Noncanonical complexes of mammalian eEF 1 À with various deacylated tRNAs

The formation of noncanonical complexes of two eEF1A isoforms with different types of deacylated tRNAs was examined and apparent dissociation constants were measured. A higher affinity to tRNA was found for the tissue specific isoform of elongation factor eEF1A2, as compared with that of eEF1A1. For the first time the formation of noncanonic complex of eEF1A with initiator tRNAi Met was found to be possible. A specific role of some tRNA sites in the interaction with eEF1A was discovered.

In tro duc tion.Pro tein biosynthesis in volves two general stages, namely the aminoacylation of tRNAs and mRNA trans la tion on ri bo somes.Trans la tion is thought to be a very com plex pro cess, es pe cially in higher eukaryotes.This pro cess is char ac ter ized by a high level of struc tural and func tional compartmentalization. Chan nel ing is one of the mech a nisms, ex plain ing po tential profit of compartmentalization in pro tein biosynthesis [1].It was sug gested that tRNAs are never free in the cy to plasm and all the time are bound to some part ners like elon ga tion fac tor 1A (eEF1A), as eEF1A*GTP*aa-tRNA com plexes, aminoacyl-tRNA syn the tas es (aaRS) or ri bo somes [2].Ac cord ing to the chan nel ing con cep tion there is a di rect trans fer of tRNA from Å-site of ri bo some to aaRS and aminoacyl-tRNA from aaRS to À-site of the ri bo some with out dis so ci a tion into cy to plasm [2].Re cently were have pro posed that eEF1À may as sist not only as a carrier of aa-tRNA but also may ex e cute a non-ca non i cal func tion of the deacylated tRNA trans fer from ri bo some to aaRS in the form of eEF1A*GDP*tRNA ter nary com plex [3].The ex is tence of the lat ter in vi tro has been shown by sev eral tech niques.
Ear lier we con firmed the for ma tion of eEF1A*GDP*tRNA Phe non-canonicalal ter nary com plex by flu o res cence po lar iza tion method [4,5], and de tected sim i lar in ter ac tion for eEF1A1 and eEF1A2 with tRNA Ser [6] by na tive gel band-shift as say.This means that the ex is tence of such type of com plexes may be a com mon fea ture of both eEF1A isoforms as well as dif fer ent types of tRNA.
There are two isoforms, eEF1A1 and eEF1A2, of trans la tion elon ga tion fac tor in higher eukaryotes.eEF1A1 is pres ent in al most all tis sues ex cept neu rons and mus cle cells.eEF1A2 func tions only in neu rons and mus cles where it ap pears and re places eEF1A1 dur ing ontogenesis im me di ately af ter the birth of organ ism [7].Un til now any sig nif i cant dif fer ences in func tion ing of these two isoforms were not found.
In the pres ent work we stud ied the for ma tion of non-canonicalal com plexes of eEF1A1 and eEF1A2 with tRNAs spe cific for Phe, Tyr, Asp, Leu, Ile, Met, Lys, and ap par ent dis so ci a tion con stants (K d ) were measured for each com plex.More over, we first showed the possi bil ity of non-ca non i cal ter nary com plex for ma tion be tween eEF1A1 or eEF1A2 and ini ti a tor tRNA i

Met
. A role of spe cific base pairs in tRNA struc ture that in flu ence its in ter ac tion with eEF1A was stud ied us ing a mutational anal y sis.We used in vi tro tran scribed tRNA 3 Lys with mu ta tions in Ò-stem and ac cep tor stem for this purpose.
T7 RNA-poly mer ase was a kind gift of Prof. M.A.Tukalo.
Nu cle o tide transferase was pu ri fied from yeasts as de scribed in [8].
