347

Biochimica et Biophysica Acta, 407 (1975) 347--356 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98425

PHYSICAL AND CODING PROPERTIES OF POLY(5-AMINOURIDYLIC ACID} AND OF 5-AMINOURIDINE-CONTAINING TRINUCLEOTIDES

W. HILLEN and H.G, GASSEN

Fachgebiet Biochemie der Technischen Hochschule Darmstadt, D 61 Darmstadt, Petersenstrasse 15 (G.F.R.) (Received April 29th, 1975)

Summary This report concerns the synthesis of poly(5-aminouridylic acid) and of 5-aminouridine-containing trinucleotides. Starting from 5-aminouridine the nucleoside 5'-phosphate was prepared enzymatically with carrot phosphotransferase whereas the nucleoside 5'-diphosphate was prepared chemically and polymerised with polynucleotide phosphorylase. The aminouridine-containing trinucleotides were prepared by known enzymatic procedures. Besides an increase of stability in the secondary structure poly(nh2SU) forms a triplestranded complex with poly(A) and stimulates the poly(Phe) synthesis like poly(U). In contrast to U-nh2SU-U, the triplet containing the 3'-terminal aminouridine does not stimulate the binding of Phe-tRNA to 70-S ribosomes. This behaviour is discussed with respect to the influence of a modification on the stacking geometry of a codon and the base pairing scheme between the 5'-nucleotide of the anticodon and the 3'-nucleotide of the codon.

Introduction In the first position of the anticodon of many tRNAs a modified uridine nucleoside is found. If uridine is replaced by 2-thiouridine or by an analogue of s2U further modified in the 5-position a restriction of codon-anticodon recognition occurs. Sekiya et al. [1] demonstrated that in a cell-free system for hemoglobin synthesis yeast tRNA3 Glu which contains a 2-thiouridine derivative in the first position of the anticodon recognizes only G-A-A but not G~A-G. On the contrary, modification in the 5-position of uridine seems to amplify the recognition pattern, since uridine 5-oxyacetic acid, occuring in the 5'-position of the Escherichia coli tRNA t set anticodon, pairs with uridine in addition to adenosine and guanosine [2]. We have shown that a replacement of uridine by s 2U to give the triplet U-U-s2U abolishes the template activity in the Phe-tRNA ribosome-binding assay [ 3].

348 If polyuridylic acid is modified in the 5-position, in most cases the thermal stability of the secondary structure is increased as well d o c u m e n t e d in the series poly(clSU) < poly(U) < poly(brSU) < poly(iSU) [4]. It is difficult, however, to draw a simple relationship between the size of the substituent and the increase in the Tm value, since poly(eSC) shows a lower Tm value compared to p o l y ( m s C) [ 5]. Besides an effect on the stability of the base stacking, 5-substitution should influence the geometry of the stacking and thus the secondary structure of the polynucleotide. Evidence for this was given by X-ray diffraction studies of modified nucleosides. It was shown that the exocyclic group is preferentially located above the aromatic ring system of the neighbouring base [6]. Up to now, however, it has not been possible to deduce a t h e o r y for the influence of 5-substitution on the geometry of base stacking. Most 5-substituted polyuridylic acids are active as templates for the binding of aminoacyl-tRNA to ribosomes [7]. If, however, a trinucleoside diphosphate is used as a template, modification in the middle position of the codon results in increased codon activity, whereas a modification of the terminal uridines causes a loss of activity as has been shown for the G-U-U Val-tRNA system by Griinberger et al. [8]. In the G-U-U tRNA val system the results are, however, difficult to interpret because of the existence of isoaccepting tRNAs. We therefore selected the U-U-U t R N A phe E. coli system and replaced uridine by 5-aminouridine. This nucleoside was selected since it had been shown with poly(A) versus poly(h 6A) that the exocyclic amino group has a profound influence on the stacking pattern in homopolynucleotides [9]. In the following we report on the template activities of poly-5-aminouridylic acid and of 5-aminouridine-containing trinucleoside diphosphates in comparison to their physical properties in an attempt to clarify the function of 5-substituted uridines in codon-anticodon recognition. Materials and Methods 5-Bromouridine and uridine-5'-diphosphate were commercial preparations from Zellstoffabrik Mannheim, Germany. Poly(U), poly(A), ribonuclease A (EC 2.7.7.16), deoxyribonuclease I (EC 3.1.4.5), ribonuclease T1 (EC 2.7.7.26), snake venom phosphodiesterase (EC 3.1.4.1) and alkaline phosphatase (EC 3.1.3.1) were obtained from Boehringer, Mannheim. Micrococcus luteus and E. coli MRE 600 cells were purchased from the Merck Company, Darmstadt, Germany. [ 14 C] ADP, [ a H] UDP, [ 3H] phenylalanine and [ 3H] valine were supplied by the Radiochemical Centre, Amersham, U.K. Polynucleotide phosphorylase (EC 2.7.7.8) from M. luteus was prepared by known procedures [10]. Isolation of 70-S ribosomes, preparation of 30-S and 50-S subunits and activation of the subunits was done according to the methods of Gros and Matthaei [11] and Zamir et al. [12]. tRNA from E. coli was charged with [ a H ] P h e (spec. act. 1 Ci/mmol) and [ 3H]Val (spec. act. 1 Ci/mmol) following known procedures [ 13]. Phosphotransferase was isolated from carrots according to Brunngraber and Chargaff [14]. (NH4)2SO4-precipitation was used instead of DEAE-cellu-

