The uridine in “U-turn”: Contributions to tRNA-ribosomal binding

1999 5: 503-511RNA S S Ashraf, G Ansari, R Guenther, et al. The uridine in "U-turn": contributions to tRNA-ribosomal binding.

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The uridine in “U-turn”: Contributions totRNA-ribosomal binding

S. SALMAN ASHRAF, 1 GHAZALA ANSARI, 1 RICHARD GUENTHER,1

ELZBIETA SOCHACKA, 2 ANDRZEJ MALKIEWICZ, 2 and PAUL F. AGRIS 1

1Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695, USA2Institute of Organic Chemistry, Technical University, 90-924 Lodz, Poland

ABSTRACT

“U-turns” represent an important class of structural motifs in the RNA world, wherein a uridine is involved in an abruptchange in the direction of the polynucleotide backbone. In the crystal structure of yeast tRNA Phe, the invariant uridineat position 33 (U 33), adjacent to the anticodon, stabilizes the exemplar U-turn with three non-Watson–Crick inter-actions: hydrogen bonding of the 2 9-OH to N 7 of A 35 and the N 3-H to A 36-phosphate, and stacking between C 32 andA35-phosphate. The functional importance of each noncanonical interaction was determined by assaying the ribo-somal binding affinities of tRNA Phe anticodon stem and loop domains (ASLs) with substitutions at U 33. An unsubsti-tuted ASL bound 30S ribosomal subunits with an affinity ( Kd = 140 6 50 nM) comparable to that of native yeast tRNA Phe

(Kd = 100 6 20 nM). However, the binding affinities of ASLs with dU-33 (no 29-OH) and C-33 (no N 3-H) were significantlyreduced (2,930 6 140 nM and 2,190 6 300 nM, respectively). Surprisingly, the ASL with N 3-methyluridine-33 (no N 3-H)bound ribosomes with a high affinity ( Kd = 220 6 20 nM). In contrast, ASLs constructed with position 33 uridineanalogs in nonstacking, nonnative, and constrained conformations, dihydrouridine (C2 9-endo), 6-methyluridine ( syn )and 29O-methyluridine (C3 9-endo) had almost undetectable binding. The inability of ASLs with 6-methyluridine-33 and29O-methyluridine-33 to bind ribosomes was not attributable to any thermal instability of the RNAs. These resultsdemonstrate that proton donations by the N 3-H and 29OH groups of U 33 are not absolutely required for ribosomalbinding. Rather, the results suggest that the overall uridine conformation, including a dynamic (C3 9-endo . C29-endo)sugar pucker, anti conformation, and ability of uracil to stack between C 32 and A 35-phosphate, are the contributingfactors to a functional U-turn.

Keywords: anticodon conformation; anticodon stem/loop analogs; ASL; nucleoside modifications; ribosome;translation; tRNA Phe U-turn

INTRODUCTION

Transfer RNAs have the general and well-recognizedshape defined by the first X-ray-derived crystal struc-ture of an entire RNA, that of yeast tRNAPhe (Kim et al+,1973)+Certain nucleotides within the sequence of yeasttRNAPhe and other tRNAs are invariant and associatedwith structural motifs that are common to other RNAsas well+ Uridine at position 33 is one of the most con-served residues in tRNAs; 97% of the 2,716 tRNA genessequenced have a uridine at position 33+All eucaryotictRNAs have the invariant uridine at position 33, U33,except for initiator tRNAs from higher eucaryotes and

some tRNAs from the genus Candida (Sprinzl et al+,1998)+ Based on the crystal structures of yeast tRNAPhe

and tRNAAsp, U33 in the anticodon domain and pseudo-uridine, c, at position 55 in the TcC loop are intrinsi-cally involved in sharp turns of the phosphodiesterbackbone (Quigley & Rich, 1976)+ These turns, desig-nated uridine- or U-turns, were initially observed onlyin tRNAPhe and tRNAAsp, but examples of U-turn motifshave been recently reported in numerous other RNAmolecules (Pley et al+, 1994; Doudna, 1995; Huanget al+, 1996; Stallings & Moore, 1997)+ Thus, the U-turnmay be an important and abundant structural motif inthe RNA world (Jucker & Pardi, 1995)+

In yeast tRNAPhe, the exemplar anticodon U-turn isresponsible for an abrupt change in the direction (;1808)of the RNA’s phosphodiester backbone just prior to thethree anticodon nucleotides+ The stability of the turnhas been attributed to three noncanonical interactions

