conceptJ in biochemiftry
Edited by: WILLIAMM. SCOVELL Bowling Green State University Bowling Green. Ohio 43403
RNA's as Catalysts A New Class of Enzymes George M. McCorkle and Sidney Anman Department of Biology, Yale University, New Haven, CT 06520
In this review we will discuss the two polyrihonucleotides whose recently discovered activity as biological catalysts has upset the long-standing dogma that all enzymes are proteins. One of these is the RNA subunit of a bacterialenzyme, rihonuclease P, which cleaves transfer RNA molecules to produce the 5' ends of functional transfer RNA's (1-3).This essential enzyme is ubiquitous among living organisms. The other is an RNA sequence embedded in the nuclear precursor transcript of the large ribosomal RNA subunit of the protozoan Tetrahymena. This "intervening sequence" or IVS, we now know, is the prototype of a group of similar sequences widely distributed among eukaryotes and recently found in a prokaryotic bacteriophage as well (2,4).These
Figure 1. The nucleotide sequence and presumed secondary structure of a reoresentalive ribonuclease P substrate. The subshats is the orecursor to an E colr SJPPIBSSOI IRNA Th s SUPPIBSSOI tRNA (des~gnatecsu3+ m genet c nomenc ature) ~sderlvedfrom tRNAT" The prec,rsor molec~ies ca ed pTyr me sketch snows pTyr wlth the rRNA molely in the Cloverleaf cOnf#g~ratlon and the nucleotides in the mature tRNA numbered. No nucleotide modifications are shown. The arrow at position 1 of the mature tRNA sequence is the Site of cleavage by ribonuelease P. The boxed nuclectides are not part of the 3' terminus of the mature tRNA and are removed by a different processing enrvme. The CCA seouence at oasitions 83-85 is found in ail tRNA aene " transcripts in E. mlibut is not present in all the tRNA gene transcripts of some other organisms, such as bacteriophage T4.
two molecules may be representative of still other catalytic RNA's: Recently, a viroid RNA and another small infections RNA molecule that is associated with plant viruses have been shown to mediate their own site-specific cleavage, and a growing body of evidence implicates several small &Aear RNA's in the splicing of eukaryotic messenger RNA's (2). We will not discuss these latter molecules in detail. Since the 1920's, hundreds of enzymes have been obtained in the pure crystalline state, and thousands more have been well characterized biochemically. All have been isolated by assavine catalvtic activitv throueh successive ~urification step& iithouiprior knowiedge ofthe hiochemieal nature of the catalyst. But until the characterization of the two RNA's considered here, every one of these enzymes had turned out to be a protein. I t is hardly surprising, therefore, that the principles of biological catalysis were developed from a solid groundwork of protein chemistry and from insights based on the known crystal structures of protein enzymes. The rate acceleration of biochemical reactions and the remarkahle speriiiciry of enzyme-substrate interartions (including rhuae of protein enzymes acting on nucleic acid substrates) were satisfactorily explained interms of the chemical properties of the 20 amino acids and the conformational versatility of polypeptide chains. Now, however, discussion of biological catalysis must be expanded to include the reactions catalmed bv RNA (5).Will RNA catalvsis be confined to nucleic acidsubstrates, or perhaps only i o RNA substrates? What is the precise nature of the "active sites" that RNA folding will allow? Finally, terms such as "intramolecular catalvsis" and "self-s~licina" are now needed to describe someRNA-catalyzed;eactfons, such as the Tetrahymena example described below, in which the catalytic molecule is also a product of the reaction. Rlbonuclease P In all organisms, functional transfer RNA molecules are formed from lareer nrecursor transcrints hv a series of orocessing steps. ~ l % o n h e a s P e creates t h e 5; ends of mGure transfer RNA's by making a single endonucleolytic cleavage in the precursor molecule (Fig. 1).This enzyme must recognize and cleave correctlv all of the 60 or so transfer RNA precursor transcripts, whch vary widely both in length and in their primary sequence around the site of cleavage. Even more remarkably, the ribonuclease P enzymes froma variety of organisms'(e.g., gram-negative and gram-positive bacteria, yeast, silkworms, and mammals including humans) all can process correctly precursor transfer RNA molecules from any other organism that has been examined. This is so Volume 64
