cationic species, such as spermidine and putrescine, merit investigation as well. T he divalent cations lead and barium have also been present during crystal growth of certain tRNAs (10, 43). Interestingly, the R N A - p r o t e i n stoichiometry in the crystallization drop plays a critical role in the crystallization of the A s p R S - t R N A A~p complex, where only a very narrow range of stoichiometries promoted growth, but not of the citrate-grown form of the G l n R S - t R N A GI~ complex (14, 41; Table 2, note). A t R N A to protein molar ratio of 2.4:1 was found to be critical to the initial growth of the best crystals of the AspRS:tRNA A~pcomplex. T he sensitivity of crystal formation to the RNA:protein stoichiometry in this system is further exemplified by the fact t h a t larger crystals of improved usefulness for diffraction studies were obtained only by seeding into a second crystallization drop which now contained a 3:1 ratio of tRNA:protein (M. Ruff, personal communication). Th e AspRS enzyme is dimeric and binds two molecules of tRNA; these results therefore indicate t hat the optimal RNA:protein stoichiometry for crystal growth need not reproduce t ha t exhibited by the normally functioning enzyme in solution. Conversely, growth of crystals of the GtnRS enzyme complexed to t RN A GIn using sodium citrate as the precipitating agent is insensitive to the RNA:protein stoichiometry in the range of at least 1:1 to 2:1 (14). This monomeric enzyme binds a single tR NA molecule in solution. Another variable that has been shown to be critical to the growth of useful RNA:protein cocrystals is the presence or absence of other ligands of the protein. Aminoacyl-tRNA synthetases, for example, possess binding sites for ATP, AMP, amino acid, and Mg2+-pyrophos phate in addition to tRNA. With the G l n R S - t R N A Gh~
81
complex, no crystals are obtained in the complete absence of additional ligands. Rather, crystal growth of isomorphous forms occurs in the presence of ATP, A T P and glutamine, or A MP and glutamine (14; J. Perona, M. Rould, and T. Steitz, unpublished observations). Any of these ligands may be freely soaked in and out of the crystals without disruption of the lattice. Conversely, no ligands were found to be required for growth of the AspRS:tRNA A~p crystals (15). Additional ligands may aid crystal growth through the stabilization of otherwise flexible protein structural domains. The limitation of experience thus far to two R N A protein complexes limits the extent to which one can state many useful generalizations. As with proteins, the most important aspects to consider are the solubility behavior, ligand-binding properties, and homogeneity of a given complex. Thus, both complexes crystallize in the presence of ammonium sulfate as precipitating agent, at similar pH's, buffer concentrations, and macromolecule concentrations, but there is nevertheless no reason to believe t hat these conditions are in any way indicative of those t hat will be successful in other cases. Crystals of RNAs obtained in the absence of protein have grown from solutions containing either ammonium sulfate or organic solvents as the precipitating agents and encompass a wide range of growth conditions (8-13, 43). In conclusion, it is clear t hat while the efforts involved in obtaining suitable material and growing crystals can be considerable, the potential rewards, in terms of detailed structural information about the mechanisms of recognition and discrimination of RNAs by proteins, are extremely large. T he structure of the G l n R S - t R N A Gin complex has been determined to a resolution of 2.8 (44) and t hat of the A s p R S - t R N A A~p complex has recently been phased, with model-building in progress (M. Ruff, personal communication). A complete understanding of the complex structure-function relationships underlying the critical biological roles of R N A -p ro te in interactions will be possible only when the detailed structures of many more such complexes are elucidated.
ACKNOWLEDGMENTS I thank Thomas Steitz, Dieter Soll, Peter Moore, Joyce Sherman, and Alex Szewczak for useful discussions and permission to cite results prior to publication. The author was supported in part by NIH Grant GM-22778to Thomas Steitz.
REFERENCES 1. Inouye, M., and Dudock, B. (Eds.) (1987) Molecular Biologyof RNA--New Perspectives,AcademicPress, San Diego. 2. Cech,T. R., and Bass, B. L. (1986)Annu. Rev. Biochem. 55,599629. 3. Altman, S., Baer, M., Gold, H., Guerrier-Takada, C., Kirsebom,