Reprint requests to: Paul F+ Agris, 128 Polk Hall, Department ofBiochemistry, NCSU Box 7622, North Carolina State University,Raleigh, North Carolina 27695-7622, USA; e-mail: agris@bchserver+bch+ncsu+edu+

RNA (1999), 5:503–511+ Cambridge University Press+ Printed in the USA+Copyright © 1999 RNA Society+

503




involving U33+ Two of the interactions are hydrogenbonds from the 29-OH of U33 to the N7 of A35 and fromthe N3-H of U33 to the phosphate of A36 (Fig+ 1)+ Thethird interaction is the continuation of base stackingthat begins on the 59 side of the anticodon stem andterminates in the loop with U33 stacked on the phos-phate of A35 (Fig+ 1; Quigley & Rich, 1976)+ Althoughconsiderable effort has been invested in elucidatingthe role of the ubiquitous U33 in the anticodon U-turn(Uhlenbeck et al+, 1982; Bare et al+, 1983;Wittenberg &Uhlenbeck, 1985; Dix et al+, 1986; Schnitzer & von Ah-sen, 1997; von Ahsen et al+, 1997), examination of eachof the three proposed stabilizing factors has not yieldeda clear understanding of the intramolecular interactionsrequired for a functional U-turn+ Recently, von Ahsenet al+ (1997) reported that the 29-OH of U33 was essen-tial for the interaction of the Escherichia coli tRNAPhe

anticodon stem and loop (ASL) with the ribosome+How-ever, these results contradicted a previous study ofyeast tRNAPhe in which only a marginal decrease inribosomal binding was observed when U33 was changedto 29-O-methyluridine (Uhlenbeck et al+, 1982)+ To de-termine the properties of U33 that are important to theanticodon U-turn structure/function relationship duringmRNA decoding, we have designed and synthesized aset of ASLs with specific nucleotide substitutions at U33

and have analyzed their relative ribosomal bindingaffinities+

RESULTS

Design of “U 33-variant” ASLs andexperimental strategy

Six ASLs were synthesized with various substitutionsat the position of U33 (Fig+ 2)+ Each substitution wasdesigned to test the functional importance of one ormore of the interactions contributed by U33 to the U-turnstructure (Table 1)+ Two of the ASLs were designedsuch that nucleoside-33 lacked the ability to donate aproton from the 29-OH: 29-O-methyluridine (ASL-Um33)and 29-deoxyuridine (ASL-dU33)+Others were designedto negate proton donation from the N3-H position: cyt-idine (ASL-C33), N3-methyluridine (ASL-m3U33), and6-methyluridine (ASL-m6U33)+ The latter precludes pro-ton donation from N3 to the phosphate of A36 becausethe N-glycosidic bond of m6U33 takes the syn confor-mation both in the mononucleoside (Felczak et al+, 1996)and within the ASL+ The syn, C39-endo conformation ofm6U33 in the ASL was determined by NMR spectros-copy (R+ Cain and P+F+ Agris, pers+ comm+)+ Two ASLswere designed to force the dynamic U33 sugar pucker(;50% C39 endo in solution) to either the C39-endoconformation by methylation (ASL-Um33; Kawai et al+,1992) or the C29-endo conformation by elimination (ASL-dU33; Basti et al+, 1996)+ An additional ASL containingdihydrouridine (ASL-D33) was designed to have theC29-endo sugar pucker, but unlike ASL-dU33, would re-tain the ability to donate a proton from the 29-OH+ Di-hydrouridine is known to be highly constrained to theC29-endo conformation as a mononucleoside (Sundaral-ingam et al+, 1971) and within tRNAs and oligomers(Dalluge et al+, 1996, 1997; Stuart et al+, 1996)+ Partic-ular nucleoside substitutions, such as m6U33 and D33,were also designed to affect the stacking interactionsthat characterize position 33+ The syn conformation ofm6U33 and the nonplanar, nonaromatic character of D33

would negatively affect stacking interactions+ Thus, nu-cleosides substituted for U33 were chosen for their abil-ities to disrupt local noncanonical H-bonds in the loopor affect nucleoside conformation and stacking inter-actions+ These substitutions could, however, have af-fected structure beyond that of the anticodon loopand would, in turn, affect the assessment of ribosomebinding+