To tal tRNA iso la tion.To tal tRNA was ob tained from bo vine liver us ing di rect deproteinization of tissue ho mog e nate with phe nol fol lowed by chro ma tog raphy on DEAE-cel lu lose [9].In di vid ual tRNA sam ples were iso lated form to tal tRNA ac cord ing to the pro cedure that in cludes chro ma tog ra phy on BD-cel lu lose and sub se quent chromatographies on Spherogel-TSK DÅÀÅ 5PW, Ñ3 RPSC 5u Ultrapore and Hypersil 5C4 ("Beckman") col umns us ing HPLC Gold sys tem.Ini ti -a tor tRNA un like elongator one may be aminoacylated by to tal aaRS from E.coli with [ 14 C]-methionine that was used for its de tec tion.All in di vid ual tRNA sam ples were la belled in 3'-end by [a-32 P]-ATP us ing yeast nucle o tide-tRNA transferase as described in [10].
eEF1A1 was pu ri fied from rab bit liver as de scribed in [4], and eEF1A2 was pu ri fied from rab bit mus cles by sim i lar method, omit ting only a gel fil tra tion step.
Band re tar da tion as say in poliacrylamide gel.The pro tein-RNA com plex for ma tion was stud ied by band re tar da tion as say in polyacrylamide gel un der na tive con di tions.In cu ba tion of elon ga tion or ini ti a tion factors (50-2000 nÌ) 200 mÌ GDP and 5000 ñðm of radio ac tively la belled tRNA was per formed in 10 Fl mixture con tain ing 20 mM Tris-HCl pH 7,5, 50 mM KCl, 5 mM MgCl 2 , 2 mM DTT and 10% Glyc erol.In cu ba tion was done dur ing 15 min at 37 0 Ñ, af ter that sam ple was loaded on na tive 5% polyacrylamide gel (acrylamide/N,N-methylenebisacrylamide ra tio -29/1) pre pared in 25 mM Tris/bo rate buffer pÍ 7.5, con taining 0.5 mM EDTA and 5% Glyc erol.Elec tro pho re sis was per formed on minigel slabs dur ing 2 hours at 4 î Ñ with 100 V con stant volt age.Then gel was dried, exposed on ÎN²ÊÎ-RF1B ("AGFA") film for 18 hours at +4EC.The ra dio au to graphs obtained were analyzed with "GelProAnaliser" program.XL-1-Blue com pe tent cells were trans formed by liga tion mix tures as de scribed in [11].Pos i tive clones were found af ter ampiciline and X-Gal se lec tion.
Re com bi nant DNAs were iso lated us ing Qiagen Maxiprep Kit.The se quence of each in sert was confirmed us ing au to matic sequenser ABI 377 ("Ap plied Biosystem", USA).
Pre para tive T7 poly mer ase me di ated syn the sis of tRNA in vi tro.The con struc tions ob tained ear lier were di gested by BshNI that cre ates 3'terminus of tran script lack ing two last nu cleo tides A and C in tRNA sequence.Thus, the size of tRNA be came 74 nu cleo tides in stead of nor mal 76.In vi tro tran scrip tion of tRNA was per formed by T7-RNA poly mer ase in the mix ture, con tain ing 40 mM TrisHCl pH 7,9, 6 mM MgCl 2 , 10 mM DTT, 10 mM NaCl, 2 mM spermidine.ATP, GTP, CTP and UTP con cen tra tions were ad justed cor respond ing to the ra tio of each nu cle o tide pres ent in the se quence.Af ter syn the sis be ing com pleted, the mix ture was treated with DNAse I. Next.we pu ri fied transcribed tRNA by chro ma tog ra phy on DEAE cel lu lose (in buffer, con tain ing 100 mM NH 4 Ac, 10 mM MgCl 2 , 100 mM NaCl), and elec tro pho re sis in 10% polyacrilamide gel (19:1) in the pres ence of 7 Ì urea.A band, cor re spond ing to the tran scrip tion prod uct, was ex cised from gel and tRNA was ex tracted into water af ter over night in cu ba tion on shaker at 37EC.Dena tur ation-renaturation of tRNA was done by heat ing till 72EÑ fol lowed by slow cool ing.Ac tiv ity of each tran script was ex am ined in aminoacylation re ac tion (data not shown).All tran scripts were la belled by [ 32 Ð]ATP using yeast nucleotide-tRNA transferase as mentioned above.