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L., Lawrence, N., Lumelsky, N., and Vioque, A. (1987) in Molecular Biology of RNA--New Perspectives (Inouye, M., and Dudock, B., Eds.), Academic Press, San Diego. 4. Moras, D., Comarmond, M. B., Fischer, J., Weiss, R., Thierry, J. C., Ebel, J. P., and Giege, R. (1980) Nature (London) 288,669. 5. Robertus, J. D., Ladner, J. E., Finch, J. T., Rhodes, D., Brown, R. S., Clark, B. F. C., and Klug, A. (1974) Nature (London) 250, 546. 6. Schevitz, R. W., Podjarny, A. D., Krishnamachari, N., Hughes, J. J., and Sigler, P. B. (1979) Nature (London) 278,188. 7. Steitz, T. A. (1990) Q. Rev. Biophys. 23,205. 8. Giege, R., Moras, D., and Thierry, J. C. (1977) J. Mol. Biol. 115, 91-96. 9. Ladner, J. E., Finch, J. T., Klug, A., and Clark, B. F. C. (1972). J. Mol. Biol. 171,207-215. 10. Woo, N. H., Roe, B. A., and Rich, A. (1980) Nature (London) 286, 346-351. 11. Johnson, C. D., Adolph, K., Rosa, J. J., Hall, M. D., and Sigler, P. B. (1970) Nature (London) 226, 1246-1247. 12. Abdel-Meguid, S. S., Moore, P. B., and Steitz, T. A. (1983) J. Mol. Biol. 171,207-215. 13. Dock-Bregon, A. C., Chevrier, B., Podjarny, A., Moras, D., deBaer, J. S., Gough, G. R., Gilham, P. T., and Johnson, J. E. (1988) Nature (London) 335, 375-378. 14. Perona, J. J., Swanson, R., Steitz, T. A., and Soll, D. (1988) J. Mol. Biol. 202,121-126. 15. Ruff, M., Cavarelli, J., Mikol, V., Lorber, B., Mitschler, A., Giege, R., Thierry, J. C., and Moras, D. (1988) J. Mol. Biol. 2 0 1 , 2 3 5 236. 16. Normanly, J., Ogden, R. C., Horvath, S. J., and Abelson, J. (1986) Nature (London) 321,213-219. 17. Masson, J.-M., and Miller, J. H. (1986) Gene 47,179-183. 18. Moore, P. B., Abo, S., Freeborn, B., Gewirth, D. T., Leontis, N. B., and Sun, G. (1988) in Methods in Enzymology (Noller, H. F., Jr., and Moldave, K., Eds.), Vol. 164, pp. 158-174, Academic Press, San Diego. 19. Remaut, E., Stanssens, P., and Fiers, W. (1981) Gene 15, 81 93. 20. Studier, F. W., Rosenberg, A. H., and Dunn, J. J. (1990) in Methods in Enzymology, Vol. 185, in press. 21. Moffatt, B. A., and Studier, F. W. (1987) Cell 49,221. 22. Nishimura, S., Shindo-Okada, N., and Crain, P. F. (1987) in Methods in Enzymology (Wu, R., Ed.), Vol. 155, pp. 373-379, Academic Press, San Diego. 23. Tanner, N. K. (1989) in Methods in Enzymology (Dahlberg, J. E., and Abelson, J. N., Eds.), Vol. 180, pp. 25-41, Academic Press, San Diego. 24. Swanson, R., Hoben, P., Sumner-Smith, M., Uemura, H., Watson, L., and Soll, D. (1988) Science 242, 1548. 25. Yarus, M. (1972)Nature New Biol. 239,106-108. 26. Milligan, J. F., Groebe, D. R., Witherell, G. W., and Uhlenbeck, O. C. (1987) Nucleic Acids Res. 15, 8783-8798.
27. Milligan, J. F., and Uhlenbeck, O. C. (1989) in Methods in Enzymology (Dahlberg, J. E., and Abelson, J. N., Eds.), Vol. 180, pp. 51-62, Academic Press, San Diego. 28. Sampson, R., and Uhlenbeck, O. C. (1988) Proc. Natl. Acad. Sci. USA 85, 1033-1037. 29. Behlen, L. S., Sampson, J. R., DiRenzo, A. B., and Uhlenbeck, O. C. (1990) Biochemistry 29, 2515-2523. 30. Hall, K., Sampson, J., Uhlenbeck, 0. C., and Redfield, A. (1989) Biochemistry 28, 5794-5801. 31. Gewirth, D. T., and Moore, P. B. (1988) Nucleic Acids Res. 16, 10,717-10,732. 32. Leinfelder, W., Zehelein, E., Mandrand-Berthelot, M.-A., and Bock, A. (1988) Nature (London) 331,723-725. 33. Bjork, G. R., Ericson, J. U., Gustafsson, C., Hagervall, T. G., Jonsson, Y. H., and Wikstrom, P. M. (1987) Annu. Rev. Biochem. 56, 263. 34. McPherson, A. (1982) The Preparation and Analysis of Protein Crystals, Wiley, New York. 35. Giege, R., Dock, A. C., Kern, D., Lorber, B., Thierry, J. C., and Moras, D. (1986) J. Crystal Growth 76,554-561. 36. Renaud, M., Bollack, C., and Ebel, J. P. (1974) Biochimie 56, 1203. 37. Vioque, A., Arnez, J., and Altman, S. (1988) J. Mol. Biol. 202, 835-848. 38. Crothers, D. M. (1979) in Transfer RNA: Structure, Properties and Recognition (Schimmel, P. R., Soil, D., and Abelson, J., Eds.), pp. 163-176, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 39. Normanly, J., and Abelson, J. (1989) Annu. Rev. Biochem. 58, 1029-1049. 40. Gueron, M., and Leroy, J. L. (1978) Anal. Biochem. 91,691-695. 41. Lorber, B., Giege, R., Ebel, J-P., Berthet, C., Thierry, J.-C., and Moras, D. (1983) J. Biol. Chem. 258, 8429-8435. 42. Puglisi, J. D., and Tinoco, I. (1989) in Methods in Enzymology (Dahlberg, J. E., and Abelson, J. N., Eds.), Vol. 180, pp. 304-325, Academic Press, San Diego. 43. Labanauskas, M. J., Norvell, C., Anderegg, J. W., Beeman, W. W., Rubin, J., Rao, S. T., Sundaralingam, M., Young, J. D., and Bock, R. M. (1972) Biochem. Biophys. Res. Commun. 49, 9. 44. Rould, M. A., Perona, J. J., Soll, D., and Steitz, T. A. (1989) Science 246, 1135-1142. 45. Quigley, G. J., Teeter, M. M., and Rich, A. (1978) Proc. Natl. Acad. Sci. USA 75, 64-68. 46. Furdon, P. J., Guerrier-Takada, C., and Altman, S. (1983) Nucleic Acids Res. 11, 1491-1505. 47. Reznikoff, W., and Gold, L., (Eds.) (1986) Maximizing Gene Expression, pp. 345-363, Butterworths, London. 48. Nakajima, N., Ozeki, H., and Shimura, Y. (1981) Cell 23, 239249. 49. Szewczak, A. A., White, S. A., Gewirth, D. T., and Moore, P. B. (1990) Nucleic Acids Res. 18, 4139-4142.