349

lose chromatography. 5-Aminouridine-5'-diphosphate (pp nh 2 s U) was prepared following the procedure of Liihrmann et al.;the overall yield was 17% starting from 5-bromouridine [15]. The reaction mixture for the polymerisation of the nucleoside diphosphates contained: 200/~l 160 m M pp n h 2 S U (NH~), 100/~I 1 M Tris • HCI p H 9.5, 10 ~l 1 M MgCl2, 10/~l 40 m M K-EDTA, 200/al polynucleotide phosphorylase (14 mg/ml, spec. act. 150 units/rag) and 80 #l H20. After incubation for 3 h at 37°C the mixture was heated to 80°C for 5 rain to denature the polynucleotide phosphorylase. The mixture was deproteinated by tripleextraction with phenol followed by five extractions with ether. The polynucleotide was then separated from its m o n o m e r by gel filtrationon a Sephadex G-50 column (1 X 50 cm). The material which appeared in the exclusion volume of the column was precipitated by adjustment of the p H to 2.5, redissolved in a minimal amount of 2 M N H 4 O H and purified again by passage over Sephadex G-50. Purity and chain length (>100 nucleotides)was determined with the aid of the nucleoside analyzer [16]. The yields varied between 15 and 30%. The trinucleoside diphosphates were prepared according to previously published procedures with the exception of U-nh 2s U which could not be prepared by the method of Gassen [17], since arninouridine is only poorly soluble. Therefore, in a polynucleotide phosphorylase-catalyzed reaction, G-Un h 2 S U was synthesized starting from G-U and pp nh2 s U, then the 5'-guanylic acid was split off with RNAase T I and the resulting dinucleoside phosphate U-nh 2s U was elongated with ppU. The purity of the compounds was examined by paper electrophoresis at p H 2.5 and 7.5 and by paper chromatography in two different solvents. Sequence determinations were done by enzymatic or chemical hydrolysis followed by identification of the products with the nucleoside analyzer. The determination of ultraviolet spectra and the p K values was performed spectrophotometrically using a Cary model 15 spectrophotometer. The p H values were determined inside the cell using a glass micro-electrode. The hyperchromicities after thermal denaturation or enzymatic digestion were calculated according to ( A D / A ° -- 1) X 100. The melting curves were recorded automatically in a Gilford 2000 apparatus at 294 n m for poly(nh2SU) and at 260 n m for complexes with poly(A). The C D spectra were determined with a Cary 61 spectrophotometer equipped with a thermostated cell holder. The temperature was measured inside the cell with a thermocouple. The binding of poly(nh2SU) to 70-S ribosomes was measured by exchange against prebound poly([ 3H] U). Prebinding was done in a mixture of 16 m M MgCI2, 50 m M KCI, 10 m M Tris. HCI (pH 7.5) containing 2.6 A260 m'U = 50 pmol reactivated 70-S ribosomes [12], and 5 nmol poly([3H]U) at 0°C for 5 min (total vol. 100 #l). Following addition of the unlabelled polynucleotide the mixture was incubated for an additional 10 rain at either 0°C or 37°C, diluted with 5 ml washing buffer (15 rnM MgCl2, 10 m M Tris • HCI p H 7.5) and washed over Selectron B A 85 filters,which were pretreated with 0.5 M N a O H [18]. The filterswere rinsed three times with washing buffer, dried and counted in 2 ml toluene/0.4% diphenyloxazole. The stimulation of the binding of labeled aminoacyl-tRNA to 70-S ribosome by the polynucleotides or trinucleoside diphosphates was measured by a

