The enzymatic synthesis of poly 4-thiouridylic acid by polynucleotide phosphorylase from Escherichia coli

BIOCHIMICA ET BIOPHYSICA ACTA

2371

BBA 96454

T H E ENZYMATIC SYNTHESIS OF POLY 4-THIOURIDYLIC ACID BY POLYNUCLEOTIDE P H O S P H O R Y L A S E FROM E S C H E R I C H I A COLI j . SIMUTH*, K. H. SCHEIT** AND E. ~[. GOTTSCHALK

Max Planck-lnstitut /iir t:.xperimentelle Medizin, dbteilung Chemic, G6ttingen (Germany) (Received October 6th, 1969) (Revised manuscript received December 23rd, 1969)

SUMMARY

4-Thiouridine 5'-diphosphate (saUDP) was found to be a substrate for polynucleotide phosphorylase from Escherichia coli. s4UDP gave /J-phosphate exchange (Kin = 0.26 raM; Vmax = O.18 jumole/ml per 20 rain) and was polymerised to poly 4-thiouridylic acid (poly s4U) (Kin = o.17 mM; Vmax I. 5 mjumoles/o.I ml per 15 rain). In the presence of phosphate poly s4U was degraded by polynucleotide phosphorylase to saUDP. Poly s4U was isolated on a preparative scale and characterized by means of sedimentation velocity analysis in an ultracentrifuge. Spectral properties of s4U and complex formation of poly s4U with poly A were investigated. =

INTRODUCTION

Recently the copolymerisation of UDP and 4-thiouridine 5'-diphosphate (s4UDP) by polynucleotide phosphorylase from Micrococcus lysodeikticus (M. luteus) was reported 1. This enzyme did not catalyze the polymerisation of saUDP to measurable amounts of poly 4-thiouridylic acid (poly s4U). Since the synthesis of poly s4U seemed of considerable importance, the action of polynucleotide phosphorylase from Escherichia coli on saUDP was investigated. The reason for the choice of this enzyme has been the much less pronounced primer dependency.

MATERIALS AND METHODS

s4UDP and E35SIs4UDP were prepared as described elsewhere 1,2. [aH]UDP was purchased from Schwarz BioResearch (Orangeburg, N.Y., U.S.A.). Polynucleotide phosphorylase (EC 2.7.7.8 ) with a specific activity of 165/*moles UDP per h per mg protein was prepared from E. coli according to the literature 3. Protein concentration was measured following the method of LOWRY et al. Ultraviolet absorption spectra were recorded with a Zeiss PMQ II or Cary 14 spectrophotometer. A Gilford model 2000 in connection with a Beckman DUR spectrophotometer and a linear Abbreviations: s4UDP, 4-thiouridine 5'-diphosphate; poly s4U, poly 4-thiouridylic acid. * Present address: Slovak Academy of Sciences, Biological Institute Bratislava, Czechoslovakia. ** To whom inquiries should be directed.

Biochim. Biophys. dcta, 204 (197o) 371-38o

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j. SIMUTH et al.

calibrated Gilford 2o 7 thermosensor was used to obtain ultraviolet absorption-temperature profiles. Radioactive samples were measured in a Tri-Carb Model 4312 liquid scintillation counter in standard scintillation mixture.

Phosphate exchange The assay was carried out as reported in the literature 3. Experimental details are given in the text.