Thermal denaturation of synthetic ASLs

To confirm that the substitutions of the various nucle-osides for U33 in the ASLs had little or no effect onoverall RNA structure, we analyzed the thermal stabil-ity of each of the seven ASLs+ Thermal denaturations ofthe ASLs monitored by UV spectroscopy (Fig+ 3) re-vealed that none of the substitutions had any signifi-cant effect on the melting temperature (Tm) of themolecules (Table 2), with one exception+ Incorporation

FIGURE 1. The three-dimensional X-ray crystallographic structureof the anticodon loop of yeast tRNAPhe showing the anticodon U-turn(Quigley & Rich, 1976)+ The two noncanonical hydrogen bondsinvolving the invariant uridine at position 33 (U33) are highlightedand numbered+ j1 : H-bonding from 29-OH (U33) to N7 of A35;j2 : H-bonding from N3-H (U33) to A36-phosphate+ The stacking in-teractions of Cm32, U33, and phosphate-35 are within the shadedarea of the figure that is labeled as j3 + Bases of the anticodon triplet(Gm34, A35, and A36) and the hypermodified tricyclic nucleoside Y37are also labeled+ Cm: 29O-methylcytidine,Gm: 29O-methylguanosine,Y: wyebutosine+ Oxygen atoms colored dark blue are the pro-Rp-phosphate oxygen atoms of A35 and A36 that are important for effi-cient ribosomal binding (Schnitzer & von Ahsen, 1997)+

504 S.S. Ashraf et al.




of the nonplanar D33 appeared to have destabilized theASL stem, as well as the loop (Fig+ 3)+ Dihydrouridinedramatically affects local conformation within RNA bytransference of the C29-endo conformation to 39-adjacent nucleosides (Dalluge et al+, 1996, 1997; Stuart

et al+, 1996)+ Other substitutions of U33 did not alter theTm of the ASLs, which varied little (62–64 6 0+6 8C)+However, some differences in stability were detected attemperatures (20–40 8C) that preceded the major tran-sition, in particular for ASL-m6U33 (Fig+ 3)+ Analysis of

FIGURE 2. Nucleotide sequence and secondary structure of yeast tRNAPhe anticodon stem-loop domain (ASL) and themodified uridines used in this study+ The uridine at position 33 (U33, boxed) of the ASL was substituted with C, Um, dU,m3U,m6U, or D to produce various position 33-substituted ASLs+

TABLE 1+ Anticodon stem-loop domains (ASLs) used in this study and their expected structural properties+

ASLNucleoside

at position 33Availabilityof 29-OH

Availabilityof N3-H

Availabilityof basestacking

Expectedsugar pucker

Expectedbase pucker

Baseconformation

ASL-U33 Uridine 1 1 1 C39 . C29 endoa planar antiASL-m3U33 3-methyl-uridine 1 2 1 C39 . C29 endoa planar antiASL-C33 Cytidine 1 2 1 C39 . C29 endoa planar antiASL-dU33 Deoxyuridine 2 1 1 C29 endob planar antiASL-Um33 29O-methyl-uridine 2 1 1 C39 endoc planar antiASL-m6U33 6-methyl-uridine 1 2 2 C39 . C29 endod planar synASL-D33 Dihydro-uridine 1 1 2 C29 endoe nonplanar, anti

puckered

aThe sugar pucker of three of the nucleosides (U, C, and m3U) at position 33 are expected to be dynamic, but predom-inantly 39 endo (C39 . C29 endo)+ Sugar puckers of bdeoxyuridine (dU) in the anticodon loop (Basti et al+, 1996) andeDihydrouridine (D) (Sundaralingam et al+, 1971) have the C29 endo conformation+ cMethylation of the 29-OH of uridines isknown to strongly constrain sugar pucker to the C39 endo conformation (Kawai et al+, 1992)+ dSugar pucker of m6U inASL-m6U33 was determined to be .50% C39 endo (data not shown)+

Uridine conformation in functional U-turn 505




the pretransition as well as the major transition over atenfold range of RNA concentrations indicated the Tm

for pretransitions and that for all the major transitionswere temperature independent+ Thus, the pretransi-tions were not due to hairpin–duplex equilibrium, andwithin the concentration range used, a unimoleculardenaturation was being observed+ We believe that thepretransition region of the thermal denaturation profileis indicative of the denaturation of loop interactions inthe ASL+ Thus, except for D33, incorporation of modifieduridines only caused changes in the structure and/ordynamics of the ASL loop+ Attempts to detect anythermodynamic differences in the major transition (stemdenaturation) by curve-fitting the profile to a two-state

model did not yield any significant differences amongsix of the seven ASLs (Table 2)+