350 modification of the Nirenberg-Leder procedure [19]. Details are listed in the legends to the respective figures. Results

The synthesis of poly-5-aminouridylic acid required high concentrations of polynucleotide phosphorylase and the yields were rather low (20%). This may have been caused by the hydrophylic group in the 5-position of the base, whose negative influence on the yields was also shown in the synthesis of poly-5-hydroxycytidylic acid [20]. The synthesis of the trinucleoside diphosphates showed no differences to normal trinucleoside diphosphates in either yields or reaction rates. Fig. 1 displays the ultraviolet-spectra of the polynucleotide at pH 7 and 12; the spectrum at pH 2 could not be measured due to precipitation of the polymer. The pK values of the monomeric units and the respective polynucleotide are listed in Table I. The pK value for the deprotonation of the N3-hydrogen resembles the pK values for poly(U), whereas the pK value for the protonation of the 5-amino group is increased from the nucleoside to the polymer. Fig. 2 shows the melting profile of the polynucleotide and Fig. 3 the difference spectra which allow calculation of the hyperchromicities at different wavelengths. The thermal stability of the secondary structure of the polynucleotide is increased after addition of 10 mM or 20 mM Mg2÷. The measured thermal hyperchromicity of ~1% is rather high for a pyrimidine polynucleotide and the Tm value at 0.01 M MgC12 is raised to 36°C (see

II

poyln(2h5U) pH ?

220

260

300

Fig. 1. U l t r a v i o l e t s p e c t r a o f p o l y ( n h 2 5 U ) t h e p H v a l u e w a s m e a s u r e d i n s i d e t h e cell.

380

a t p H 1 2 . 0 a n d 7.0. T h e s p e c t r a w e r e r e c o r d e d w i t h a C a r y 1 5 ;

351 TABLE I pK-VALUES OF 5-AMINOURIDINE COMPOUNDS T h e p K v a l u e s w e r e d e t e r m i n e d s p e c t r o p h o r o m e t r i c a l l y . T h e p H v a l u e w a s m e a s u r e d i n s i d e t h e cell w i t h a glass m i c r o - e l e c t r o d e , p H a d j u s t m e n t s w e r e m a d e w i t h 1 0 M N a O H o r 1 0 M HC1 Substance

pK 1

PK2

n h 2 5U p n h 2 5U p p n h 2 5U p o l y ( n h 2 5U)

2.7 3.0 4,0 4.1

9.1 9.6 9.8 9.4

Az94-E. 0.z..

0,2-o--o--o- poly nh2SU) lOmMl~

~'oi~

36

s'o

8'0 T

[%]

Fig. 2. Melting profiles of p o l y ( n h 2 5 u ) in 0 . I M NaCI, 0 . 0 5 M s o d i u m c a c o d y l a t e p H 7 . 0 (o o); and after a d d i t i o n of 1 0 m M MgCI 2 (o o). Raising the Mg ~ c o n c e n t r a t i o n to 20 mM did n o t change t h e profile further.

1,Ol 30

/x""~'--~

o.t--.. /\,

2;0

--.o-o- poly (U} --x--x-poly { nh2 SU)

\

2~0 "°"

3~o

\"~,

[rim]

Fig. 3.. Difference spectra o f p o l y ( U ) (o o) and o f p o l y ( n h 2 $ U ) ( X X) m e a s u r e d in 10 m M Trls • HCI p H "/.5, 2 0 m M MgCl 2 and 50 m M KCI b e f o r e and after h y d r o l y s i s w i t h R N A a s e A at 20°C.