Crystallization of RNA-Protein Complexes J o h n J. P e r o n a Department of Pharmaceutical Chemistry, 926 Medical Sciences Building, University of California, San Francisco, California 94143
The widespread occurrence of RNA-protein interactions in critical cellular processes makes an understanding of the structural basis for recognition and sequence discrimination of paramount importance. Until recently, however, the difficulty in obtaining large quantities of homogeneous RNA had made structural investigations of many interesting species by biophysical methods impossible. Summarized here are the recent applications of several in vivo expression systems to the overproduction of a number of RNA species. The potential usefulness in crystallization of RNA synthesized in vitro by T7 RNA polymerase is also discussed. Together with advancements in separation methods by HPLC, these two methods make a much wider range of systems accessible to structural analysis. A comparison of the successful cocrystallizations of two complexes of arninoacyl-tRNA synthetases with their cognate tRNA substrates is presented. © 1990 Academic Press, Inc.
RNA plays a central and increasingly recognized role in many aspects of cellular structure and function. RNAs possess both functional and structural roles in the transmission of genetic information from DNA to prot e i n - - a s messenger molecules, as components of the splicing apparatus and of the ribosome, and as small adaptor molecules (tRNA) which provide the critical link between nucleotide and amino acid sequence information (1). Retroviral genomes are composed entirely of RNA, demonstrating the ability of this molecule to serve also as an organism's storehouse of genetic information. Certain RNAs also function enzymatically in self-splicing and tRNA maturation reactions (2, 3). The crystal structures of several tRNAs have been determined at atomic resolution (4-6; R. Basavappa and P. B. Sigler, personal communication). From these structures it is clear t h a t large RNAs are in general likely to possess compact, globular conformations containing singlestranded loop regions as well as helical sections. In tRNA, the tertiary fold is maintained in part via hydrogen bonding and hydrophobic stacking interactions between nucleotide bases in parts of the polynucleotide chain t h a t are distant from each other in primary se1046-2023/90 $3.00
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
quence. Additionally, the sugar-phosphate backbone possesses considerable flexibility, which is likely to play a critical role in the interactions of the RNA with proteins. The stereochemical rules governing these proteinnucleic acid interactions are at present largely unknown and constitute a major area of biophysical investigation (7). X-ray crystallographic methods currently offer the only well-established means for obtaining atomic-resolution views of RNA-protein interaction. However, the preparation of large quantities of homogeneous RNA and its subsequent cocrystallization with protein have proven to be very challenging steps t h a t have thus far limited investigation to a small number of systems. Crystals of RNA t h a t diffract to medium resolution or better have thus far been reported only for a variety of transfer RNA species (8-11, 43), for a fragment of 5 S RNA (12), and recently for a 14-bp helical RNA duplex for which the structure has been determined at high resolution (13). In most of these cases, the RNA was obtained either by purification directly from large quantities of bacterial or yeast cells (8-11, 43) or by chemical synthesis in solution (13). The limitations inherent in these methods (requirements for high intracellular abundance and difficulties in reproducibility for the former; size limitations for the latter) do much to explain why the current database of available RNA structures is so limited. Described here are several bacterial expression systems, originally developed for the overproduction of proteins, that have been applied to the in vivo synthesis of multimilligram quantities of a number of RNA species. The usefulness of in vitro synthesized RNA in the preparation of high-resolution diffracting crystals is also discussed. Together, these systems hold the promise of significantly enlarging the number of RNA-protein complexes t h a t may become amenable to crystallographic study. The in vivo overproduction of glutaminespecific tRNA via an engineered bacterial strain was instrumental in the recent growth of crystals of the Escherichia coli glutaminyl-tRNA synthetase:tRNAa.n complex (14). A comparative analysis of the crystallization conditions used for this complex with those utilized 75
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in the crystallization of the yeast aspartyl-tRNA synthetase:tRNA Asp complex (15) is presented. Although due regard for the structural properties and solution behavior of RNA is warranted, in general the approaches t hat have been used for successful crystallization of RNA and of RNA-protein complexes have been quite similar to those used in many laboratories over the past generation for the crystallization of proteins.