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Precursor domain
H Mature domain
OH
Figwe 2. TWOpossible mechanismsfor the cleavage reaction catalyzed by the RNA subunit of ribonuciease P. (above, left) in this hypothetical mechanism of hydrolysis, the reaction is catalyzed by a Mg-HP complex that is initially bound to a phosphate of the RNA subunit of ribanuciease P. Mg2+ Is formally Shown as hexacoordinated, but may well be tetracoordinated as indicated by the parentheses around the two equatorial water iigands. in the tap panel a Water moiecuie from the solventthat will participate in hydrolysis is positioned by a hydrogen bond to an 0 or N atom in the RNA subunit. in the middle and bonom oanels the tRNA DrecurJor substrate is bound bu the water molecule ahached to me RNA subumt al r banuclsare P, and passes thro~gh a hansltm state prlor to cleavage a l me upnrsam precursor olqonucleotde ano the add llon of Or1 to i s 5' terminal phosphate Aner lhe react on steps shown here, a solvent water chain between the axial iigands of MgZ+re-cocks the enzyme for the next cyde. The M1 notation is an arbitrary notation utilized in experiments in the authors' lab. (Reprinted wim permission from ref 7. Copyright 1986 American Chemical Society.) (above, right) A general scheme for h~droivsisof IRNA Orecursors bv the RNA subunit of ribonucleare P. The hatcned oodndary ndlcater the s~rfaceof the RhA subunot Band 8' are baser Tne phorphadfestsroond connect ng the precusor and mature ooma ns of me precursor tRhA smstrats 8% dsponsd (Reprmed wth perm sslon from ref 8 Copyright 1985 by the AAAS.)
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despite the sequence divergence of more than 50% for instance, between the RNA subunits of the E. coli and B. subtilis enzymes, and an even greater divergence between subunits of the bacterial and eukaryotic rihonuclease P enzymes, as judged by their hybridizaiion with bacterial RNA and DNA probes. (It is thought that ribonuclease I'activities are present in both mitochondria and nuclei of eukaryotes, but their RNA components have not yet been rigorously characterized.) The orecise molecular basis of this remarkable cleavage specificity is not known. I t must include, a t the least. conserved features of the three-dimensional structures of both the enzyme and its precursor transfer RNA suhstrates. The complete active enzyme from E. coli (i.e., the holuenzyme) rontains both protein and HNA subunits. In viro and in vitw (in huffers withmagnesium ionconcentrationsin the physiological range of 5-1U mM), both types of subunits are reauired for ratalvtic activitv. When it war first orwed that thd holoenzyme cbntained bbth RNA and proteh, the obvious inference was that the protein subunit did the catalytic work and the RNA subunit conferred stability or substrate specificity on the enzyme complex. However, rigorous analysis of the roles of the two subunits had to await the development of an assay system in which enzyme activity could be reconstituted from pure, inactive subunits. The first investinations with such a svstem revealed that the protein alone Lad no detectable hydrolytic activity, and this ied to the idea that the RNA subunit, also, might be anecessarv component of the active site. subsequently, in experimentsto determine optimal conditions for reconstitution of activity, purified RNA and protein subunits from both the E. coli and B. subtilis enzymes were assayed separately and in all comhinations (3). The protein subunits from either genus, alone, had no activity under any conditions. Astonishingly, however, a t magnesium ion concentrations substantially higher than those normally used for assay of the enzyme from E. coli (60 mM vs. 5-10 mM), or in the presence of 10 mM magnesium plus 1 mM spermidine, the RNA subunits of both eenera processed precursor substrates correctlv, in the T o eliminate any possibiliiy that a total absence of protein contaminant had been co-purified with the RNA subunits and might be responsible, the experiments were repeated using an unproressed transcript of the cloned gene fur the RNA rubunit, prepared in vitro. These experiments established beyond doubt that the HNA component of rit~onuclease P contained the catalytic site of theenzyme. If the protein subunit was not the catalyst, then what was