Enzymatic polymerisation Standard assay. The incubation mixture contained o.I M Tris-HC1 buffer (pH 8.3) ; 2 mM MgC12; [3H~UPD (specific activity I mC/#mole) 5" l°5 counts/rain per ml; [35SJs4UDP (specific activity 6. 4-1o 5 counts/rain per #mole) 5' lO5 counts/rain per ml; and 15 enzyme units polynucleotide phosphorylase per ml. The substrate concentration is specified in the corresponding figures. Aliquots were withdrawn from the incubation mixture at certain time intervals, spotted on paper strips (2 cm × IO cm, W h a t m a n 3-MM), and the reaction stopped b y addition of a drop of acetic acid. The paper strip was dried and chromatographed in ethanol-I M ammonium acetate (I :I, b y vol.). The origin was cut out and the radioactivity of the absorbed polymeric material measured. Isolation o[ polynmleotides. The enzymatic reaction was stopped by addition of o.i ml I ~o aqueous cetyltrimethylammonium bromide solution to I ml of incubation mixture. The precipitate was collected by centrifugation, the supernatant discarded and the remaining pellet dissolved in 0.5 ml methanol. Traces of insoluble material were removed by centrifugation. An equal volume of i °/o NaCIO a in acetone (w/v) was added to the clear methanolic solution. The resulting precipitate was collected by centritugation, dissolved in a minimal volume of I °/o (w/v) aqueous NaC1Q and the sodium salts of polynucleotides precipitated by addition of excess ethanol. For further purification the crude mixture of polynucleotides was chromatographed on either Sephadex G-2oo, G-75 or G-25. Phosphorolysis. The incubation mixture contained: o.I M Tris-HC1 buffer (pH 8.3); 2 mM MgC12; 0.02 #mole poly s4U; 4 #moles (approx. lO4 counts/rain) KH2s2po 4 and 1.5 enzyme units polynucleotide phosphorylase, in a total reaction volume of o.I ml. After certain incubation times at 37 ° aliquots (25/~1) of the incubation mixture were chromatographed in isobutyric acid-I M NH4OH-o.I M E D T A (50:30:0.8, by vol.) on W h a t m a n 3-MM paper. The paper chromatograph was cut in narrow strips of equal size and the radioactivity measured in toluene scintillation fluid.

RESULTS AND DISCUSSION

s4UD P fl-phosphate exchange reaction Incubation of a normal nucleoside 5'-diphosphate with polynucleotide phosphorylase in the presence of inorganic phosphate leads to an exchange of the flphosphate residue. Whether this is due to an independent enzymatic activity or phosphorolysis of formed polynucleotides is still an open question 4,5. s4UDP gives /5-phosphate exchange (see Fig. ia). The exchange with s4UDP occurred at much lower concentrations than with UDP which showed that an UDP contamination was Biochim. Biophys. Acta, 2o 4 (197 o) 371-38o

373

ENZYMIC SYNTHESIS OF POLY s 4 U a

3 tO-2

~Olmole~~2151. . . . . [32p] S4UDP

t*

25 ml 20 rain

s UDP

"

b

s 4 UDP phosphate exchange

1t. 102

UDP

#mole substrote

!Vlq(m120mn.lJmoe l)

,.J!

01

0.25ml

. . . . . . .5. . . . . .

10 [-s4 UDP]-I (mM-l)

Figs. I. a and b. s 4 U D P /~-phosphate exchange. The incubation mixture contained o.I M T r i s HC1 buffer (pH 8.3); 2 mM MgClz; o.38ffM (approx. 5.IO4 counts/min) KH232PO4; 1.2 e n z y m e units polynucleotide phosphorylase; U D P or s 4 U D P as specified in the figures. Reaction volume 0. 5 ml, incubation temperature 37 °. Each incubation mixture was assayed after 2o min.

not responsible for the incorporation of E~2P]phosphate into nucleotide material. From a Lineweaver-Burk plot of the kinetic data of s4UDP/5-phosphate exchange, K m = 0.26 mM and v.... = 0.18 ffmole/ml per 20 min were obtained (Fig. Ib).