Binding of ASLs to 30S ribosomal subunits

The ability of the variously U33-substituted ASLs to bindto the poly(U)-programmed 30S ribosomal subunit wasassessed and compared to that of the unsubstitutedASL-U33 (Fig+ 4)+ Dissociation constants (Kd) for theASLs binding to 30S ribosomal subunits were calcu-lated from binding isotherms using nonlinear regres-sion (Table 3)+ The Kd for the yeast tRNAPhe ASL-U33

(140 6 50 nM) was in good agreement with the previ-ously reported Kd for the E. coli tRNAPhe ASL binding to

FIGURE 3. UV-monitored thermal denaturations of the various position 33-substituted ASLs in comparison to ASL-U33+A: ASLs with U33 substitutions (m3U,m6U, and C) that disrupt N3-H bonding in comparison to that of unsubstituted ASL-U33+B: ASLs with U33 substitutions (dU and Um) that prevent H-bonding from 29-OH, as well as the ASL containing a nonplanarbase (ASL-D33), in comparison to that of the unsubstituted ASL-U33+ The perpendicular gray shading indicates the averageTm of the ASLs (62+6 6 0+6 8C)+ UV-monitored denaturation profiles of the ASLs were obtained as described in Materials andMethods+





30S subunits (130 6 40 nM; von Ahsen et al+, 1997)+Binding of the unsubstituted ASL-U33 to the ribosomewas also comparable to that of the native yeast tRNAPhe

(Kd 5 100 6 20 nM)+ In comparison to the unsubsti-tuted ASL-U33, ASLs lacking the ability to donate pro-tons for H-bonding from either the N3-H (ASL-C33) orfrom the 29-OH (ASL-dU33 and ASL-Um33) had sub-stantially reduced Kds, 2,190 6 300 nM, 2,930 6 140nM, and 13,700 6 4,600 nM, respectively+ ASL-m6U33,in which the syn conformation negated proton donationfrom the N3 to the A35-phosphate, also bound verypoorly to 30S subunits (Fig+ 4)+ In fact, we could notcalculate an accurate Kd (.300 3 1027 M) for the bind-ing of the ASL-m6U33+ Surprisingly, ASL-m3U33, whichlacked the N3 proton for H-bonding, bound to 30S ri-bosomal subunits with a Kd (220 6 20 nM) that wascomparable to that of ASL-U33 (Table 3)+ Ribosomalbinding could not be detected for the ASL-D33, in whichD33 had protons available for H-bonding at both the29-OH and N3, but had a nonplanar base and a highlyconstrained C29-endo sugar pucker+

DISCUSSION

Stability of the anticodon U-turn within the X-ray crys-tallographic structure of yeast tRNAPhe is dependent onU33 contributing two noncanonical hydrogen bonds andstacking interactions (Quigley & Rich, 1976; Fig+ 1)+ Totest the functional importance of these H-bonds andstacking interactions, six ASLs were synthesized withsubstitutions at U33 designed to disrupt the individualcontributions of the nucleoside to the U-turn+ This ap-proach of site selective incorporation of natural andnonnatural modifications into synthetic ASLs has provenvery useful for addressing specific structure/function-related questions (Uhlenbeck et al+, 1982; Bare et al+,1983; Wittenberg & Uhlenbeck, 1985; Dix et al+, 1986;Chen et al+, 1993; Dao et al+, 1994; von Ahsen et al+,1997)+ In fact, we have recently shown that a specific,single-atom substitution (U34 r s2U34) in the anticodonstem-loop of tRNALys is sufficient to restore ribosomalbinding of an otherwise biologically inactive ASL (Ashrafet al+, 1999)+

In this report, the degree to which each of the sixASLs bound the ribosome in comparison to that of theunsubstituted ASL-U33 was used as a measure of thefunctional importance of U33 H-bonding and stackinginteractions+ However, in this approach we and others(Uhlenbeck et al+, 1982; Bare et al+, 1983;Wittenberg &Uhlenbeck, 1985; Dix et al+, 1986; Schnitzer & von Ah-sen, 1997; von Ahsen et al+, 1997) have assumed thatthe various substitutions at U33 negate specific uridine