its function in the holoenzyme? The finding that a high magnesium ion concentration or spermidine could replace the strongly basic protein suhunit in an active complex suggested that the protein served as a structural counter-ion to provide electrostatic shielding; even so, i t is worth noting that the catalytic rate (kc,,) in the standard assay using tyrosine precursor tRNA as suhstrate was twofold higher in the presence of the protein suhunit than in the prescnce of magnesium or spermidine. Finally, in vitro processing of precursor transfer RNA's hy the RNA subunit alone is more efficient when the CCA sequence found at the 3' terminusof mature transfer RNA's is present than when it is absent, whereas the holoenzyme cleaves equally well in either case ( 6 ) .The protein subunit thus has a demonstrable effect on the rate of processing of various substrates. The exact chcmistry of the rihonuclease P reaction is not knownai yet, nor cnnthe nature of theactivesite beinferred in more than very general terms. The RNA subunit retains some residual cataiytic activity even if as many as 120 nncleotides are removed from its 3' end, just as some protein enzymes can lose a substantial number of amino acids from one terminus without destroying- the structural integrity . . of the catalytic site. The reaction requires the native structure of the RNA suhunit in association with either the protein subunit, a high concentration of magnesium ions, spermidine or polyethylene alvcol. In fact. rates of reaction of the RNA subunit in v i t r o h the absence of protein) can approximate those expected in vivo provided that 100 mM magnesium, 5%polyethylene glycol or 10% 2-methyl-24-pentanediol are in the reaction mixture. In addition to the structural reauirement for a divalent cation (Ca, Sr, or Mg will function in this regard), there is a basal requirement for 5-10 mM magnesium, presumably for coordination with molecules a t the active site. The structure of the RNA component appears to be strongly conserved in spite of wide differences in primary sequence. The reaction does not reanire a free 3'-hvdroxvl on the RNA suhunit and is prohahl; a site-specific h y d r o h i s (Fig. 2). I t has not vet been determined whether the hvdroxvl n h e o p h i l e that attacks the phosphoester bond is herivld from a water molecule in solution or from the surface of the RNA. There is no transient covalent intermediate formed between enzyme and substrate durina the reaction, or other breaking and re-joining of bonds in t i e RNA subunit itself. The reaction requirements will he considerrd in more detail below in comparison with the reaction mechanism of the Tetrahymena intervening sequence. ~
lntervenlng Sequence of the Tetrahymena Ribosomal RNA Precursor The other catalvticRNA of interest here was discovered in the course of research on RNA splicing in the protozoan Tetrahvmena. The nuclear transcript of the laree ribosomal RNA &bunit contains a 413-nucieotide intervening sequence (intron or IVS) separating the two parts (exons) of the functional ribosomal RNA. One of the first steps in processing this transcript is the removal of the IVS and the joining of the two exons of the mature ribosomal RNA. About five years ago, while searching for the enzyme responsible for splicing.'rhomas Cerh and his associates found that the reaction took place in vitro in a mixture containing only magnesium chloride, ammonium acetate, and guanosine, but without either added protein or an external energy source such as ATP to drive the ligation reaction. Either a tightly hound protein enzyme had survived the stringent steps used to purify the precursor RNA or-reasoned the authors in their r e p o r t t h e reaction was auto-catalytic; that is, the RNA molecule. the onlv macromolecule in the reaction mixture, was itselfboth catalyst and suhstrate. The following year the result was confirmed under conditions
- f 9
A A UCUp, GpUAA
UCUpUAA
)'
precursor
ligated exons
Figure 3. Self-splicing reactions of the intervening sequence (IVS) of the Tehahymenaribosomal RNA precursor. (1)Guanoslneaddstome 5' end of the IVS, releasing the 5' exon. (2) The 5' exon adds to me 3' exon, ligatingthe two and releasing the IVS as a linear molecule. Bath reactions proceed by lransesterificstion. (Reprinted with permission from ref 9. Copyright 1985 by M.I.T.)