Polymerisation o/s4UDP In contrast to polynucleotide phosphorylase from M. lysodeikticus the enzyme isolated from E. coli polymerises s4UDP. The kinetics of this enzymatic reaction were followed by measuring the incorporation of [~sS]s4UMP into poly s4U. The obtained kinetic data are represented in a Lineweaver-Burk diagram giving K m = o.17 mM and Vmax= 1.5 mfmoles/o.i ml per 15 rain, v.... (UDP) = I #mole/o.I ml per 15 rain. There is a striking difference between vmax (UDP) and Vmax (s4UDP), the latter being smaller by a factor of 108. K~, (s4UDP) is about 3 times smaller than K m

(UDP). s 4 UDP-potymerisotion

2.103

V~I ( IJmole-I, 15 rain. 0.1 rnp)

Km 1.7"10-4 M

Vma× 1.5,10-3 IJtnole 0.1m1.15rn~

Is 4 UDP] -~ ( r a M - ' )

Fig. 2. s 4 U D P polymerisation.

E x p e r i m e n t a l details of the assay procedure are given under

MATERIALS AND METHODS.

The copolymerisation of U D P and s4UDP in a ratio I : I led to results the interpretations of which are also relevant for the action of polynucleotide phosphorylase on s4UDP. Fig. 3a shows that the kinetics of [3HIUMP incorporation into polynuBiochim. Biophys. Acta, 2o 4 (197 o) 3 7 1 - 3 8 o

j. SIMUTH et al.

374 /umole 3H-UM P incorpora~sdm M a

UDP

0.?

f

__

•

s4UDP

0.5

0.3

0.1 0 1

2

3

4

h

~30 m

absor bance

b

75

UMP =1.1 s4LIMP

t5 !i ab

A2~

1~

125

1~

rn~n

. . . .

m~'

Fig. 3. a and b. Copolymerisation of U D P and s4UDP, a. E x p e r i m e n t a l details as described u n d e r MATERIALS AND METHODS. I n c u b a t i o n mixtures: O - Q , o.5 mM [3H]UDP; Q - Q , 0.5 mM [3H~UDP and 0. 5 mM s4UDP, b. Sephadex G-25 c h r o m a t o g r a p h y of the incubation mixture, Colu m n size: i.o cm x 61 cm; eluent: 0.05 M Tris-HC1 buffer (pH 7.5), containing o.I mM dithiothreitol; flow rate, 0.2 ml/min, c. Ultraviolet a b s o r p t i o n s p e c t r u m of poly (s4U, U) at p H 7.

cleotides is different for a 5 mM UDP solution and a mixture of 5 mM UDP and 5 mM s4UDP. The rate of E~H]UDP polymerisation in the presence of s4UDP was slower but the extent of incorporation was the same. The copolymer formed in this reaction was isolated by Sephadex chromatography. The ratio UMP/s4UMP calculated from the ultraviolet absorption spectrum (Fig. 3c) was found to be I . I (Fig. 3b). This analysis showed that the composition of polynucleotides corresponded to that of the incubation mixture. The kinetics of the copolymerisation of UDP and s4UDP can be explained by means of the individual Km and vma~ values obtained from the kinetic analysis of the corresponding homopolymerisations. Since the K m for both substrates is about the same but the vmax is much higher for UDP than for s4UDP, the rate of synthesis oi the copolymer should be slower than that of poly U. It would, however, be faster than that of poly s4U if a 3'-terminal UMP reacts faster with s4UDP than a 3'-terminal s4UMP. Biochim. Biophys. Acta, 204 (197 o) 371-38o

375

ENZYMIC SYNTHESIS OF POLY s 4 U

Phosphorolysis o/poly s4U Polynucleotide phosphorylase catalyzes the degradation of polynucleotide chains to nucleotide 5'-diphosphates in the presence of inorganic phosphate. Since s4UDP was polymerised to poly s4U by this enzyme, poly s4U should also be phosphorylized. Fig. 4 represents the experimental evidence of the phosphorolysis of poly s4U. Paper chromatography of the incubation mixture showed the appearance of a E32p]phosphate containing species having the same RF value as s4UDP.

3.I0 3 2 I

T50

~

¢

~

4

3.103

A330

10C

lSO 1~

°"°'°

I!

,....

s/"UDP PO~ ?