Re sults.Com par i son of the af fin ity of dif fer ent tRNA spe cies for eEF1A1 and eEF1A2.As men tioned ear lier, tRNAs seem to be never free in a cell and all the time are bound to some part ners -eEF1A, in the form of eEF1A*GDP*aa-tRNA com plex, aaRSs or ri bosomes.eEF1A was sug gested to be a part ner of deacylated tRNA, guid ing it from the ri bo some to aaRS dur ing the tRNA chan nel ing cy cle [3].We reported ear lier the for ma tion of ter nary com plex of eEF1A1*GDP with tRNA Phe for which an ap par ent K d was mea sured by flu o res cence po lar iza tion [4], whereas the dis so ci a tion con stants for ter nary complexes of eEF1A1*GDP and eEF1A2*GDP with tRNA Ser were estimated by band shift assay [6].
We stud ied the for ma tion of non-ca non i cal ter nary com plexes for GDP-bound form of eEF1A1 and eEF1A2 with tRNAs of dif fer ent specificities, namely tRNA Asp (Fig. 1), tRNA Ile (Fig. 2), tRNA Tyr Mutation variants of tRNA 3 Lys gene.
Construction of tRNA3 Lys gene cloned into pUC18 plasmid.
tRNA Met , tRNA Leu , tRNA Pro (Fig. 3), by the band re tarda tion tech nique.The ap par ent K d of pro tein-tRNA com plexes was es ti mated to be equal to the con cen tration of eEF1A re tain ing a half of the la belled tRNA.The amount of free tRNA and bound with eEF1A was mea sured by densitometry.As shown in Fig ures 1-3, the K d val ues for eEF1A1*GDP com plexes with tRNAs of var i ous specificities are in 200-750 nÌ range, at the same time the K d for eEF1A2*GDP*tRNA com plexes are in 50-400nÌ range.In ter est ingly, for all tRNAs stud ied the sta bil ity of eEF1A2*GDP*tRNA ter nary com plex is 2-4 fold higher than that of the eEF1A1 me di ated ter nary com plexes.Only one ex ception was ob served for tRNA Phe which has the same affin ity for the both isoforms of elon ga tion fac tor.All data for the ter nary com plexes for ma tion are shown in ta ble 2, K d val ues were measured in this and previous studies [4,6].It is worth not ing that K d value for the com plex between eEF1A1 and tRNA Phe pre sented here in ta ble 2 differes con sid er ably from the value ob tained in the pre vi ous work.It is nec es sary to take into ac count that our re sults were ob tained in non-equi lib rium con ditions in contrasr to the pre vi ous ones ob tained at equilib rium by another method.
In this work we showed the for ma tion of non-canon i cal com plexes for eEF1A and 8 deacylated tRNAs of dif fer ent specificities that is in fa vour of the deacylated tRNA chan nel ing pro cess.It is known that eEF1A binds all aminoacyl-tRNAs with a high and near equal af fin ity.The dif fer ence in K d val ues observed for com plexes be tween eEF1A isoforms and dif fer ent deacylated tRNAs can be equal ized by dif ferent im pact of aminoacid res i dues re sult ing fi nally in sim i lar sta bil ity of the eEF1A*GTP*aa-tRNA ter nary com plexes.
Thus, the for ma tion of eEF1A*GTP*aa-tRNA ter nary com plexes with equal ther mo dy namic char ac ter is tics may be im por tant for their in ter ac tions with ri bo some [12].It was pro posed, that such a mech a nism of com pen sa tion ex ists for prokaryotic aa-tRNAs [13].On the other hand, it was sug gested that the pri mary struc ture of tRNA it self influ ences the in ter ac tion of re spec tive aminoacyl-tRNA with prokaryotic elongation factor EF1A [14].