METHODS (a) I n Vivo O v e r p r o d u c t i o n of R N A
Overproduction of RNA in vivo can offer the advantage of obtaining a product t hat corresponds to the natural, biologically active molecule. Work in this area has thus far emphasized the use of bacterial expression systems, originally developed for the overproduction of proteins, to obtain multimilligram quantities of t R N A and 5 S RNA species. Both constitutive and inducible expression vectors have been employed; Table 1 lists a n u m b e r of these systems with an indication of the yields th at have thus far been obtained. Methods of cell growth and induction, preparation of crude RNA fractions, and subsequent separations on high-resolution chromatographic resins vary among the different systems, and the reader is referred to the references for a detailed description of individual protocols. H e r e the emphasis is on a comparative assessment and a description of common issues facing the biochemist who may wish to use a similar method for expression of a novel RNA. One general problem t h a t must be confronted when expressing RNA i n vivo is t h a t the overproduced product may be detrimental to cellular function. For example, expression levels of tRNAs cloned as operons onto mul-
ticopy plasmids have often not been high. T h e reason for this may be t h a t there is a negative control exerted on t RN A gene expression or, alternatively, th a t transformants containing such plasmids are unstable. Inducible systems provide a means to circumvent this problem by allowing cell growth to proceed to the mid-logarithmic stage before expression of the RNA is turned on. After induction, it is often the case t hat the cells no longer grow or divide at normal rates; nonetheless, large quantities of RNA are still produced. Considerable overproduction of RNA using inducible systems has been achieved in two cases (Table 1). Overexpression ofE. coli t RN A Ginwas achieved on expression vector PLc28, which utilizes the k leftward operator and promoter sequences to stimulate transcription of two tandem t RN A Gin genes in a temperature-inducible fashion (14, 19). T h e host strain used for expression harbors a defective X prophage carrying the ci857 temperaturelabile repressor gene, which represses the operon at 28°C but not at 42°C. T h e expression of tRNA~ in was carried out by temperature induction, but it should be noted t h a t the system allows also for induction via addition of naladixic acid: r e c A - s t i m u l a t e d protease activity is thereby induced, leading to cleavage of the c I repressor (47). Extremely high levels of expression of tR N A GIn have been obtained with this system; up to 70% of the small RNA in the crude RNA extract corresponds to this isoacceptor, an overproduction of 80- to 100-fold (Table 1). T he high levels obtained are undoubtedly due partly to the presence of two copies of the gene, which derive originally from the isolation of a transducing phage carrying a segment of the E. coli chromosome containing a total of seven t RN A genes (48). Importantly, no evidence for the existence of higher molecular weight transcripts was found when extracts from cells carrying the plasmid were analyzed. This indicates th a t the cellular enzymes responsible for correct processing of the pre-
TABLE
1
I n Vivo Expression Systems for Overproduction of RNA
Expressionvector
Inducibility
PL promoter in PLc28
Temperature
E. coli tRNA~in
IPTG
5 S RNA fragment
In vivo T7 RNA
Species expressed
polymerasesystem E. coli sup F tRNAw~ lac or tac promoter in
pBR322-derived vectors lpp promoter in pEMBL vectors rrnB ribosomalcistron in pBR322-derivedvector
IPTG
E. coli tRNAL~u(UAG)and
Yields
Ref.
1 mg pure RNA per gram cells (60-70% in extract) 4 mg ptJre RNA per gram cells 35-40% of small RNA in crude extract
(14)
Not characterized
P. Moore,personal communication
D. SoU,personal communication (16)
tRNAT M mutants Constitutive
E. coli synthetic
Not characterized
(17)
Constitutive
suppressor tRNAs 5 S RNA
Up to 7.5 mg pure
(18)
..RNAper gram cells
CRYSTALLIZATION OF RNA-PROTEIN COMPLEXES cursor tRNA to its mature form were able to cope with the increased rates of synthesis. However, all tRNAs and many other RNAs of interest also contain a variety of modified bases that are produced via post-transcriptional reactions carried out by highly specific modifying enzymes (33). Overexpression of these RNAs then raises the question of whether the material produced is in fact fully modified, because it seems quite possible that the modification enzymes might not be able to keep up with the increased rates of RNA synthesis. This issue has not yet been addressed in a rigorous manner. However, trial purifications of tRNA2atn on reversed-phase chromatographic resins showed that closely spaced peaks of glutamine-accepting activity, of roughly equivalent size, were eluted. The most likely explanation for this phenomenon is that the peaks represented differently modified forms of this tRNA Species (14). In the case of tRNA~ in the base modifications are all small in size, and their underrepresentation, if present, might in any case be unlikely to prove refractory to crystal growth (see below). In general, however, correct processing and proper base modification are issues that are likely to be import a n t in the in vivo expression of many RNAs. They may prove to be particularly troublesome in the heterologous expression in E. coli of RNAs from other, higher organisms. The second inducible system that has been utilized for the in vivo overproduction of RNA is that originally developed by Studier et al. (20), which employs T7 RNA polymerase to direct the expression of cloned genes. In this system, genes for the RNAs of interest are cloned behind the T7 promoter sequences in vectors derived originally from pBR322. The expression strain carries bacteriophage DE3, a derivative of phage },, as a lysogen. This phage carries a DNA fragment containing the lacU V 5 promoter followed by the gene for T7 RNA polymerase (20). Induction is achieved by addition of IPTG to growing cultures, which stimulates transcription of the polymerase from the lysogenic sequences. The expressed polymerase then transcribes the RNA gene of interest from the plasmid vector. An additional feature of this system is that a further level of control is possible through the use of T7 lysozyme, a natural inhibitor of T7 RNA polymerase (21). The T7 lysozyme is expressed from a second plasmid which is compatible with that carrying the RNA gene. This system has been used recently for the expression of several RNAs in high yield (Table 1) and is the system of choice when the toxicity of the gene product demands carefully regulated expression. Using this method the level of expression in crude extracts of the suppressor tRNA wyr(Table 1) is observed to be about half that obtained for tRNA G1~1overproduction from the PLc28 vector. This difference might be due to a number of factors: for example, decreased processing rates of the precursor form of the tRNA or an increased cell toxicity associated with overexpression of the suppressor tRNA species.
