Volume 7 Number 4 1979

Nucleic Acids Research

Detennination of base pairing in Escherichia coli and Bacillus stearothermophilus SS RNAs by infrared spectroscopy B.Appel and V.A.Erdmann Max-Planck-Institut fur Molekulare Genetik, Abt. Wittmann, Ihnestr, 63-73, D-1000 Berlin, 33 and J.Stulz and Th.Ackermann Institut fur Physikalische Chemie II, Universitit Freiburg, D-7800 Freiburg, GFR

Received 3 July 1979 ABSTRACT The extent of base pairing in Escherichia coli and Bacillus stearothermophilus 5S RNAs was determined by infrared spectroscopy. From the infrared spectra taken at 200 and 520C it is concluded that E. coli and B. stearothermophilus 5S RNAs possess a large number of base pairs (Table I). Comparison of our results with those previously published using other methods leads to the conclusion that the structures of prokaryotic 5S RNAs involve a large number of tertiary interactions, in which the base pairing is not necessarily solely of the Watson-Crick type. INTRODUCTION

5S RNA is an essential constituent of the large ribosomal subunits of pro- and eukaryotic ribosomes and has intensively been studied (reviewed in ref. 1). Currently the primary structures of 13 pro- and 23 eukaryotic 5S RNAs are known (2). In recent years considerable efforts have been invested in the problem of 5S RNA secondary and tertiary structure. The primary aim of different approaches has been to obtain data from which the extent of single or double strandedness in 5S RNA could be deduced. The results so far obtained suggest that in E. coli 5S RNA there are 28-50 base pairs (1). Recently we have finished the oligonucleotide binding studies, which permitted the identification of single stranded regions in E. coli and B. stearothermophilus 5S RNAs (3). To complement these data we have turned to infrared spectroscopy to estimate the amount of base pairing in both 5S RNA species. IR-spectroscopy is especially suited for the determination of base pairs in RNA (4-10), and it has recently been shown that with four specific tRNAs the number of base pairs can be estimated with a C Information Retrieval Umited 1 Falconberg Court London Wl V 5FG England

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Nucleic Acids Research precision of 10% (5). The results obtained from the experiments reported in this communication suggest the involvement of 20 A-U and 36 G-C base pairs in E. coli 5S RNA secondary and tertiary structure, and of 17 A-U and 29 G-C base pairs in B. stearothermophilus 5S RNA secondary and tertiary structure. The relatively high amount of base pairing observed for E. coli 5S RNA by IR-spectroscopy is in agreement with two recent reports in which the same RNA was analyzed by Raman spectroscopy (11,12). MATERIALS AND METHODS

E. coli (A19) and B. stearothermophilus (strain 799) 5S RNAs were isolated as previously described (13). Prior to measurements the 5S RNAs were lyophilized twice from 99.75% D20 and then dissolved in D20 buffer at pD 7.5. The measurements were either in 10 mM Tris-DCl or in 0.4 M phosphate buffer as indicated in the legends of the Figures and Tables. For the measurements the RNA concentrations were adjusted to between 30 and 60 A260 units per ml. The specific absorption of E. coli 5S RNA (M.W. 40 778) was determined to be 19.18 A260 units per mg and that of B. stearothermophilus 5S RNA (M.W. 39 746) as 18.84 A260 units per mg. UV-absorption was recorded with a Zeiss PMQ II or Unicam SP 1800

spectrophotometer. Infrared measurements were carried out with a Perkin Elmer 325 spectrometer, using the technique described previously by Schernau and Ackermann (5). The spectra were digitized in steps of 2 cm-1 in the range of 1800 - 1500 cm-1 and normalized to the extinction coefficient based on molar concentration of the monomer units. The extent of base pairs was estimated by comparing simulated spectra, based on standard component spectra, with experimental ones. As reference components we used a) for unpaired regions of the RNA (5'-3')ApA, (5'-3')UpU, (5'-3')CpC and 5'GMP and b) for Watson-Crick type base pairs poly(A) *poly(U) and poly(C)-poly(G). The concentration of Mg++ ions was determined by atomic absorption measurements using a Perkin Elmer 305 atomic absorption spectrophotometer.

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Nucleic Acids Research RESULTS Since the estimation of base pairing in RNA by infrared spectroscopy is dependent upon the availability of corresponding standard reference spectra, the IR spectra of the following substances had to be measured: Poly(A)-poly(U), poly(C)-poly(G), ApA, UpU, CpC and GMP (Figure 1). The polynucleotide spectra served as standards for the helical parts and GMP, and the three dinucleotides as standards for the single stranded regions of the RNA. The IR-spectrum of E. coli 5S RNA taken at 200C is shown in Figure 2. In order to determine the extent of base pairing, to which this experimental curve corresponds, a curve fitting

Figure 1. Infrared spectra a) of model compounds for single strandedness; ApA (-), UpU (- -41, CpC (-.- ) and GMP (-- -) and b) of double-helical complexes poly(A)- poly(U) ) and poly(G)-poly(C) ( - -). For experimental details (see Materials and Methods.