that excluded any possibility of a Tetrahymena protein heing present, namely, by use of aprecursor fragment, containing the IVS and the adjacent sections of both exons, which had been transcribed in vitrofrom clonedDNA. This experiment demonstrated conclusively that the activity was inherent in the RNA itself. This, however, was only the beginning of the story of this astonishing molecule. As we shall see, a series of catalytic events that occur after the self-splicing reaction provided imoortant evidence about the catalvtic site and mechanism. when this was combined with the e;idence from the splicing reactionitself. it was oossihle to reconstruct theevents a t the catalytic site in consiberahle detail. An important clue to the nature of the self-splicing reaction is that a guanosine, not encoded by the DNA sequence of the gene, is added to the 5' terminus of the intervening sequence during its excision from the precursor transcript. This explains the requirement for guanosine in the in vitro reaction and also explains why no external energy is needed for ligation of the ribosomal RNA exons following excision of the IVS. As shown in Figure 3, both steps occur by exchange of phosphate esters, with no net gain or loss of honds. The 3'hydroxyl of the free guanosine molecule attacks the phosphate a t the 5' splice junction, transrerring the G to the 5' end of the IVS and leaving a 3'-hydroxyl a t the end of the 5' Volume 64 Number 3 March 1987
223
exon. The latter then serves as the nucleophilefor thesecond transfer reaction, which ligates the two exons and frees the
.In., its "self-splicing" mode, the IVS lacks one quality rell, 7C
quired of a catalyst, namely that i t act on many substrate molecules in a second-order reaction without itself being altered or consumed. There are few precedents for a biomolecule that is simultaneously both substrate and enzyme. (A closely analogous example is the proenzyme from which histidine decarboxylase is derived by post-translational modification in an unusual, intramolecular, single turnover event (lo).) Yet, although the IVS emerges from the selfsplicing reaction altered, its catalytic capability is not lost. Just three years after the discovery of self-splicing, i t was demonstrated in another remarkable paper that the IVS can function, after all, as a true enzvme under certain conditions in vitro ( 4 ) . Let us examine the-details, depicwd in Figure 4. After its excision from the precursor, the IVS undereoes a cascade of additional reactiom: first, i t cyclizes in a transesterification reaction that joins its 3'-hydrnxyl t o a phosphate near its 5' end, releasing a 15-nucleotide fragment from the 5' end (Fig. 4, reaction 1). The bond formed in this reaction is labile and undergoes a slow hut specific hydrolysis (Fig. 4, reaction 3). When re-opened, it again cyclizes in the same manner. releasine a second fraement four nucleotides long (Fig. 4,'reactionh). site-specific hydrolysis again occurs a t the same labile bond to yield the final stable product, L-19, a linear molecule lacking the first 19 nucleotides of the excisedRNA (Fig. 4,reaction 5 ) .This truncated IVS acts as a true enzyme 'inlnt'ermolecula~reactions in vitro. With
Gow
\ .... Subwquent reactions of !he interrening sequence (iVS)aner selfsplicing. (11Major cyclization reaction. The 3'-terminal guanosine ofthe linear IVS (L IVSI adds between the 15th and 10th nucleotides, forming a circular molecule and releasing a 15-nudeotide fragment, the 15-mer. (2) Minor cyclization reaction. Same as (1) except that 3'-termlnal guanosine adds between the 19th and 20th nucieotldss.Thls reaction is discussed in ref 9. (3) Specific hydrolysis of circular IVS (C IVS).C IVS opens at the site of cyciiratian, leaving 3' hydroxyl and 5' phosphate ends. (4) Cyclization of L-15 IVS. site. 151 of The L-15 cvcliles at the secondam. wclization . . .Soecific . ~.hvdroivsi~ , C' VS. ana oga~s to (3) React ons I . 2. and 4 proceed by lransesleriiicatim (Ue~mledworn permission lrom re1 9. Copyright 1985 Dy M.1.T.I Figure 4.
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RNA substrates containing oligopyrimidine sequences a t their 3' ends, it can perform intermolecular ligation reactions and can polymerize polynucleotides of appreciable length. In the reverse reaction the IVS possesses the qualities of a site-specific ribonnclease that can recognize a pyrimidine sequence of three or more nucleotides and cleave a t its 3' end. The self-snlicine reaction (Fie. 3) as well as the subsequent reactions oftthe excised I ~ ((Pig. S 4) occur a t a single "active site" in the folded structure of the molecule. RNARNA interactions govern both sets of reactions; i t is in this resnect that the reaction differs most from that which a enzyme might mediate. At the S e n d of the IVS (Fig. 5)isa "ruitlr seauence" tCCVCGACCG) that can base-uair with a-sequence spanning the 5' exon-intron junciion (CUCUCUAAA) to align it for the initial self-splicing reaction and perhaps (although the evidence is as unccar) to align the 3' intron-exon junction for the second phosphotransfer, which ligates the two exons and frees the IVS. After the IVS has been excised, the "guide sequence" is also instrumental in the processing reactions which subsequently remove additional nucleotides from its 5' end. Conformational changes occur to re-align new tripyrimidine sequences on the 5' side of the sites of cyclization until there are no more such sequences left to serve as substrates within the active site. These translocations are reminiscent of the ratchet mechanism bv which messenger RNA is thoueht to move, three nucleotides a t a time, through the rib&ome during protein synthesis. The correctness of the "guide sequence" model is supported by the fact that the stahle final molecule, L-19,acts in vitro in a formal sense as an RNA polymerase, specifically with polycytidylic acid and (to a lesser extent) with polyuridylic acid substrates. In addition, it run cntalye nth& i&ermulecttlar splicin: reactions using pyrimidine-rirh oligonurleotides as 5' exons. Thus. after its eacisiun from the ribosomal RNA nrecursor, the' IVS becomes a true enzyme capable of mebiatiug intermolecular reactions. The anestion arises: Does this versatile polymerase/endonuclease have a natural role within the cell? We do not know. There is no evidence to sueeest that the intricate structure of the active site evolved f o ; k y purpose other than the precise and efficient maturation of the ribosomal RNA. Nevertheless, the subsequent cascade of self-processing reactions and the in vitro intermolecular catalytic activity of L-19 dramatically demonstrate the efficiency of catalysis by an RNA enzyme on RNA substrates. The Tetrahymena IVS is the best characterized represeutative of a large class of RNA splicing activities which are found widely dispersed among living organisms. RNA's of this class, termed Group I because they share a common set of conserved sequence elements, include mitochondria1 mRNA and rRNA introns from yeast and fungi and also others from such diverse sources as the chloroplasts of higher plants and the E. coli bacteriophage T4. A second class, Group 11. comes from manv of the same sources hut has a dit'ie;ent set of consrrved~sequcnces.Roth Croup I and ( ; I O I I ~ I I intnms are capnl~leof self-splicing, bur with merhanistic differences: As we haw seen, Group 1 inrrons require guanosinr and the excised 1VS furms a circle; Gruup I1 introns do not require guanosine and the excised IVS forms a branched "lariat" srructure similar to those formed by the excised introns of nurlear RNA orecursors. The circulnr IVS of the Croup Ireaction is produced because the &eophila attacking the nhosnhoester bond is a 3'-hvdroxvl: "lariats" introns are prod;ced by t i e Group I1 and nuclear ~ R N A because the attackine nucleo~hileis a 2'-hvdroxvl within the intron. Some repres&tatives of both ~ r o u pI a i d Group I1 are self-splicing in vitro like the Tetrahymena IVS while others are not; the reason for this difference is notentirely clear hut may be due to the absence in vitro of a protein cofactor that is required in vivo to stabilize the RNA. ~
~~
.
~~
4 Splicing
J
--
Ligated exons
L - -IVS
Conformotional change ( tronslocotion ) CA G A A-U U -A
4
CA
Major cyclization C A G A A-U
A-U A-U
-4 Minor cyclizotion C' I V S
A-U
UcUo"J
u,,
~-
Oligonucleotide binding
~-
Reverse
-
cycltzotion
-
UCU-L'
Figure 5. The single active site model for self-splicingand other self-processing reactions mediated by the TetrahymenaIVS RNA. Lower case letters, exons: capital lettern. IVS. The pre-rRNA is shown with the 5' exon paired with the internal guide sequence. The critical oligopyrimidine binding site within the internal guide s% guence is boxed. It is part of the active site for trsnsesterification.The labile phosphodiester bondat the 3' splice site is depicted as e strained band. Followingsplicing, the oligopyrimidine binding site is unoccupied, so a local conformational change can bring another tripyrimidine sequence into the binding site. The UUU precedingthe major cyclization site Is the only remaining pyrimidine that can bind to the GGA. The CCU precedingthe minor cyclizationsite could presumably bind to 8 portion of the internal guide sequence adjacent to the GGA, as shown. In either case the 3' terminal guanosine residue of the IVS RNA can occupy the guanosine binding site and undergo transesterificationto produce a circular IVS RNA (C IVS or C' IVS). Cyclization produces a snained GpA or GpU bond. Followingcyclization, the oligopyrimidine binding site is again unoccupied and available for binding Uipyrimidines such as UCU, which can attack the cyclization junction and linearize the IVS RNA (3.(Reproducedwith permission, from the Annuel Review of Biochemistry Vol. 55. Copyright 1986 by Annual Reviews inc.)