7-1
Preparation of a crude RNA extract may be carried out by any of the well-established means for carrying out bacterial cell lysis. However, a crude extract containing a small RNA of interest may be obtained as well by a direct phenol extraction of cells (14). In this method, actual lysis does not occur; rather, the action of the phenol on the cell walls of the bacteria is sufficient to liberate low-molecular-weight macromolecules, including small RNAs. The resulting extract contains little high-molecular-weight nucleic acid or protein and is therefore less likely to contain potentially deleterious nuclease activity. Subsequent purifications of tRNA from such an extract are further optimized by a short incubation at high pH to induce chemical deacylation, eliminating the possibility of altered retention of acylated and deacylated tRNAs on the same chromatographic matrix. Incubation of the crude extract at 4°C in the presence of 1 M NaC1 further serves to precipitate any higher molecular weight RNA that was obtained in the phenol extraction. Prior to application on expensive preparative-scale H P L C columns, binding of the crude tRNA fraction at 0.2 M NaC1 to a DEAE-cellulose resin, followed by batch elution at 1 M NaC1, serves to further clarify the extract and to remove potential contaminants that could damage the H P L C resin (14). The polyanionic character of the sugar-phosphate backbone of RNA and the presence of the hydrophobic bases suggest that both anion-exchange and reversedphase resins will be useful in obtaining separations. This was borne out in early separations of bulk E. coli tRNAs, which utilized matrices such as DEAE-Sephadex, RPC5, and BD-cellulose (22). More recently, a number of H P L C resins that produce considerably higher-resolution separations in a shorter time have been developed (23). In the case of the expression of tRNA2aln (Table 1, Ref. (14)), the levels of expression were such that a single H P L C anion-exchange column (DEAE 5PW, Waters) was sufficient to increase the purity of the material to 90-95%. Recently, excellent separations of crude tRNA enriched for the selenocysteine-inserting tRNA set species (32) have been obtained on a C4 reversed-phase H P L C support (Phenomenex W-Porex 5-C4) (J. Perona and D. Soll, unpublished results). The use of Sephacryl S-200 as well as various Sephadex size-exclusion columns has provided useful large-scale separations of 5 S RNA and of enzymatically prepared fragments of this molecule (18). The central role of tRNA in ensuring the faithful transmission of genetic information has sometimes been invoked as a rationale for suggesting that its overexpression is likely to be deleterious to the host cell. This is because of the complex equilibrium that exists between the approximately 60 different tRNA species and the 20 aminoacyl-tRNA synthetase enzymes responsible for their correct acylation. The existence of these parallel synthetase-tRNA systems in vivo is likely to aid in suppressing deleterious noncognate interactions (25). Over-
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JOHN J. PERONA
expression of a single species can therefore upset this delicate balance (24), possibly leading to misincorporation of amino acids into cellular proteins and ensuing deleterious effects on cell viability. Similarly, because the individual nucleotides that specify the identity of tRNAs are often shared among several species (39), the expression of tRNA mutants may also result in misrecognition events. Mutant species may also suffer from incorrect processing of 5' and 3' flanking sequences, as well as potentially decreased thermodynamic stability and resistance to nucleases. Despite these potential difficulties, the stable expression of a number of synthetic suppressors in vivo has been achieved (Table 1), although the levels of their expression have not been rigorously characterized. It is noteworthy that the yields of both intact 5 S RNA and a 5 S RNA fragment are considerably higher than those Obtained for tRNA, by both inducible and constitutive overexpression (Table 1). Although careful regulation of the levels of ribosomal RNA in E. coli occurs as an integral part of ribosome assembly, one reason for the increased levels of expression relative to tRNA may be that overproduction of one component of the particle does not cause deleterious effects on the overall assembly pathway. It is possible that heterologous expression in E. coli of RNAs performing functions not present in bacteria (for example, small nuclear RNAs) will be favored by the absence of a normal cellular process with which they could interfere. In general, many of the considerations applicable to the expression of proteins in E. coli will also be relevant to the expression of RNA. Among these considerations are the strength and inducibility of the promoter, the copy number of the plasmid, and the presence of an efficient terminator following the gene (47). These features will be of equal relevance whether the RNA gene is derived synthetically or is obtained via cloning from a natural source. The expression of RNA, of course, does not benefit or suffer from any of the features governing translational regulation of expression. Thus far, the problem of intracellular aggregation of RNA leading to the formation of inclusion bodies, a major problem in the expression of some proteins, has not been reported. Crystals of overexpressed RNA have been grown of a fragment of the constitutively overproduced intact 5 S RNA (12), as well as of the complex of tRNA Ginwith the cognate glutaminyl-tRNA synthetase (14). Methods for growth of RNA-protein crystals are discussed below.