TABLE 2+ Thermal denaturation analysis of variously substituted and ASL-U33+

ASL Tm (8C)a DHVH (Kcal/mol)a DSVH (Cal/molpK)a DGVH (Kcal/mol)a

ASL-U33 63+5 6 0+7 256+0 6 1+9 2166+4 6 5+9 24+40 6 0+1ASL-m3U33 62+6 6 0+5 257+3 6 2+2 2170+7 6 6+5 24+4 6 0+2ASL-C33 62+7 6 0+4 256+0 6 0+5 2166+8 6 1+6 24+3 6 0+04ASL-dU33 63+1 6 0+4 258+2 6 2+6 2172+9 6 7+4 24+6 6 0+3ASL-Um33 61+9 6 0+6 262+6 6 0+4 2187+0 6 1+1 24+7 6 0+1ASL-m6U33 61+6 6 0+6 259+3 6 2+2 2177+2 6 6+8 24+4 6 0+2ASL-D33

b no data no data no data no data

aMelting point (Tm) and other thermodynamic parameters (DH, DS, and DG) were extractedfrom the major thermal transition (;50–65 8C) (Fig+ 3)+

bDenaturation of ASL-D33 did not yield data from which Tm or other parameters could beextracted (Fig+ 3)+

FIGURE 4. Binding of variously substituted ASLs to programmed30S ribosomal subunits in comparison to ASL-U33+ Ribosomal bind-ing assays were conducted as described in Materials and Methodsby using 32P-labeled ASLs and poly(U)-programmed 30S ribosomalsubunits+ The fraction of the ASL bound to ribosomes was normal-ized to the fraction of ASL-U33 bound to 30S subunits according topreviously published methods (von Ahsen et al+, 1997)+

TABLE 3+ Binding affinities (Kd) of variously substituted ASLs andASL-U33 to poly(U)-programmed 30S ribosomal subunits+

ASL Kd (3 1027 M)

ASL-U33 1+4 (6 0+5)ASL-m3U33 2+2 (6 0+2)ASL-C33 21+9 (6 3+0)ASL-dU33 29+3 (6 13+6)ASL-Um33 137+0 (6 46+0)ASL-m6U33 .300ASL-D33 Could not be detected





contributions to the U-turn+ In fact, it is possible thatsome or all of the substitutions also sterically distort theanticodon loop conformation, which would lead to re-duced ribosomal binding+Yet,Uhlenbeck and colleagues(1982) have reported only marginal effects on ribo-somal binding by fully modified ASLs with the samesubstitutions at position 33 as ours+ Thus, these sub-stitutions probably do not grossly disturb the modifiedanticodon loop structure+ Even in the absence of nat-ural modifications, thermal denaturations of the six ASLswith U33 substitutions strongly indicated that all the ASLs,with the exception of ASL-D33, were as stable as theASL containing U33 (Table 2; Fig+ 2)+ In addition, incomparing the degree to which the ASLs bound theribosome, we and others also assumed that the ASLsare all binding at the same site on the 30S subunits+ Toconfirm this, we assessed the abilities of the ASLs toprotect two 16S rRNA P-site nucleotides, G926 andG1338, from chemical modification (Fig+ 5; Moazed &Noller, 1990)+ ASLs with m3U33, C33, and dU33 pro-tected nucleosides G926 and G1338 from kethoxal re-activity, as did ASL-U33 and the complete yeast tRNAPhe

molecule+ However, the ASLs that exhibited little (ASL-Um33) or no (ASL-m6U33 and ASL-D33) binding to theribosome demonstrated little or no protection of thetwo 16S P-site nucleotides (Fig+ 5)+ Hence, ASLs thatbound the ribosome in our filter binding assays were in

fact binding at the same tRNA P-site in 30S ribosomalsubunits+

One of the three interactions of U33 that stabilizes theanticodon U-turn in the yeast tRNAPhe structure is thehydrogen bonding of its 29-OH to the N7 of A35+ Thisinteraction could be important to tRNAs that have apurine at anticodon position 35, that is, roughly half ofall tRNAs+ When hydrogen bonding of the 29-OH atposition 33 was disrupted by substituting dU33 and Um33

for U33, the ASLs’ abilities to bind the ribosome werereduced to 5% and 1% of that of the unmodified ASL,respectively+ These results are in agreement with thatof von Ahsen et al+ (1997), and we concur with theirconclusion that the 29-OH of U33 is important for ribo-somal binding+ Yet ASL-C33, ASL-m6U33, and ASL-D33,all of which had the 29-OH groups available, also showedsignificantly reduced or hardly detectable ribosomalbinding+ Perhaps the important contribution of the 29-OHof U33 to the functional U-turn is some property otherthan the ability to hydrogen bond+ Deoxynucleosides(as in ASL-dU33) are highly constrained to the C29-endo conformation (Basti et al+, 1996) and 29-O-methylnucleosides (as in ASL-Um33) are highly constrained tothe C39-endo conformation (Kawai et al+, 1992)+ Un-altered ribose, particularly those of uridines, have adynamic sugar pucker that favors the C39-endo con-formation (Smith et al+, 1992)+ Therefore, we propose