352 T A B L E II Tm VALUES FOR THE POLYNUCLEOTIDES T h e T m values for p o l y ( n h 2 SU) and p o l y ( n h 2 5U)-containing c o m p l e x e s w e r e measured in 0.1 M NaCI 0 . 0 5 s o d i u m c a c o d y l a t e at the Mg 2 ÷ c o n c e n t r a t i o n s indicated. Polynucleotide

Mg 2+ ( m M )

T m (°C)

Poly(nh2 5U) P o l y ( n h 2 5U) P o l y ( A ) • 2 p o l y ( n h 2 SU) P o l y ( A ) • 2 p o l y ( n h 2 SU)

0 10 0 10

14 36 53 63

Table II). The clearest indication for a stable secondary structure is displayed by the difference in total hyperchromicity at 7°C and 20°C, 44.5% and 10.5%, respectively. Fig. 4 shows the CD-spectra of the nucleotide and the respective polynucleotide at different temperatures. The large increase in the positive CD band should indicate a strong stacking interaction among the heterocyclic bases. Two sets of temperature dependent curves can clearly be distinguished for the CD spectra. The change below 20°C may be attributed to the breakdown of the secondary structure. If the molar ellipticity is plotted against the temperature, a sigmoid curve is obtained indicating a cooperative melting of the secondary structure (Fig. 4). Poly(nh2 s U) forms a triple stranded complex with poly(A)

12--

0] 10-3 e lo-3 r ----

8--

poly( nh2 Su) nh 2 SLI

~

10 O:

'

'\

4--

10 0

~i °

"'

~,°°/ " II....

~ - _

Vi, ~io

Y°

x [~",'3

-8--

-12--

Fig. 4. Circular d i c h r o i s m spectra o f p o l y ( n h 2 S U ) and its m o n o m e r i c c o m p o n e n t s in 0.1 M NaCI, 0 . 0 5 M s o d i u m c a c o d y l a t e p H 7 . 0 at 20°C, T h e spectra w e r e r e c o r d e d w i t h a Cary 61.

353 TABLE III STIMULATION OF POLY(Phe) SYNTHESIS The assay was performed with 50 pmol 70-S ribosomes for 20 rain at 37°C in a total volume of 100/~1. The mixture contained 50/~1 Mix+, 20 #1 [3H] Phe-tRNA (40 A260 nm-U/ml, 1.8% charged), 10 ~tl poly(N) (1 m g / m l ) and 10 /~1 S-100. Mix: 50 mM Tris • maleic acid (pH 5.1--7.3), 200 mM NH4CI, 30 mM magnesium acetate 25 mM K-ATP, 1 mM K-GTP, 75 mM potassium phosphoenol pyruvate, 1 mM dithiothreitol and pyruvate kinase (50 #g/ml). Following incubation a 50 #1 aliquot was applied to GF/A filters. They were incubated for 5 rain at 80°C in 109b trichloroacetie acid and rinsed with 5% trichloroacetic acid, ethanol, ethanol/ether 1 : 1 and ether. Template

pH

Poly(U) Poly(nh2 SU) Poly(U) Poly(nh2 SU) Poly(U) Poly(nh2 5U) Poly(U) Poly(nh 2 5U) PolF(U) Poly(nh2 5U )

Poly(Phe) synthesized (pmol) 0.4 0.6 9.0 10.2 11.4 12.8 11.6 13.6 11.6 10.8

5.1 5.6 6.0 6.7 7.3

with a one:step melting and thermal stability comparable to the poly(A) • 2 poly(U) complex (Fig. 5). Addition of 10 mM MgC12 shifts the Tm value by 10°C (Fig. 6). Poly(nh2 s U) is not bound to 70-S ribosomes in the absence of tRNA Phe. Whereas poly([ 3H] U) can be exchanged by poly(U) at 0°C and at 37°C, poly(nh22 U) can not replace prebound poly([ 3H] U). No difference can be found between poly(U) and poly(nh2 s U) in the stimulation of the binding of Phe-tRNA to 70-S ribosomes and in the template activity for poly(Phe) synthesis (Fig. 7). With trinucleoside diphosphates containing nh2 s u the tern-

A2+I 104

~

_

N=U

05

I

100%

50%

0%

Fig. 5. Complex formation between poly(A) and poly(nh2 $ U) in 0.1 M NaCI, 0.05 M sodium cacodylate pH 7.0 at 10°C. The formation of the poly(A) • 2 poly(U) complex is shown for comparison.