(b) Crystallization of RNA-Protein Complexes (i) General considerations. Once a sufficient quantity of purified RNA has been obtained, its cocrystallization with protein can be attempted. For an initial scanning of potential growth conditions, the quantity of RNA available need not be large; as little as 5 mg can suffice for these trials, as a considerable proportion of
the molecular mass of the complex will be formed by the protein. The method of choice, for its adaptability to rapid scanning of a wide variety of conditions, is that of vapor diffusion using hanging drops (34). When a novel crystallization is being attempted, it is probably best at first not to demand absolute homogeneity of the sample. In general, a purity of about 90% is sufficient for initial crystallization trials. The need for a high degree of homogeneity may be less stringent in the case of cocrystallization with a protein that binds the RNA specifically. For example, glutaminyl-tRNA synthetase will specifically bind and cocrystallize with tRNA Gl" despite the fact that between 5 and 10% of the tRNAs present in the crystallization drop are noncognate species (14). However, if a reasonable initial scanning of conditions does not produce suitable crystals, then improvement of the homogeneity of the samples can be one of the most important steps in the continuation of the project. The crystallization of the yeast aspartyl-tRNA synthetase: tRNA Asp complex provides a case study in the importance of sample purity, as discussed below (35). It is convenient to store the RNA in a way that will simplify its subsequent use in crystallization trials. In the case of tRNA Gin, the procedure is to store the final purified material (approximately 30-40 mg from a 10liter cell growth) as an ethanol precipitate in aliquots of approximately 5 mg. When needed, the RNA in each aliquot is recovered, resuspended in distilled water at high concentration, and lyophilized as separate pellets of approximately 0.5 rag. Each pellet then provides material sufficient for scanning 20-30 separate conditions. If proper precautions to eliminate nuclease contamination are followed, the RNA may be stored for many years in this manner without harm. Determination of RNA concentration from uv absorption is not straightforward, because the absorbance of an RNA in its native state is hypochromic relative to that when it is in a disordered, random coil conformation. Large hypochromic effects are thus indicative of a structure containing large amounts of base pairing and hydrophobic stacking; e.g., tRNAs exhibit an effect of approximately 30% (40). The importance of the RNA:protein stoichiometry as a variable in crystallization (41; also see below) emphasizes the importance of an accurate determination. In the case of tRNA, differences due to length and nucleotide sequence are small, and standard values of the extinction coefficients and hypochromicity that are likely to be accurate within 10% are available (40). For other RNAs, the extinction coefficient may be determined (for a known sequence) by complete hydrolysis of the molecule and measurement of the absorption of the nucleotide mixture or, alternatively, may be calculated based on the assumption that base interactions occur only between neighboring nucleotides (42). For large RNAs the experimental measurement will probably give a more accurate determination. In light of the potential
CRYSTALLIZATION OF RNA-PROTEIN COMPLEXES importance of the RNA:protein ratio to crystallization and the difficulty in quantitating RNA concentration, the best approach to exploring this parameter in a novel system is to test a range of stoichiometries larger than that otherwise considered necessary. High-quality crystals of RNA have proven to be quite difficult to obtain, largely because the nature of the material gives rise to sample heterogeneities which can be quite difficult to detect (35). Sequence heterogeneities within an RNA sample may be due to its susceptibility to nucleases or to nicking of the backbone as a consequence of alkaline hydrolysis or metal ion-catalyzed cleavage. Proper precautions in the preparation of samples can minimize these effects. However, heterogeneity can also occur at the conformational level due to the intrinsic flexibility of the polynucleotide backbone. It is also important to be aware that the conformation of RNA will vary as a consequence of differing conditions of salt and ionic strength (38). In the case of tRNA, which is by far the most intensively studied species, various classes of unfolded structure occur at low ionic strength in the absence of magnesium ions. Some tRNAs are even known to adopt alternative stable structures. To obtain a more homogeneous population of species prior to crystallization attempts, protocols such as heating of the material to 60°C in the presence of 10-20 mM magnesium ions, followed by slow cooling, may be carried out. However, the early observation that PheRS can promote a conformational change in tRNA Phe from an inactive to an active form suggests t h a t protein binding may well serve to stabilize a single conformation of the RNA (36). This provides reason for optimism that the crystallization of RNA-protein complexes may suffer less from the problem of RNA conformational variability. (ii) Crystallization of in vitro synthesized RNA. A second means of obtaining multimilligram quantities of RNA utilizes the technology of in vitro transcription of RNA genes by T7 RNA polymerase, as developed by Uhlenbeck (26, 27). The methodologies involved have been well described, and here the potential usefulness of this material for the preparation of high-resolution diffracting crystals is discussed. A number of technical difficulties associated with the synthesis of RNA by this method bear directly on the issue of its applicability for this purpose. It appears that RNA synthesized in vitro by T7 RNA polymerase will take on a native, biologically active conformation. The transcript most fully analyzed, that of yeast tRNA Phe, has been examined by aminoacylation activity and thermal melting profile (28), precise lead cleavage at a defined site in the D-loop (29), and proton N M R studies (30). All of these investigations support the conclusion that the molecule adopts a fully native conformation, despite the complete absence of modified nucleotides. Thus, it appears that the accuracy of incor-
79