FIGURE 5. Protection of 16S P-site nucleotides (G1228 and G926) from kethoxal reactivity by variously substituted ASLsand ASL-U33+ Poly(U)-programmed 30S ribosomal subunits (10 pmol) were incubated with buffer alone (lane 1) or 50 pmolof one of the various ASLs (lanes 2–8)+ Lane 2: ASL-U33; lane 3: ASL-m3U33; lane 4: ASL-C33; lane 5: ASL-dU33; lane 6:ASL-Um33; lane 7: ASL-m6U33; and lane 8: ASL-D33+





that the conformational dynamics of the U33 sugarpucker provided by the 29-OH is functionally more im-portant than its ability to hydrogen bond to the N7 ofposition 35 purines+ The need for a conformationallydynamic sugar pucker of U33 would be important to alltRNAs and not just those with a position 35 purine andsuggests that nucleoside conformation and/or dynam-ics is critical for a functional U-turn+

The hydrogen bond from U33 N3-H to the phosphateof nucleoside-36 also stabilizes the yeast tRNAPhe anti-codon U-turn+ This H-bond could be important to theribosome binding of all tRNAs and important for thestructure of all anticodon U-turns+ ASLs containing C33

and m6U33, both of which preclude N3-H bonding tophosphate-36, bound the ribosome with affinities thatwere 5% and ,0+5% of that of the ASL-U33, respec-tively+ These results are in accord with a recent studyin which substitution of pro-Rp-phosphate-36 withRp-thiophosphate (which disrupts N3-H bonding tophosphate-36) reduced ribosomal binding to about 6%of that of controls (Schnitzer & von Ahsen, 1997)+ Thus,the interaction N3-H of U33 with phosphate-36 is impor-tant for a functional U-turn+

However, our experiments yielded one result thatseemingly contradicted this conclusion+ ASL-m3U33

lacked the N3-H but was bound by the ribosome al-most as well as ASL-U33 (Fig+ 4; Table 3)+ Methylatedpyrimidines stack better than their unmodified counter-parts (Sowers et al+, 1987; Wang & Kool, 1995)+ N3-methyluridine, though unable to form a hydrogen bondfrom N3, would engage in stacking interactions to ahigher degree than the unmodified uridine+ The thirdcontribution of U33 to the stability of the U-turn in thecrystal structure of yeast tRNAPhe is its stacking inter-actions with C32 and the phosphate of nucleoside-35(Fig+ 1)+ In the previously cited study of individual thio-phosphate substitutions (Schnitzer & von Ahsen, 1997),Rp-thiophosphate substitution of pro-Rp-phosphate-35resulted in a reduction of ribosomal binding even moresignificant than substitution of phosphate-36+ Althoughunexplained at the time, we believe that the decreasein ribosomal binding was probably due to the fact thatsubstitution of the phosphate-35 oxygen with the lesselectronegative Rp-thiophosphate sulfur reduced theeffectiveness of the ion-induced dipole interaction in-volving uracil-33 on phosphate-35 (Quigley & Rich,1976)+ ASLs with Um33 and dU33 had the ability to hy-drogen bond through N3-H, yet they bound the ribo-some poorly+ Their strongly constrained C39-endo andC29-endo sugar puckers, respectively, could havechanged the orientation of the N-glycosidic bond,thereby disrupting the stacking interactions betweenC32, U33, and phosphate-35+ In contrast, the enhancedstacking of m3U33 between C32 and phosphate-35 com-pensated for the lack of H-bonding from N3 to thephosphate-36, thus stabilizing the ASL-m3U33 and main-taining the conformation of the U-turn+ The lack of ri-

bosomal binding by both ASL-m6U33 and ASL-D33

further demonstrated the importance of stacking onphosphate-35+ Although both ASLs had the 29-OHgroups, the syn conformation of the m6U33 and thepuckered rather than the planar character of D33 pre-vented the bases from stacking between C32 andphosphate-35, as well as forming the N3-H bond tophosphate-36+ Thus, stacking of U33 between C32

and phosphate-35 may be a very important contribu-tion of U33 to the functional U-turn+