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Nucleic Acids Research Figure 2. Infrared spectra of E. coli 5S RNA a) at 200C and b) at 520C. Figure 2c represents the infrared spectrum of the 520C sample after cooling to 2ooC. Other experimental details

20|

52EL

are given in the text.

20 C

160

1800

1500

cm1 method was applied. The principles of this method are shown as an example in Figure 3, in which 23 (Figure 3a) and 29 (Figure 3b) G-C base pairs were assumed for E. coli 5S RNA, while the amount of A-U base pairs was varied from 7 to 19. The number of G-C and A-U base pairs were varied in a similar manner until the computer presented an IR-spectra which agreed best with the experimental curve of E. coli 5S RNA. The E. coli 5S RNA 200C spectra (Figure 2) agreed best with simulated spectra consisting of 20 A-U and 36 G-C base pairs per 5S RNA (Table I). We have previously shown that E. coli 5S RNA exhibits a twoa

C

A-U

23 7 s15 19

t E

1046

J b

V

n r

>

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Figure 3. Simulated infrared spectra for E. coli 5S

RNA in which the number of G-C base pairs were assumed to be 23 (a) and 29 (b). The number of A-U base pairs were varied between 7 and 19 per 5S RNA.

Nucleic Acids Research Table I

Number of base pairs in E. coli and B. stearothermophilus 5S RNAs at 20 and 525C Number of base pairs per 5S RNA

200C

5S RNA

E. coli

stearothermophilus

520C

A - U

G

C

20 ± 2

36

2

t

29

17

2

±

2

A -U

G- C

2

30

7 i 2

25

16

2

±

2

phase melting behaviour in which the first plateau was reached at 520C (14). Since in analogy to tRNA, it may be assumed that melting up to the first plateau is primarily due to the unfolding of the tertiary structure, we repeated the IR-measurement at 520C (Figure 2). The evaluation of the IR-spectrum, by the curve fitting method, lead to the conclusion that E. coli 5S RNA consists at 520C of 16 A-U and 30 G-C base pairs (Table I), and that these base pairs are probably the ones involved in secondary structure. Renaturation of this 5S RNA by cooling to 200C, and remeasuring the IR-absorption yielded the spectrum shown in Figure 2c, which is nearly identical to the one in Figure 2a, suggesting that the 52 0C procedure did not irreversibly denature the RNA. This conclusion was also supported by polyacrylamide gel electrophoresis experiments (data not shown). For comparative purposes the IR-spectra of another prokaryotic 5S RNA, namely that from B. stearothermophilus, were also analyzed (Figure 4). Again the spectra were taken at 20 and 520C. Although B. stearothermophilus 5.$ RNA does not..show a two-phase melting behavior the 520C IR-spectrum was taken with the assumption that at this temperature, similar to E. coli 5S RNA, most of the tertiary structure would have melted. The results obtained after application of the curve fitting method are summarized in Table I and led to the conclusion, that B. stearothermophilus 1047

Nucleic Acids Research Figure 4. Infrared

spectra of B. stearothermophilus 5S RNA a) at 200C and b) at 520C. Figure 4c represents the infrared spectrum of the 520C sample after cooling to 200C. Other experimental

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1

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1900

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500

details

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1600

5S RNA consists at 200C of 17 ± 2 A-U and 29 ± 2 G-C and at 52°C of 7 ± 2 A-U and 25 ± 2 G-C base pairs. It is known that Mg++ contributes significantly to the structure of nucleic acids, we therefore repeated these measurements with E. coli and B. stearothermophilus 5S RNAs, which had been extensively dialyzed (48 hrs) against a Mg++-free buffer. The IR-spectra obtained were identical to those shown in Figures 2 and 4. Atomic absorption analysis of these RNAs showed that E. coli 5S RNA contained 3.7 and B. stearothermophilus 5S RNA 6.7 tightly bound Mg++ ions per RNA molecule. The tightness of the magnesium binding suggests that they are involved in stabilizing the RNA structure. The ability to simulate IR-spectra with the aid of a computer makes it possible to test proposed 5S RNA structural models for their validity. We used this method to analyze all published 1048