poration of ribonucleotides by the enzyme under the conditions of large-scale in vitro transcription is quite high. Nonetheless, the fidelity of the enzyme has been a source of concern, based upon the observation that NMR linewidths of 5 S RNA molecules seemed to be unduly broad (31). This suggested the possibility of a high rate of misincorporation leading to a heterogeneous population of species. Recently, however, this issue has been reexamined (49). The conclusion is that RNA synthesized by T7 RNA polymerase from identical DNA templates either in vitro or in vivo gives NMR spectra of comparable quality. Broadened linewidths obtained from the original preparations have been traced in many cases to aggregation of the material as a consequence of the presence of 5' overhanging guanine residues. Therefore, transcripts containing such residues should probably be avoided in crystallization attempts unless conditions under which aggregation does not occur are found. Of potentially serious concern is the fact that the T7 RNA potymerase often produces transcripts that are lengthened at their 3' termini by one or occasionally two or more nontemplate encoded nucleotides. This observation, coupled with the fact that large amounts of abortive transcripts ranging in length from two to six nucleotides are always synthesized, emphasizes strongly the need for purification of the desired transcript prior to its use in crystallization attempts. For preparative-scale quantities of material, anion-exchange and reversed-phase H P L C are the methods of choice. Additionally, it will be important to characterize the full-length products obtained via gel electrophoresis, using a separation length appropriate for distinguishing transcripts differing in size by a single nucleotide. Apparently, the extent of 3'terminal heterogeneity cannot be predicted in advance for any given transcript, and reaction conditions for minimization of this effect have thus far not been found (27). Growth of high-quality crystals of RNA produced in this manner may thus in some cases depend upon the adequate separation of transcripts of lengths differing by only a single nucleotide, in sufficient yield for crystallization trials. In the case of tRNAs, aminoacylation of the mixture of full-length and extended transcripts will result in the charging of only properly terminated species. The acylated tRNAs will then be separated more readily from the uncharged extended molecules on HPLC, where the resin and conditions can be chosen to exploit the interaction of the amino acid moiety with the column. The lability of the aminoacyl linkage at high pH should be borne in mind when such a separation is attempted. A final consideration relates to the requirement of T7 RNA polymerase for a specific promoter sequence, rich in G residues at positions +1 to +6, for efficient initiation of transcription (27). It may therefore be difficult to obtain multimilligram quantities of RNAs possessing unfavorable 5'-terminal sequences. This problem can be
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JOHN J. PERONA
circumvented in the case of t R N A transcripts by synthesizing a precursor molecule instead of the mature species. The precursor contains an extended 5' flank of 1015 nucleotides possessing the consensus T7 promoter sequence. The correct 5' terminus can then be obtained via in vitro enzymatic processing using the catalytic M1 RNA of RNase P, which can itself be produced by T7 RNA polymerase transcription in vitro (37). It is important to be aware that the synthesis of some mutant tRNAs by this method may not be possible, since the rate and accuracy of RNase P cleavage are affected by the structure of the mature species (46). A number of mutant t R N A Gin species have recently been synthesized and characterized successfully using this methodology (M. Jahn and D. Soll, personal communication). High-resolution diffracting crystals containing unmodified R N A produced by in vitro transcription have recently been grown of several mutants of E. coli, tRNA2aln, complexed with glutaminyl-tRNA synthetase (J. Arnez and T. A. Steitz, personal communication). The high similarity in structure demonstrated between native and synthesized RNAs provides reason for optimism that this technique will continue to bear fruit in the near future.
(iii) Crystallizations of aminoacyl-tRNA synthetase: tRNA complexes. Table 2 summarizes the crystallization conditions used in the preparation of crystals of two aminoacyl-tRNA synthetase:tRNA complexes, the only high-resolution diffracting crystals of RNA-protein complexes thus far reported (14, 15). Examination of the conditions used reveals both similarities and differences and highlights specific issues that are relevant to the preparation of crystals of other RNA-protein complexes. A major consideration relates to the means by which large quantities of material were made available for the crystallization attempts. Both GlnRS and tRNA Gin have been overexpressed using engineered bacterial strains, leading to the facile production of multimilligram quantities of both macromolecules (14). The high levels of expression facilitated the development of rapid purification methods, and crystals have been readily obtained and reproduced without difficulty. By contrast, AspRS and t R N A Asp were each obtained via very large-scale purifications from large quantities of yeast cells (8, 15, 35). Gradual improvement in the procedures used, necessitated as a consequence of difficulties with yields and reproducibility, has resulted in a succession of crystal forms of increasing quality grown
TABLE 2
Crystallization of Two Aminoacyl-tRNA Synthetase:tRNA Complexes Conditions
Y e a s t A s p R S - t R N A Asp
E. coli G l n R S - t R N A GI~
RNA:protein stoichiometry Precipitating agent Starting concentration Final concentration M a c r o m o l e c u l e c o n c e n t r a t i o n s (initial) Protein RNA Temperature M g 2+ ions Buffer Other substrates
2.4:1 AmSO4 25% 62%
1:1 AmSO4 24%
10 m g / m l 4.8 m g / m l 4 °C 5 mM MgCle 40 mM T r i s - m a l e a t e / N a O H None
6.7-10.0 m g / m l 2.6-3.9 m g / m l 17°C 20 mM MgSO4 80 mM P i p e s 4 mM A T P 4 mM A M P + 60 mM g l u t a m i n e 4 mM A T P + 60 mM g l u t a m i n e 7.4
pH
7.5
48%
C r y s t a l properties
Diffraction limit Space group Cell d i m e n s i o n s
Solvent c o n t e n t No. c o m p l e x e s / a s y m m e t r i c u n i t
2.7 .~ p21212 a = 210.4 A b = 145.3 h c = 86.0 A 69% 1
2.5 .~ C2221 a = 242.8 b = 93.5 h c = 115.7/k 70% 1
Note. C o n d i t i o n s for t h e y e a s t A s p R S t R N A A~pc o m p l e x are generally as reported (15), with s m a l l modifications (M. Ruff, p e r s o n a l c o m m u n i cation). T h e original crystal f o r m of t h e G l n R S : t R N A GInc o m p l e x was grown f r o m solutions c o n t a i n i n g s o d i u m citrate as t h e p r e c i p i t a t i n g a g e n t (14); t h e form reported here was u s e d for t h e s t r u c t u r e d e t e r m i n a t i o n (44).