How can we ex plain the fact that two isoforms have dif fer ent af fin ity for the same tRNA?There is no clear an swer to this ques tion.It was found that eEF1A1 has more ex tended con for ma tion in so lu tion than eEF1A2 [15], that, prob a bly, may be a rea son for such dif ferences.Un for tu nately, the re sults ob tained al low us to sug gest only the ex is tence of some dif fer ences in function ing of two EF1A isoforms in the tis sues where they are expressed.
For the fist time, we showed here the abil ity of two eEF1A isoforms to form the non-ca non i cal ter nary com plexes with ini ti a tor tRNA i Met (Fig. 4).Ac cord ing to Fig. 4 these com plexes are formed hav ing a sim i lar sta bil ity as com pared with the elongator tRNAs.Thus, K d for the com plex eEF1A1*GDP*tRNA i Met is ap prox imately 370 nM and for the com plex eEF1A2*GDP*tRNA i Met is 250 nM.The ini ti a tor tRNA in ter acts with both isoforms of elon ga tion fac tor even better than some elongator tRNA, in clud ing tRNA e Met .The for ma tion of non-ca non i cal com plexes of eEF1A with the ini ti a tor tRNA was nei ther ex pected nor predicted [16].More over, it was shown that Met-tRNA i Met did not act as an elongator tRNA in a cell-free pro tein syn the sis sys tem.Be sides, tRNA i Met has two antideterminants for eEF1A: one is the ac cep tor stem base pair -A1:U72, sec ond, the Ò-stem base pairs -A50:U64 and U51:A63 [17].On the other hand, Met-tRNA i Met of plants and yeasts has A64-phosphoribosilation as an other antideterminant [18].Prob a bly, the in ter ac tion be tween eEF1A and Met-tRNA i Met does not take place be cause of the ab sence of con tact be tween pro tein and aminoacid res i due (which is ori ented in elongator Met-tRNA e Met at an other an gle rel a tively to the ac cep tor stem).We sup pose, that the pair U51:A63 in T-stem of tRNA i Met is not strong enough as an antideterminant for the elon ga tion fac tor, that may be a pos si ble ex pla na tion for the ex is tence of non-ca non i cal eEF1A*GDP*tRNA i Met com plex.We be lieve that for ma tion of the eEF1A1/eEF1A2*GDP*tRNA i Met com plex could be im por tant for the eEF1A me di ated chan nel ing of ini ti ator tRNA af ter its re lease from the E-site of ri bo some  to methionyl-tRNA synthetase.Be sides, the abil ity of eIF2, g-sub unit of which is ho mol o gous to eEF1A, to in ter act with deacylated tRNA i Met and tRNA e Met but not with other elongator tRNAs [19] may be con sid ered as an al ter na tive pos si bil ity for the ini ti a tor tRNA channel ing.The com par i son of K d val ues for the eIF2 complexes is shown in Ta ble 3 to gether with the cor respond ing val ues for eEF1A1 and eEF1A2.As can be seen, K d val ues for the eEF1A non-ca non i cal ter nary com plexes are much higher than K d val ues for re spective eIF2*GDP com plexes, but we be lieve that this does not pre vent eEF1A from in ter ac tion with all deacylated tRNAs in or der to de liver them to aaRSs.Thus, all the re sults ob tained are in fa vour of the tRNAs chan nel ing during translation at the elongation and initiation steps.
De ter mi na tion of the G:U base pair sig nif i cance in 51:63 and 50:64 po si tions of tRNA for its in ter ac tion with eEF1A.The sites re spon si ble for in ter ac tion with tRNA have been found re cently in prokaryotic EF1A [13].It was also found that G:C base pair is more of ten pres ent at 51:63 po si tion of tRNA than A:U base pair.Among mam mals only tRNA (QUU) Asn , tRNA (UGA) Ser and tRNA i Met have A:U base pairs at 51:63 po si tion, and tRNA Phe and tRNA Tyr have G:U.