Earlier studies by Uhlenbeck and coworkers usingyeast tRNAPhe anticodon domains containing naturallyoccurring nucleoside modifications revealed that theinvariant uridine at position 33 is not important for ri-bosomal binding (Uhlenbeck et al+, 1982; Bare et al+,1983; Dix et al+, 1986)+ Substitution of the uridine atposition 33 to any nucleoside, including D, Um, or dU,was tolerated with only marginal effects on ribosomalbinding+ For example, substitution of U33 to Um33 in theASL containing the naturally modified nucleosides re-duced the Kd for poly(U)-programmed 30S ribosomalsubunits to 30% of controls (Uhlenbeck et al+, 1982)+The disparity between results reported here and byvon Ahsen et al+ (1997) and that reported by Uhlen-beck et al+ is most likely due to the presence of thenatural modifications in the ASL/tRNA used by Uhlen-beck et al+ We believe that in ASLs (or tRNAs) con-taining naturally occurring nucleoside modifications,substitutions at position 33 are tolerated because thenucleoside modifications help maintain the overall con-formation of the anticodon U-turn+ In fact, the ASL usedby Uhlenbeck et al+ had pseudouridine at position 39,wyebutosine at position 37, and 29-O-methylguanosineat position 34, all of which contribute to the conforma-tional rigidity of the anticodon loop (Davanloo et al+,1979; Davis & Poulter, 1991; Kawai et al+, 1991; Ko-walak et al+, 1994)+ Further support for the role of mod-ified nucleosides in conformational restraint of yeasttRNAPhe anticodon stem and loop comes from NMRstudies of native yeast tRNAPhe as well as unmodifiedASLs+ Under conditions in which spectra of tRNAPhe

(with naturally modified nucleosides) exhibited the U-turnsignature phosphate (Gorenstein & Goldfield, 1982),31P-NMR analysis of the ASL failed to show the ex-pected phosphorus signal (data not shown)+ The failureto observe the U-turn phosphate in the unmodified ASLis probably indicative of a dynamic unmodified anti-codon loop with an equilibrium between various con-formations+ In fact, we have previously reported NMRdata demonstrating that the anticodon loop of the un-modified ASL is dynamic (Chen et al+, 1993), whereasthe methyl groups in the anticodon loop of the fullymodified native tRNAPhe are considerably more re-stricted than methyl groups in other regions of the mol-ecule (Schmidt et al+, 1987)+ Our results and that of vonAhsen et al+ (1997) demonstrate that, unlike ASLs con-taining modified nucleosides, the functional conforma-





tion of the anticodon U-turn of unmodified ASLs isextremely sensitive to changes in U33 conformation+ Infact, we have demonstrated that two site-selectivelyplaced, simple methylations at positions 37 and 40 aresufficient to transform a nonbinding DNA analog oftRNAPhe ASL with dU at position 33 into a functionalribosome binder (Dao et al+, 1994)+

The data reported here confirm and elucidate resultsfrom previous studies demonstrating a requirement forthe 29-OH group (von Ahsen et al+, 1997) as well as theN3-H and the importance of the pro-Rp-phosphate ox-ygen at position 35 (Schnitzer & von Ahsen, 1997)+ Inaddition to explaining these previous observations, weclearly show (with an array of ASLs variously substi-tuted at U33) that the U33 nucleoside conformation, im-portant for effective stacking of the uridine between C32

and phosphate-35, is a critical determinant for a func-tional U-turn in the anticodon+ Proper U33 nucleosideconformation/dynamics may not only be critical for afunctional anticodon U-turn, but also for U-turn motifsin RNA structures in general+

MATERIALS AND METHODS

Synthesis of ASLs and preparation of 30Sribosomal subunits

Unsubstituted ASLs and ASLs containing C, Um, dU, m3U,m6U, or D at position 33 were synthesized using standardsolid phase chemistry for RNA synthesis and then purified byion-exchange HPLC (Ogilvie et al+, 1988; Agris et al+, 1995)+Synthesis of the m6U phosphoramidite and structural analy-sis of the m6U containing ASL are described elsewhere (E+Sochacka, G+ Czerwinska, R+ Cain, R+ Guenther, G+ Ansari,C+ Yarian, P+F+ Agris & A+ Malkiewicz, in prep+)+ Denaturingpolyacrylamide gel electrophoresis and HPLC analyses con-firmed the purity of ASLs to be greater than 90%+Modificationof the ASLs was confirmed by determining nucleoside com-position (Gehrke et al+, 1982)+ Small ribosomal subunits (30S)were prepared and activated as previously described (Eric-son et al+, 1995)+