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Comparison of Ribonuclease P and Tefrahymena IVS
Reactlons Most of our knowledee of RNA catalvsis comes from studies of the ribonuclease"~and ~ e t r a h i m e n aIVS reactions. Both reactions, as well as the other known examples of RNA catalysis, share these characteristics: None require an external source of energy. All depend on the integrity of the native structure of the RNA molecule, which can he maintained by one or more of the followina: a orotein co-factor (as in ribonuclease P), magnesium o;other divalent cations, or polyamines such as spermidine. Whether intramolecular (the Tetrahymena IVS) or intermolecular (ribonuclease P), these reactions all have an RNA molecule as substrate. The only reaction mechanisms so far observed are phosphoester transfer reactions and specific hydrolysis. The transfer (transesterification) reactions can emolov . . as the attacking" nucleophile either an available 3'-terminal hydroxyl or a 2'hvdroxvl from within the RNA molecule.. vieldine.. resuec. ti5,ely as product either a cyrlized IVS (Tetrohyneno precursor ribosom:~lHNAJor an IVS in the form ot'a branched "lariat" (Group 11 andnuclear mRNA introns).
-
Several points are worth noting: 1. Both ribonuclease P and the Tetrahymena N S create
RNA products having 5'-phosphate and 3'-hydroxyl termini. Such termini are characteristic of functional ribonucleic acids. Bv contrast. deeradative reactions such as nonenzymatic alkaline hydroiysis or attack by protein 5'-hvdroxvl ribonucleases such as T, normallv- produce . and 2',3'-cyclic phosphatk termini. 2. Complementary antiparallel RNA-RNA base-pairing at the active site is the unique contribution that an RNA enzyme can make to the specificity and mechanism of catalysis. The nine-nucleotide guide sequence of the Tetrahvmena IVS. discussed above. is critical t o the selection of the 5' splice site and also to the positioning of the 3'-OH terminus of the 5' exon for attack on the 3' splice site. I t is not yet clear, however, that this will be a universal feature of the active sites of RNAenzvmes. Ribonuclease P , for example, must recognize and cleave a t a single specific site the precursors of more than 60 transfer
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Journal of Chemical Education
RNA's, which do not share a single common sequence around the site of cleavage. Ribonuclease P is thought to recognize the three-dimensional structure of the pretransfer RNA's, hut i t is also possible that nucleotides highly conserved at certain locations within the pretRNA sequence may also play a part in the specific positioning of the 5' cleavage site. 3. The role in vivo of the protein co-factor in rihonuclease P and in several of the Group I and Group 11 self-splicing activities remains to he clarified. In vitro, the protein subunit of ribonuclease P can he replaced by magnesium ions or polyamines which can provide structural stability, leading to the inference that this is the role of the protein in vivo as well. There are also questions to he answered about the possible roles of proteins associated with some of the splicing activities. Why are some of these activities functional in vitro in the absence of protein, such as the Tetrahymena IVS, while others are only functional in vivo? I t has been hypothesized that some of the IVS's, which are several times larger than the 400-nucleotide Tetrahymena IVS, require a protein co-factor for stability. 4. A final point is that in Tetrahymena and other systems such as plant virus satellite RNA the particular bond a t which catalysis is to occur has been made especially labile, presumably by strain resulting from RNA structure. I t will be interestine to see if the 5' cleavaee sites of precursor transfer RNA's which are substrates for rihonucleaie P are sirnilarlv strained orior to catalvsis. and if so, what elements of tertiary produce this lahility.
structure
Literature Cited 1. Altman. S. o t al. "RNA~proceasingNudeeses" snd "RNA-pmc~asingNucleaaes. A Supplement". In Nuclmses; Linn, S. M.; Roberts, R. J., Edn.: Cold Spring Harbor Lahoratory,ColdSpring Harbor,NY, 1 9 8 5 2. C8ch.T. R.; Bars, B.L.Ann.Rsu.Biochsrn. 1966.55.599. 3. Guerrier~Takada,C. et d.Cell 1983.35.849. I. Zsug, A. J.: Coch, T. R. Seianca 1989,231,470. 5. Lewin. B. M. Genes, 2nd ed.; Wiloy: New York, 1985. 6. Guerrier-Takada, C. et a 1 Cell 1984 38,219. 7. Guerrior-Takada. C. et el. Biochemistry 1986.25.1509. 8. Mar3h.T.L.:Pace. N. R. Science 1985,229.79. 9. Sullivan. F. X.: Coch. T. a. Cell 1985.42.639. 10. Rocsei. P.A.;Snell,E.E.A n n R r u . Biochem. 1984.53.357.