10

'103 2h

20

30 cm

0 10-20 30 40 50

fraction

Fig. 4. P h o s p h o r o l y s i s of poly s4U. E x p e r i m e n t a l details are given u n d e r MATERIALS AND METHODS. Fig. 5. S e p h a d e x G-2oo c h r o m a t o g r a p h y of poly s4U. S e p a r a t i o n of a crude poly s4U m i x t u r e isolated b y m e a n s of c e t y l t r i m e t h y l a m m o n i u m precipitation. C o l u m n size: 2 cm × 67 cm; 4-ml fractions; eluent: o.i m M dithiothreitol.

Properties o/poly s4U Isolation of poly s4U on a preparative scale was achieved in a very convenient way by precipitating the formed polynucleotides as cetyltrimethylammonium salts directly from the incubation mixture. The practically water-insoluble cetyltrimethylammonium polynucleotides collected by centrifugation were converted to the corresponding sodium salts and proved to be free of unreacted substrate. This polydisperse poly s4U mixture was subjected to Sephadex G-2oo chromatography (Fig. 5) thus separating long-chain from short-chain poly s4U. Poly s*U present in the first fractions of the breakthrough peak of the Sephadex G-2oo chromatography was subjected to a sedimentation velocity analysis in an ultracentrifuge. This investigation furnished a sedimentation coefficient s°20,~ = 3.85 S for poly s4U (Fig. 6a) and the polynucleotide chain distribution curve (Fig. 6b). From the latter it followed that only io % of the poly s4U chains possessed sedimentation coefficients below 1. 5 S. The following experiments have been done exclusively with the above-described poly s4U fraction. Biochim. Biophys. Acta, 2o 4 (197 o) 371-38o

376

j. SIMUTHet al. o

c,%

1012 s 2G,w /

°2;0.....

/ /

/ / 0 5 A b s o r ' b o n c e ( 3 3 0 re,u)

5

Is

10

15

lO-'3 20,w

s

Fig. 6. a and b. Sedimentation velocity analysis of poly s4U. Sedimentation experiments were performed in a Spinco Model E ultracentrifuge equipped with a photoelectric scanner and a multiplexer. Double-sector cells with i 2 - m m optical p a t h and sapphire windows were used. Sedimentation coefficients were calculated by the m o v i n g - b o u n d a r y methods, a. S e d i m e n t a t i o n coefficient of poly s4U. The ultracentrifuge was r u n at 36 ooo rev./min and 2o °. S2o,w values were calculated from photoelectric scanner tracings (33 ° raft). Solvent: 0.05 M NaC1 containing o.i mM dithiothreitol and I mM E D T A . b. Integral sedimentation distribution of poly s4U. Speed of run: 36 ooo rev./min; t e m p e r a t u r e : 20°; solvent: 0.o 5 M NaC1 containing o.i mM dithiothreitol and I mM E D T A ; concentration of poly sqJ: 0.527 An30 my units per ml (co); distribution was calculated from photoelectric scanner tracings (33o my).

Fig. 7a shows the ultraviolet absorption spectrum of poly s4U. Compared to s4UMP the absorption maximum in the visible region is shifted to the blue, a phenomenon which is also shown by oligo ds4T nucleotides (K. H. SCHEIT, unpublished results). From the pH-dependence of the poly s4U absorption spectrum the apparent pK of s4UMP in poly s4U was found to be 8.7o, compared to 8.2o for s4UMP itself (Fig. 7b). The enzymatic hydrolysis of poly saU by pancreatic ribonuclease was accompanied by a hyperchromicity of the absorption spectrum. The difference between the absorption spectra of poly s4U before and after treatment with ribonuclease was measured and interpreted as the absorption difference spectrum between s4UMP 0.5

Absorbance a

0.,4

A340

b 0.4

0.3 E3

0.2 O2

pK

8.7

0.1 0.1

0

i

240

i

i

~

i

i

260

q

i

i

. . . . . . . . . . . . . . . . . .