Nucleic Acids Research E. coli 5S RNA structural models in this respect. The proposed secondary structural models were compared with our IR-spectrum at 520C, while the tertiary structural models were compared with the IR-data collected at 200C. The types and amounts of base pairing for the different structural models are summarized in Table II, while the spectra-simulations of these models are shown in Figures 5-9. The results obtained show, that none of the structural models simulates an IR-spectrum which is identical with the ones experimentally observed at either 200C or

520C. It is of interest to note that information on G-U base pairing can be obtained by means of IR spectroscopy (17). Randomly copolymerized poly(A,G) forms a double stranded helix with poly(U), the spectrum of which differs significantly from that of double helical poly(A)-poly(U) and also from a simulated spectrum of a stoichiometric mixture of poly(A)-poly(U), guanine and uridine. Since the ratio of A and G was about 4:1, the extrapolation of the spectral data to "pure poly(G)'poly(U)" is not possible. Therefore we have no real model compound for G-U base pairing. For our simulations of spectra for proposed 5S RNA models we used a G-C base pair in place of a G-U base pair. Even if this procedure is not entirely justified, the main spectroscopic effects caused by G-U base pairing may be correctly described. These main spectroscopic effects are: intense bands at 1695 cm-1 and at 1656 cm-1 (poly(G)hpoly(C) at 1688 cm-1 and at 1647 cm-1, a low intensity absorption between these bands and the loss of intensity of the G band near 1568 cm-1. From the intensity of the bands we may conclude that the changes of the infrared spectrum caused by one G-U base pair correspond to the changes caused by 1-2 G-C base pairs.

DISCUSSION The structures of E. coli and B. stearothermophilus 5S RNAs were analyzed by infrared spectroscopy. For this method it has been shown that the double bond region in the IR-spectrum is sensitive to secondary and tertiary structural interactions of the nucleotides (5). The frequencies and intensities of the carbonyl bands are changed by hydrogen bonding, coupling in 1049

Nucleic Acids Research Table II

Number of base pairs in proposed E. coli 5S RNA structural models

Number of base pairs

Model Proposed

Brownlee et al. Boetker and Kelling Cantor Raacke Madison Jordan DuBuy and Weissman Monier Kearns and Wong Fox and Woese Osterberg et al. Weidner et al.

(24) (25) (26) (27) (28) (29) (30) a b (31) a b c (32) a b

(33) (34)

(35) a b

Luoma and Marshall Hori and Osawa Chen et al.

Experimentally determined at 20°C at

520C

(12) (36) (11)

A - U

G - C

G - U

4 8 14 6 9 9 9 8 9 8 7 7 4 5 9 5 4 10 5 35%A

16 24 32 21 23 19 25 24 25 22 22 20 16 17 27 17 12 21 17 70%G

3 5 3 4 7 3 6 6 6 3 4 1 1 3 6 3 1 5 3 -

20 16

3630

*Includes possible G-U base pairs.

plain (15) and vertical coupling (16). If the vertical coupling effect is neglected it is possible to calculate a spectrum of a nucleic acid using stoichiometric amounts of infrared spectra 1050

Nucleic Acids Research

a

b II

J~~

i_

I.

1800

1700

cm .4160

1500

C

1800

1700

1600

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170 r0 c

1800

150

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1500

zo..

1800

17 cm10

15006

1800

1700 cm4 1600

150

Simulated infrared spectra for E. coli 5S RNA structural models (-) in comparison to the experimental spectrum recorded at 520C (...). The models are taken from the following references a (24), b (25), c (26), d (27), e (28) and f (29).

Figure 5.

from model compounds. The results reported in this study are based upon the following model compounds: a) for double strandedness: Poly(A)-poly(U) and poly(C)-poly(G) and b) for single strandedness: ApA, CpC, 5' GMP and UpU. The three dinucleotides used were chosen instead of the mononucleotides, because the spectra of random coiled nucleic acids are better fitted to them. Comparing the experimental spectra with the calculated spectra the main differences are found near 1672 cm 1 where double

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Nucleic Acids Research

ji

,~~~.2* ,< iiocm

160

So170io cmF

1800

1700 m

1600

1500

1800

1700

1600

1500

1800

1700

1600

1500

1800

1700

1600

1500

160

cm

160

1500

Simulated infrared spectra for E. coli 5S RNA structural models (-) in comparison to the experimental spectrum recorded at 520C (-..). The models are taken from the following references a (30), b (30), c (31), d (31), e (31) and f (32).