CRYSTALLIZATION OF RNA-PROTEIN COMPLEXES under similar conditions (15, 35, 41), culminating in an orthorhombic crystal t hat diffracts to 2.7 .~ resolution (15). Th e message is clear: while good expression systems do not guarantee the growth of adequate crystals, they reduce the need for such heroic efforts, which in the long run will result in a much greater number of crystallization attempts and, presumably, successes. Both complexes crystallize in the presence of magnesium ions, and this cation has also been present for many of the RNAs crystallized to date (8-13, 43). Detailed structural analysis of yeast t R N A phe has demonstrated a role for magnesium in stabilizing the structure, and binding sites were determined for a number of the ions in hydrated form, as well as for two spermine molecules (45). In fact, alternative crystal forms of tRNA Phe were grown from solutions t hat differed only slightly in the stoichiometric ratio of magnesium ions to spermine molecules (9). Clearly, the role of these cations in ensuring structural stability, thereby favoring crystallization, justifies extensive exploration of growth conditions in their presence. A variation in magnesium ion concentration of 5 mM or less can cause significant effects on crystal growth and properties. Other multiply charged cationic species, such as spermidine and putrescine, merit investigation as well. T he divalent cations lead and barium have also been present during crystal growth of certain tRNAs (10, 43). Interestingly, the R N A - p r o t e i n stoichiometry in the crystallization drop plays a critical role in the crystallization of the A s p R S - t R N A A~p complex, where only a very narrow range of stoichiometries promoted growth, but not of the citrate-grown form of the G l n R S - t R N A GI~ complex (14, 41; Table 2, note). A t R N A to protein molar ratio of 2.4:1 was found to be critical to the initial growth of the best crystals of the AspRS:tRNA A~pcomplex. T he sensitivity of crystal formation to the RNA:protein stoichiometry in this system is further exemplified by the fact t h a t larger crystals of improved usefulness for diffraction studies were obtained only by seeding into a second crystallization drop which now contained a 3:1 ratio of tRNA:protein (M. Ruff, personal communication). Th e AspRS enzyme is dimeric and binds two molecules of tRNA; these results therefore indicate t hat the optimal RNA:protein stoichiometry for crystal growth need not reproduce t ha t exhibited by the normally functioning enzyme in solution. Conversely, growth of crystals of the GtnRS enzyme complexed to t RN A GIn using sodium citrate as the precipitating agent is insensitive to the RNA:protein stoichiometry in the range of at least 1:1 to 2:1 (14). This monomeric enzyme binds a single tR NA molecule in solution. Another variable that has been shown to be critical to the growth of useful RNA:protein cocrystals is the presence or absence of other ligands of the protein. Aminoacyl-tRNA synthetases, for example, possess binding sites for ATP, AMP, amino acid, and Mg2+-pyrophos phate in addition to tRNA. With the G l n R S - t R N A Gh~
81
complex, no crystals are obtained in the complete absence of additional ligands. Rather, crystal growth of isomorphous forms occurs in the presence of ATP, A T P and glutamine, or A MP and glutamine (14; J. Perona, M. Rould, and T. Steitz, unpublished observations). Any of these ligands may be freely soaked in and out of the crystals without disruption of the lattice. Conversely, no ligands were found to be required for growth of the AspRS:tRNA A~p crystals (15). Additional ligands may aid crystal growth through the stabilization of otherwise flexible protein structural domains. The limitation of experience thus far to two R N A protein complexes limits the extent to which one can state many useful generalizations. As with proteins, the most important aspects to consider are the solubility behavior, ligand-binding properties, and homogeneity of a given complex. Thus, both complexes crystallize in the presence of ammonium sulfate as precipitating agent, at similar pH's, buffer concentrations, and macromolecule concentrations, but there is nevertheless no reason to believe t hat these conditions are in any way indicative of those t hat will be successful in other cases. Crystals of RNAs obtained in the absence of protein have grown from solutions containing either ammonium sulfate or organic solvents as the precipitating agents and encompass a wide range of growth conditions (8-13, 43). In conclusion, it is clear t hat while the efforts involved in obtaining suitable material and growing crystals can be considerable, the potential rewards, in terms of detailed structural information about the mechanisms of recognition and discrimination of RNAs by proteins, are extremely large. T he structure of the G l n R S - t R N A Gin complex has been determined to a resolution of 2.8 (44) and t hat of the A s p R S - t R N A A~p complex has recently been phased, with model-building in progress (M. Ruff, personal communication). A complete understanding of the complex structure-function relationships underlying the critical biological roles of R N A -p ro te in interactions will be possible only when the detailed structures of many more such complexes are elucidated.
ACKNOWLEDGMENTS I thank Thomas Steitz, Dieter Soll, Peter Moore, Joyce Sherman, and Alex Szewczak for useful discussions and permission to cite results prior to publication. The author was supported in part by NIH Grant GM-22778to Thomas Steitz.
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