280

300

32Q

mH

340

:

360

360

6

7

8

9 ?0

11

pH

Fig. 7. a and b. Absorption s p e c t r u m of poly s4U. a. Absorption s p e c t r u m of poly s4U in 0.05 M sodium cacodylate (pH 7.) b. Spectrophotometric titration of poly s4U. The titration has been carried out directly in a cuvette b y addition of small volumes of i M N a O H or i M HC1 to a solution of poly s4U in o.o 5 M NaC1. Isosbestic point at 32z mff; a p p a r e n t p K of saUMP in poly s4U, 8.70.

Biochim. Biophys. Acta, 204 (197o) 371-38o

377

ENZYMIC SYNTHESIS OF POLY s 4 e

and s4UMP in poly s4U (Fig. 8). The ratio A~0 m~/A260 m# = 6.60 found in the absorption spectrum after hydrolysis of poly s4U to saUMP was very close to the ratio A336 m#/A~eo m# = 7 .0 of saUMP (ref. 7), thus revealing a considerable purity of poly saU. Addition of Mg 2+ to poly saU solutions caused a significant hyperchromicity of the absorption spectrum (Fig. 9),indicating an interaction of Mg 2+ with the hetero0.2 ~04A

J

•0.11 0

~E

............................................

0

-0.1

Io~o.I o.2

0.2

0.3 0.4

0.3

o.5 0.6 0.7 o,8

0.5 ¸

0.9

250

300

mp

350

400

....... C 300

............ 400m/u

350

Fig. 8. H y p e r c h r o m i c i t y of poly s4U. i A330 mu unit p o l y s4U in 0.05 M sodium cacodylate (pH 7) containing o.1 M KC1 and o.oi M MgC12 was incubated with 5 Pg pancreatic ribonuclease for 3 h at 37 °. The difference spectrum was calculated as d e per s4UMP residue from the absorption spectrum before and after e n z y m e treatment. Fig. 9. Interaction of p o l y sau with Mg 2+. Solvent: o . i o M NaC1 in o.o 5 ]Vf sodium cacodylate (pH 7); poly s4U A330 mv: 0.35o; MgClz: o.oi M; Zle was calculated per s4UMP residue.

cyclic chromophore. Likewise s4UMP interacted with Mg 2+ as shown in Fig. I0 where the spectrophotometric titration of saUMP with Mg 2+ is represented. No interaction of s4U with Mg 2+ could be detected. Therefore a phosphate or phospho-

A330- unffs

\

0.45 ~330 0.44 0.43 0.42 0.41

1.3 0d,0 ?00

20O

3OO

4

8

12

16

20°

}Jrnole M g 2 *

Fig. IO. Interaction of s4UMP with Mg 2+. Solvent: 0.05 M Tris-HC1 buffer (pH 7); s4UMP: 1.4 d3a0 m~ units; spectrophotometric titration was carried out directly in a c u v e t t e b y additions of small v o l u m e s of I M MgC12 monitoring the absorbance at 33 ° m/z. The values are corrected for dilution. Fig. I i . A b s o r p t i o n - t e m p e r a t u r e profile of p o l y saU. Solvent: 0.05 M sodium cacodylate (pH 7) containing o.i M KC1 and o.oi M MgC1 v

Biochim. Biophys. dcta, 204 (I97 o) 3 7 1 - 3 8 o

J. SIMUTH et al.

378

diester moiety (as in poly s4U) seems to be a prerequisite of the interaction of s4UMP or poly s4U with Mg 2+. A detailed investigation has been started and will allow a more profound interpretation of this phenomenon. In the presence of o.oi M Mg 2+ poly U exhibits a cooperative transition in an ultraviolet absorption temperature profile with a Tm of 8.5 ° indicating secondary structure of low stability s,9. Under the conditions specified in Fig. II poly s4U behaved in a manner very similar to poly U displaying a transition with a Tm of 8 °.