354 A260-E - o - - o - poly (A) • 2 poiy {U ) -- -1D-

poty(A]. 2poly(nh2Su) p o l y l A ) 2poly( nh2Su),10mMMgCI2

0.5

lb

3~

s'0

Fig. 6. M e l t i n g p r o f i l e s o f p o l y ( A )

poly(A) • 2 poly(nh2 SU) at 10 m M

Jo

• 2 poly(U)

9'o (o

M g 2+ (z~

T [°C]

o), p o l y ( A )

• 2 p o l y ( n h 2 s U ) (e

o) a n d o f

~) in 0.1 M NaCI, 0.05 M sodium cacodylate p H 7.0.

x--x poly {U) EE a.o

a_ i

o--o poly ( nh 2 SU)

50-

~'0

~&0

p01ynucleotide[~9 ] Fig.7. Stimulation of Phe-tRNA binding to 70-S ribosomes by poly(U) and poly(nh25U). The assay system was that of Nixenberg and Leder [19]. - o - o - U - nh 2 5U-U e, ,~ U-U-U ~ - . o - U - U - nh 2 5U

oE

~n u]

®2

E ~-30o-bo

o~ Jo

20-

a. -1-

10-

o

~

~

I

250

~

I

~__

I

500 750 trinucleoside diphosphate [nmoles]

Fig. 8. Stimulation of Phe-tRNA binding to 70-S ribosomes by trinucleoside diphosphates containin[ nh 25 U. The stimulation by U°U-U is s h o w n for comparison.

355 G-U~U

: :

G-U-nh25U

o

L

oo~ 100"

~R z n-

-5

2B0

~0

trinucleoside diphosphclte [nmoLes] Fig. 9. S t i m u l a t i o n o f V a l - t R N A b i n d i n g t o 7 0 - S r i b o s o m e s b y G - U - U a n d G - U - n h 2 S U.

plate activity in Phe-tRNA binding depends on the position of the modified nucleoside. Although nh2SU shows full activity when placed in the middle position of the U-U-U codon, template activity is abolished when the aminouridine occupies the 3'-terminal position (Fig. 8). A similar effect can be shown in the G-U-U stimulated binding of Val-tRNA. G-U-U stimulates the binding of 10 pmol Val-tRNA to 50 pmol of 70-S ribosomes, however, the binding b y G-U-nh2 s U is reduced to 3--4 pmol (Fig. 9). Discussion In the 5'-position of the anticodon of many tRNAs quite often a basemodified uridine is found. According to the tertiary structure of t R N A Phe as proposed b y Kim et al. [ 2 1 ] , this nucleoside is i n an exposed position since it forms the top of the anticodon stack having no stacked 5'-neighbour. It may be conceivable that a modification of this nucleoside results in a change of the stacking geometry of the anticodon. Thus a modification of the w o b b l e nucleoside in the anticodon may be a tool for the fine adjustment of specificity in c o d o n anticodon complex formation and result in a selection among different possible codons. In order to examine the influence of exocyclic groups on the stacking patterns of polynucleotides and oligonucleotides we compared poly(U) with poly(nh2 s U) and 5-aminouridine containing oligonucleotides. Besides the increase in the thermal stability of the secondary structure, poly(nh 2 s U) behaves similarly to poly(U). It forms a triple-stranded complex with poly(A). In addition poly(nh 2 s U) stimulates both the binding of Phe-tRNA and the poly(Phe) synthesis. Poly(nh2 s U), however, is not b o u n d to 70-S ribosomes in the absence of t R N A Phe. This cannot be attributed to the more stable secondary structure of the polynucleotide, since even at 37°C no exchange occurs. At the present there is no explanation for this behaviour.

356

Although U-nh 2 s U-U stimulated binding of Phe-tRNA was comparable to binding stimulated by U-U-U, the trinucleoside diphosphate containing the modified nucleoside in the 3'-position was inactive. This seems to be in contradiction to the template activity of poly(nh2 s U). Since, however, a triplet is bound by only two negative charges to the ribosome, the orientation effect on the base stacking is rather low. In the third position of the codon, uridine pairs with guanosine by presumably forming the 2-C = 0 (U) -* 1-NH(G) and 3-NH(U) ~ 6-C = 0 (G) hydrogen bonds. It may be that this base pair is very sensitive to minor changes in the exact steric location of the base. Such a change in the geometry of the base stacking can be caused by the amino group in the 5-position of the 3'-terminal nucleoside. The poly(A) • 2 poly(nh 2 s U) complex displays a similar T m value as the poly(A) • 2 poly(U) complex at 10 mM Mg 2+. However, since the T~ value in the single strand is raised, compared to polyuridylate, the stacking pattern in the single strand seems to be different from that of the hydrogen bonded structure.

Acknowledgements This work was supported by grants f~om the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. References 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21

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