We stud ied the im por tance of these po si tions in tRNA struc ture for the in ter ac tion with eEF1A.The tRNA 3 Lys in vi tro mu ta gen e sis was de scribed in Methods.Mu tant and con trol tRNAs had com pa ra ble ac tivity in aminoacylation test (data not shown).We stud ied the af fin ity of mu tant tRNAs for both isoforms of eEF1A by band re tar da tion as say (Fig. 5).The re sults The values obtained in the previous studies are marked with asterisk (*) [3,5].For the eEF1A2 com plex the pres ence of G50:U64 base pair in creased K d from 30 nM to 170 nM, and G51:U63 to 100 nM, i.e. 5,5 and 3 fold respectively.Thus, we found that po si tions 51:63 and 50:64 in the elongator tRNA 3 Lys are im por tant for tRNA in ter action with eEF1A.We de tected even higher im pact of these mu ta tions on in ter ac tion with eEF1A2, when the af fin ity of tRNA de creased about 5 fold even if A50:U64 changed to more fa vour able G50:U64 base pair, and 3 fold if G51:C63 changed to G51:U63.Data are shown in Table 4.
The dif fer ences found for the K d val ues of ter nary com plexes for var i ous mu tant and wild type tRNAs as well as dif fer ent af fin ity of one tested tRNA for two isoforms of eEF1A dem on strate that both pro tein and tRNA con trib ute to the interaction.Thus, we found that for ma tion of non-ca non i cal ter nary com plexes of tRNA with eEF1A*GDP is univer sal, and seems to be an in trin sic fea ture of all elongator tRNAs.These com plexes are in her ent for both isoforms of eEF1A.We also ob served the non-canon i cal ter nary com plexes of ini ti a tor tRNA with both isoforms of eEF1A.Sur pris ingly, eEF1A2 forms such com plexes with higher af fin ity than eEF1A1 to al most all ex am ined deacylated tRNAs.We also stud ied the struc ture el e ments of tRNA re spon si ble for eEF1A bind ing and found that re place ment of G64:C50 base pairs for G64:U50 and Ñ63:G50 for U63:G50 in T-stem re sults in valu able de crease in tRNA 3 Lys af fin ity to both isoforms of eEF1A.This proves an es sen tial role of the men tioned po si tions in the for ma tion of non-canonical complex tRNA*eEF1A*GDP .We thank Novosylna O.V. for the help in pu ri fi cation of elon ga tion fac tors. This

Fig. 1 .
Fig. 1.Interaction of tRNA Asp with eEF1A1*GDP and eEF1A2*GDP visualized by band-shift assay in native PAGE.a-autoradiography of free and complexed tRNA Asp , obtained at decreasing eEF1A1 concentrations ranging from 2 mM to 0.1 mM; b -densitometric analysis of the autoradiography with GelProAnalyser software.The curve reflects decreasing in free [ 32 P]tRNA Asp intensity as function of the eEF1A1*GDP concentration.The intensity of free [ 32 P]tRNA Asp considered as 100%.c -autoradiography of free and complexed tRNA Asp , obtained at decreasing eEF1A2 concentrations ranging from 2 mM to 0.1 mM; d -the same as c but for eEF1A2.

Fig. 2 .
Fig. 2. Interaction of tRNA Ile with eEF1A1*GDP and eEF1A2*GDP visualized by band-shift assay in native PAGE.a -Autoradiography of free and complexed tRNAI Ile , obtained at decreasing eEF1A1(left part) or eEF1A2 (right part) concentrations ranging from 2 mM to 0.1mM (with smaller increment than in Fig.1).b -Densitometric analysis of the autoradiography with GelProAnalyser software.The curve reflects decreasing in free [ 32 P]tRNA Ile intensity as function of the eEF1A*GDP concentration.The intensity of free [ 32 P]tRNA Ile considered as 100%.
Fig. 4. Interaction of tRNAi Met with GDP-bound forms of eEF1A and eEF1A2.Autoradiography of native gels.

Table 3 .
Disosiation constant for the complexes of translation initiation and elongation factors with initiator or elongator tRNA Met