A260-units

A330- u n i t s

0.6

0.5

O9 0.~,

10 20 mpmoles poly A

30

10 20 mpmoles pc~y A

Fig. I2. a and b. Spectrophotometric titration of poly s4U with p o l y A. Solvent: 0.05 M sodium cacodylate (pH 7) containing o.i 1Vf KC1. K n o w n a m o u n t s of p o l y A were added to 1.o 4 A330 my units p o l y s4U monitoring the absorbance at either 33 ° m # (a) or 260 m/~ (b). All values are corrected for dilution,/~M p o l y A m e a n s / z M A M P residues in p o l y A.

A spectrophotometric titration of poly saU with poly A followed at 33 ° m # clearly indicated the formation of a helical complex Epoly s4U]2 • [poly A~ (Fig. I 2 a ) ; surprisingly, no hyperchromicity was observed during this titration at 260 m # (Fig. I2b). Therefore the absorption difference spectrum between poly A and poly s4U and Ipoly A 1. Epoly s4Ul was measured (Fig. 13). Again no hyperchromicity was found at 26o m # but instead a hyperchromic band at 28o m#. The broad structured i % Difference

/

2O

AA

I

O3

* ~0

lO

02

1

.~"

*

20

,

,

~

30

250

300

3~0

7

02

t

03

O4 OS

O~ 07

400 mp

Fig. 13. Absorption difference spectrum of [poly s4U~) • [poly A~. The difference spectrum bet w e e n [poly s4U2] • Epoly A) and the noninteracting components was measured using t a n d e m cuvettes. Solvent: o.i lV[ sodium cacodylate (pH 7). The percent difference w a s calculated using the sum spectrum of the noninteracting components obtained in a t a n d e m cell.

Biochim. Biophys. Acta, 2o 4 (197 o) 3 7 1 - 3 8 o

ENZYMIC SYNTHESIS OF POLY s4U

379

hyperchromic band in the visible region was better resolved if the difference spectrum was plotted as percent difference v e r s u s wavelength. Then three hyperchromic bands in the difference spectrum at 345, 330 and 31o m # could be distinguished. The appearance of a hyperchromic band at 31o m/, is interesting, since the absorption maxima of the mesomeric s4U anion and s4CH3U (ref. Io) are found at the same place. This seems to mean that structures similar to the mesomeric s4U anion make contributions to the absorption spectrum of the [poly s4U] • [poly A] complex. The ultraviolet absorption-temperature profile of [poly saU]2 • [poly A] at o.15 M Na + exhibited two transitions with Tm values 34 and 62 ° when measured at 33 ° m/~ (Fig. I4a ). According to the absorption difference spectrum of [poly s4U] • Ipoly AI (Fig. 13) no transition could be detected at 260 and 294 m#. At 280 m#, however, a two-step hyperchromic transition with Tm values 35 and 55 ° was found (Fig. I4b ). Since the absolute change of absorbance at 28o m # during the dissociation of the complex was very small, the estimation of Tm values in Fig. I4b was very inaccurate.

A2B0

Tin- 3s°

b

A330

021

A330

0 22

/

021

0

C

a

2 ~

?

Tm 62 o H

019

Tm

25"/.

605 °

H 25"/.

018 017

4o

o

016

0.1B

0

10 20 30 40 50 60 ~)

BOo

0.3

0

10

20

30

40

50

60

70

80 °

10 20 30 40

50 60 70 e0 °

Fig. 14. a, b a n d c. A b s o r p t i o n - t e m p e r a t u r e profile [poly s4U~] • [poly A]. a. S o l v e n t o.05 M s o d i u m c a c o d y l a t e (pH 7) c o n t a i n i n g o . i IV[ KC1; absorbance m o n i t o r e d at 33 ° m]~. b. Solvent: as described in (a) ; absorbance m o n i t o r e d at 28o m/l. c. Solvent: 0.o 5 M s o d i u m c a c o d y l a t e (pH 7) c o n t a i n i n g o . i M KC1 and o . o i M MgC12; absorbance m o n i t o r e d at 33 ° m/~.