Thermal denaturation of the ASLs

Thermal denaturations of the tRNAPhe ASLs (in 10 mM so-dium phosphate buffer, pH 7+2, 100 mM NaCl, and 0+1 mMEDTA) were monitored with UV using a Cary 3E spectropho-tometer+ Melting point temperature and other thermodynamicparameters were extracted from the major thermal transitionoccurring between 50 and 65 8C+ The data was analyzedusing Origin software and assuming a two-state model ofdenaturation+Curve-fitting for determining DSVH, DHVH, DGVH,and Tm was restricted to the major transition because of thelow temperature transition (;20–40 8C) in some ASLs+ How-ever, a two-state model of denaturation was justified becauseneither transition exhibited changes in Tm with concentrationand were independent of each other, as judged by analysis offirst derivative plots of the denaturation profiles (data notshown)+

Binding of tRNAs and ASLs to 30Sribosomal subunits

To determine the binding affinities (Kd) of ribosomes for tRNAand various ASLs, 30S subunits (10 pmol) and poly(U)(10 mg) were incubated for 20 min at 37 8C with increasingamounts of 32P-labeled ASL (up to 50 pmol) in 40 mL of CMNbuffer (80 mM potassium-cacodylate, pH 7+2, 20 mM MgCl2,100 mM NH4Cl, and 3 mM b-mercaptoethanol)+ The reactionwas incubated for another 20 min on ice, passed throughnitrocellulose filters (0+45 mm) and the filter-bound RNAwashed twice with ice-cold CMN buffer (100 mL)+ The filterswere then air-dried and counted+ Disassociation constantsand their standard deviations were determined using nonlin-ear regression analysis on four replicates of each experi-ment+ To estimate the Kd values of ASLs substituted at U33, astoichiometry of 0+5–0+7 (as obtained for ASL-U33) was as-sumed (von Ahsen et al+, 1997)+

Chemical probing of 16S P-site nucleotidesprotected by ASLs

Chemical probing for those 16S P-site nucleotides that wereprotected by the ASLs was accomplished as described(Moazed & Noller, 1990)+Modifications with kethoxal (2 mL of20-fold diluted in 20% ethanol) were conducted at 20 8C for30 min in 40 mL reaction buffer (80 mM sodium cacodylate,pH 7+2, 100 mM NH4Cl, 20 mM MgCl2, 3 mM b-mercapto-ethanol)+ Primer extension of chemically modified rRNA wasperformed by annealing 1 pmol of 32P-labeled primer to0+2 mg of rRNA in 2 mL of annealing buffer (40 mM Pipes,400 mM NaCl, 1 mM EDTA, 80% formamide) for 10 min at45 8C, followed by 10 min incubation on ice+ Reverse tran-scription was started by adding 20 mL of the primer extensionmixture (50 mM Tris-Cl, pH 8+2, 50 mM KCl, 6 mM MgCl2,10 mM DTT, 0+5 mM each of dATP, dCTP, dGTP, and dTTP,and 1 U of AMV-RT)+ The reaction was allowed to proceed for10 min at 42 8C and was then stopped by adding 100 mL of10 mM Tris, pH 8+0, 1 mM EDTA to each tube+ After phenoland chloroform extractions, reverse transcribed cDNA wasethanol precipitated, dried, and resuspended in 10 mL of 2/3diluted formamide containing Bromophenol Blue and XyleneCyanol dyes+ The samples were heat denatured (2 min at100 8C) before being subjected to gel electrophoresis (3 mLeach sample) on an 8% sequencing gel+

ACKNOWLEDGMENTS

The authors thank Guihua Liu, Robert Cain, Amy Truscello,Christine Li, and Winnell Newman+ We also thank Dr+ PaulWollenzien for helpful discussions and providing 30S ribo-somal subunits+ NIH (GM23037) and NSF (MCB9631103)grants to P+F+A+ and Polish Committee for Scientific Researchgrant (PB0506/P3/93/05) to A+M+ supported this work+

Received October 22, 1998; returned for revisionDecember 8, 1998; revised manuscript receivedJanuary 12, 1999





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The uridine in “U-turn”: Contributions to tRNA-ribosomal binding

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