Figure 6.

helical poly(A)hpoly(U) exhibits an intense, sharp band due to a vibration of the C(4)=O group of uracil (16). In naturally occurring nucleic acids it must be expected that this band is broadened by small interactions with different nearest neighbours, and as a result this band cannot be found in the spectrum of 5S RNA. All other spectral effects resulting from base pairing are very well described by our model. The bands of adenine near 1630 cm 1, guanine near 1570 cm 1 and cytidine near 1500 cm 1

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Nucleic Acids Research

b

11 X

X170

cm' 160

1500

C

5A 1800

1700

C

cm

0W

1500

Figure 7. Simulated infrared spectra for E. coli 5S RNA structural models (-) in comparison to the experimental spectrum ). The models are taken from the following recorded at 520C ( references a (32), b (33), c (34), d (35), e (35) and f (12).

of decreased intensity, the band near 1650 cm 1 corresponding to vibrations of guanine, cytidine and uracil is reduced and a new band appears near 1690 cm 1 in analogy to double helical poly(G) -poly (C). Before discussing the results obtained in this study it is important that two additional points are considered. First, a G-U base pair causes an intense band in the IR-spectrum at 1695 cm-1, which contributes to the band of nucleic acids at are

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Nucleic Acids Research

b

a

4

.1 1800

m 1600

1700

,.

1800

1500

.., 1600

1700

1500.

Figure 8. Simulated infrared spectra for E. coli 5S RNA structural models (-) in comparison to the experimental spectrum recorded at 520C (...). The model (a) was taken from reference 3 while Figure,8b shows a simulated spectrum in which 35% of A and 70% of G was assumed to be pase paired in E.coli 5SRNA ,

b

a

I

I'

6

6

I. I ~

1800

~

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~

~

-1

I I

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18

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I.

I

I'

I

I

I8 1800

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-

CM-.

1600

1500

11300

1700

Cm--'

1600

1500

Figure 9. Simulated infrared spectra for those E. coli 5S RNA structural models in which tertiary interactions have been proposed (-) in comparison to the experimental spectrum recorded at 200C (---). The models are taken from the following references a(26), b (29), and c (34). The simulated spectrum (d) is based upon Raman spectroscopy data which suggested that in E. coli 5S RNA 35% of the adenines and 70% of the guanosines are base paired (11). 1054

Nucleic Acids Research 1690 cm 1 in such a way that an overestimation of 1-2 G-C base pairs for each G-U base pair may occur (17). Second, tertiary interactions are also expected to increase the intensity of the band at 1690 cm 1 and to decrease the intensity of the band at 1630 cm 1 due to vibrations of the adenine residue and therefore result in an overestimation of both types of base pairs (18,19). Considering these two points, it is apparent that our determinations of base pairing in the two 5S RNAs at 200C will be the maximum amount possible. Under the assumption that at 520C most of the tertiary structures are melted, possible remaining G-U base pairs will only lead to an overestimation of G-C base pairs. IR-spectroscopic determination of base pairing in E. coli and B. stearothermophilus 5S RNAs is summarized in Table I. Comparison of these results (at 200C) with those previously published on the basis of other physical techniques (28-52 base pairs per 5S RNA, ref. 1) shows, that our estimations approximate those which predict a large degree of base pairing in E. coli 5S RNA. More recently a similarly high degree of base pairing for E. coli 5S RNA was also estimated by 19F-NMR (20), C-NMR (21) and Raman spectroscopy (11,12).Apparently these results are not in full agreement with oligonucleotide binding studies (22,23), which suggest a lesser degree of base pairing. A plausible explanation for this apparent discrepancy may lie in the fact that 5S RNA has a complicated and extensive tertiary structure, in which, besides Watson-Crick base pairing, other known base pairing types, including triple-base pairs, may stabilize the tertiary structure. Since it is likely that most of the tertiary interactions in E. coli 5S RNA are melted at 52°C (14) the IR-spectroscopy spectra taken at this temperature should primarily reflect secondary interactions. It should be noted that we cannot exclude the possibility that some of the tertiary interactions still remain at this temperature, since E. coli and B. stearothermophilus 5S RNA contain respectively 4 and 7 stronaly bound Mg++ per molecule. This fact and the effects of possible G-U base pairs on the determination of G-C base pairs, as discussed above, leads us to consider our 52°C results as representing the maximum amount of base pairing involved in 5S RNA secondary 1055