But principally the transition at 28o m/* corresponds to that observed at 33o m/~. It is seen from Figs. 14a and I4b that the second major transition occurred over a broad temperature range of 20 °. Mg 2+ cations had an unexpected effect on the thermal dissociation of [poly s4U]~ • [poly A] : the breadth of the transition was increased and the Tm values somewhat lowered (Fig. I4C ). The experimental data clearly show that the poly U-analog poly s4U forms a helical complex [poly s4U]2 • [poly AI, but the properties of [poly U]2" [poly A] and [poly s4U]~ • [poly A] under identical conditions differ to some extent (Table I). Biochim. Biophys. Acta, 2o 4 (197 o) 3 7 1 - 3 8 o

38o TABLE

J. SIMUTH et

al.

I

PROPERTIES OF HELICAL COMPLEXES [poly U]z" [poly A] AND [ p o l y s4U]2 • [ p o l y A]

~poty u~2. Lt)oly A I

[poly s4U]2 . [poly A]

A T of transition

2°

20 °

Hyperchromicity at 26o m #

Yes

No

Hyperchromicity at 28o rn#

Yes

Yes

Effect of Mg 2+

Stabilisation of secondary structure

N o stabilisation

59 °

62 ~)

Tm v a l u e s

of secondary structure; increased breadth of transition

The lack of hyperchromicity at 260 m# during thermal dissociation of [poly s4U]2 • [poly A] is a rather important finding, since as well as [poly U, s4U12 • [poly AJ (ref. 6), poly r(A-s4U) (F. CRAMER AND K. H. SCHEIT, unpublished results) and poly d(A-s4T) (ref. II) showed hyperchromicity in absorption-temperature profiles both at 26o and 33o m~. Furthermore whereas the transitions at 33o m/* of [poly U, s4U12 • ~poly AI and poly r[A-s4Ul exhibited hyperchromicities between 45 and 5o %, the latter dropped to 3 ° °/o in Epoly s4U]2 • [poly A]. It could well be that hydrogen bonding between adenine and 4-thiouracil as well as helical structure in Ipoly s4U]' [poly A] and poly rIA-s4U] are completely different. More information concerning this problem is to be expected from X-ray studies of [poly s4U]2 • [poly A] and poly rFA s~U] fibers. Work along this line is in progress.

ACKNOWLEDGEMENTS

Our thanks are due to Dr, F. Eckstein for stimulating discussions and to Professor F. Cramer for his constant interest.We are indebted to the Deutsche Forschungsgemeinsehaft for financial support. REFERENCES I 2 3 4 5 6 7 8 9 io ii

K. H . SCHEIT AND E. GAERTNER, Biochim. Biophys. Acta, 182 (1969) i . K. H. SCHEIT, Chem. Ber., IOI (1968) 1141. Y . I~2HIMI AND U. Z. LITTAUER, Methods Enzymol., 12 (1968) 513 . ]VI. GRUNBERG-MANAGO, i n I. N. DAVIDSON AND W . E . COHN, Progress in Nucleic Acid Research, Vol. i , A c a d e m i c P r e s s , N e w Y o r k , 1963, p. 93G. I{AUFMA.NN AND U. Z. LITTAUER, F E B S Letters, 4 (1969) 79. t~. n . SCHEIT AND E . GAERTNER, Biochim. Biophys. Acta, 182 (1969) io. I~. H . SCHEIT, Biochim. Biophys. Acta, 166 (1968) 285. M. ]12(. LIPSETT, Proc. Natl. Acad. Sci. U.S., 46 (196o) 445. E . G. RICHARDS, C. P. FLESSEL AND I. R . FRESCO, Biopolymers, i (1963) 431. K . H . SCHEIT, Tetrahedron Letters, (~967) 113. A. G. L E z l u s AND K. H . SCHEIT, European J. Biochem., 3 (1967) 85.

Biochim. Biophys. Acta, 2o 4 (197 o) 3 7 1 - 3 8 o