Nucleic Acids Research structure. Nevertheless, we consider it valid to compare the experimental IR-spectra taken at 520C with those simulated for diffetent proposed 5S RNA structural models (Fig. 5-8). This comparison shows that none of the simulated spectra is identical to the ones experimentally observed and that best aqreement at 200C is achieved with the model shown in Figure 9a and at 520C with the models shown in Figures 5e, 6b, 7c, 7f and 8b. ACKNOWLEDGEMENTS We thank Dr. H. G. Wittmann for continuous support and critical discussions and M. Digweed for critical reading of the manuscript. In addition do we like to acknowledge the support of these studies by the Deutsche Forschungsgemeinschaft. REFERENCES 1 Erdmann, V.A. (1976) In Progress in Nucleic Acid Research and Molecular Biology, Cohn, W.E., Ed., Vol. 18, pp. 45-90. Academic Press, New York 2 Erdmann, V.A. (1979) Nucleic Acids Res. 6, r29-r44 3 Wrede, P., Pongs, 0. and Erdmann, V.A. (1978) J. Mol. Biol. 120, 83-96 4 Thomas, G.J., Jr. (1969) Biopolymers 7, 325-334 5 Schernau, U. and Ackermann, Th. (1977) Biopolymers 16,

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1735-1745 Morikawa, K., Tsuboi, M. and Kyogoku, Y. (1969) Nature 223, 537-538 Tsuboi, M., Higuchi, S., Kyogoku, Y. and Nishimura, S. (1969) Biochim. Biophys. Acta 195, 23-28 Nishimura, Y., Morikawa, K., Tsuboi, M. (1973) Bull. Chem. Soc. Japan 46, 3891-3892 Katsura, T., Morikawa, K., Tsuboi, M., Kyogoku, Y., Seno, T. and Nishimura, S. (1971) Biopolymers 10, 681-698 Tsuboi, M. (1971) in: International Union of Pure and Applied Chemistry Suppl. Vol. 7, 145-177 Chen, M.C., Giege, R., Lord, R.C. and Rich, A. (1978) Biochemistry 17, No. 15, 3134-3138. Luoma, G.A. and Marshall, A.G. (1978) Proc. Natl. Acad. Sci. USA 75, 4901-4905 Erdmann, V.A., Doberer, H.G. and Sprinzl, M. (1971) Mol. Gen. Genet. 114, 89-94 Cramer, F. and Erdmann, V.A. (1968) Nature (Lond.) 218, 92-93 Howard, F.B., Frazier, J. and Miles, H.T. (1969) Proc. Natl. Acad. Sci. USA 64, 451-458 Morikawa, K., Tsuboi, M., Takahashi, S., Kyogoku, Y., Mitsui, Y., Jitaka, Y. and Thomas, G.J. (1973) Biopolymers 12, 799-816

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Ackermann, Th., Gramlich, V., Klump, H., Knable, T., Schmidt, E. and Stulz, J. (in preparation) Howard, F.B., Frazier, J., Lipsett, M.N. and Miles, H.T. (1964) Biochem. Biophys. Res. Commun. 17, 93-102 Miles, H.T. and Frazier, J. (1964) Biochem. Biophys. Res. Commun. 14, 21-28 Marshall, A.G. and Smith, J.L. (1977) J. Am. Chem. Soc. 99, 635-636 Hamill, W.D., Grant, D.M., Cooper, R.B. and Harmon, S.A. (1978) J. Am. Chem. Soc. 100, 633-635 Lewis, J.B. and Doty, P. (1977) Biochemistry 16, 5016-5025 Wrede, P., Pongs, 0. and Erdmann, V.A. (1978) J. Mol. Biol. 120, 83-96 Brownlee, G.G., Sanger, F. and Barrell, B.G. (1967) Nature 215, 735-736 Boedtker, H. and Kelling, D.G. (1967) Biochem. Biophys. Res. Commun. 29, 758-766 Cantor, C.R. (1967) Nature 216, 513-514 Raacke, J.D. (1968) Biochem. Biophys. Res. Commun. 31, 528-533 Madison, J.T. (1968) In Ann. Rev. of Biochem., Ed. Boyer, P.D. 37, 131-148 Jordan, B.R. (1971) J. theor. Biol. 34, 363-378 DuBuy, B. and Weissmann, S.M. (1971) J. Biol. Chem. 246, 747-761 Monier, R. (1974) In Ribosomes, (Eds. Nomura, M., Tissidres, A. and Lengyel, P.) Cold Spring Harbor Lab., pp. 141-168 Kearns, D.R. and Wong, Y.P. (1974) J. Mol. Biol. 87, 744755 Fox, G.E. and Woese, C.R. (1975) J. Mol. Evol. 6, 61-76 Osterberg, R., Sjoberg, B. and Garrett, R.A. (1976) Eur. J. Biochem. 68, 481-487 Weidner, H., Yuan, R. and Crothers, D.M. (1977) Nature 266, 193-194 Hori, H. and Osawa, S. (1979) Proc. Natl. Acad. Sci. USA 76, 381-385

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