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Comparison of eubacterial and eukaryotic 5S RNA structures: a Raman spectroscopic study
Comparison of eubacterial and eukaryotic 5S RNA structures: a Raman spectroscopic study H. Fabian, S. B6hm, R. Misselwitz and H. Weifle Central Institute of Molecular Biology, Academy of Sciences of the GDR, Robert-Strasse 10, 1115 Berlin-Buch, GDR
W. Hiilzer and W. Carius Pedagogic University 'Dr Th. Neubauer', 5010 Erfurt, GDR
and V. V. Filimonov Institute of Protein Research, Academy of Sciences of the USSR, 142 292 Pushchino, USSR (Received 1 April 1987) Raman spectra of eubacterial ribosomal 5S RNAs of Escherichia coli, Bacillus subtilis and Thermus thermophilis and of eukaryotic 5S RNAs of yeast and rat liver have been compared. The spectra show a very high and comparable regularity in the ribophosphate backbone as indicated by the ratio 1.67+__0.03 for Is12/I1 loo in all samples. The 5S RNAs studied have a similar degree of stacking of the G, A and pyrimidine bases. A high percentage of base-paired U residues between 43 and 66 % is indicated. Conformational alterations occurring in 5S RNAs in the presence of Mg 2+ ions between 20 and 50"C are localized mainly in the region of loop II of the molecule. The implications of these results for the 5S RNA structure are discussed. Keywords: Ribosomal 5S RNA; conformation; Raman spectroscopy
Introduction The large subunits of eubacterial and eukaryotic ribosomes contain a small ribosomal RNA designated as 5S RNA. One 5S RNA molecule is a constituent required for protein synthesis in virtually any ribosome. The chain length of 5S RNA is usually close to 120 nucleotides and, at present, the nucleotide sequence is known for the 5S RNAs of about 350 species ~. In 1982, several research groups independently arrived at the conclusion that the 5S RNA secondary structure is universal and contains five helices in all species2-4. This general secondary structure model was derived from a comparative sequence analysis and is a modification of the model proposed by Nishikawa and Takemura in 19745. Such models have to be verified experimentally by biochemical and biophysical methods. Detailed knowledge of the threedimensional structure of RNAs may be derived from Xray diffraction studies on single crystals, such as for tRNAs. However, no 5S RNAs have been obtained up to now in crystalline forms that give suitable diffraction patterns. Thus, the investigation of the structure of 5S RNA by other physical techniques is important. Among these, high-resolution n.m.r, spectroscopy is a promising approach for determining the solution structure of small RNAs. Other powerful techniques are Raman and infrared spectroscopy. Studies on tRNAs have shown that the Raman spectra provide convenient means of identifying differences in conformation between related molecules, or on molecules in different environments 6-~2. 0141-8130/87/060349-08503.00 © 1987 Butterworth & Co. (Publishers) Ltd
Several Raman lines can serve that purpose, giving information on the types and extent of base stacking, on the percentage of U residues in base-paired versus singlestranded regions, and the regularity in the ribophosphate backbone (reviewed, e.g. in Ref. 13). In contrast to numerous measurements on tRNAs, Raman spectra of only one eubacterial 5S RNA1 o,14 and two eukaryotic 5S RNAs 14"-16have been published. The present work extends these earlier studies by a detailed comparative structural analysis of three eubacterial 5S RNAs and two eukaryotic 5S RNAs. Figure I shows the proposed secondary structure models of these 5S RNAs, namely for the eubacterial 5S RNAs of Bacillus subtilis, Escherichia coil and Thermus thermophilus and for the eukaryotic 5S RNAs of yeast and rat liver. The Raman studies of these 5S RNAs were undertaken to test the generalized secondary structure model and to search for significant conformational differences between 5S RNAs of organisms from different kingdoms that may account for the non-exchangeability of eubacterial and eukaryotic 5S RNAs in reconstitution experiments with ribosomal proteins ~7,~8. The results obtained from experiments reported in this communication point to rather high structural similarities between 5S RNAs. Furthermore, the data suggest that conformational alterations in loop II of the molecule are related to the first step of the melting of some higher-order structures of 5S RNAs.
Int. J. Biol. Macromol., 1987, Vol 9, December
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Experimental Sample preparation 5S RNAs were prepared from ribosomes of E. coli 19, B. subtilis, T. thermophilus 2°, yeast (Saccharomyces cerevisiae) and rat liver 16 using standard procedures. Purified 5S RNAs were dialysed against diluted buffers, lyophilized, and dissolved in the appropriate volumes of H 2 0 or D 2 0 , respectively, in order to yield the buffer conditions of 0.004 M MgCI 2 and 0.01 M Na-cacodylate at pH7.0. The purity of 5S RNAs was checked by polyacrylamide gel electrophoresis. Raman scattering Raman spectra excited at 488 nm (200-300 mW of laser power) with an argon-ion laser (ILA-120, VEB Carl Zeiss, Jena) were recorded on a Raman spectrometer consisting of a double monochromator G D M 1000 (VEB Carl Zeiss, Jena), a photomultiplier with $20-
350
Int. J. Biol. Macromol., 1987, Vol 9, December
photocathode and direct current amplification. Sample tubes were glass microcells kept at the indicated temperature by a circulating water bath. A 90 ° geometry was used, with a slit width of 6 cm-1. Scan speed was 0.2cm-~/s at a time constant of 3 s. Spectra of high signal-to-noise ratio were obtained under these conditions without data smoothing. The intensity measurements for all resolved Raman lines are peak heights above background measured with respect to the 1100 cm - x line, since this line is insensitive to conformation and indicates the total concentration of phosphate groups 13. The peak height was determined from a baseline drawn tangent to the spectrum as for tRNAs ~o.
Results and discussion Figure 2 shows the Raman spectra for the 5S RNAs studied in H 2 0 - b u f f e r at 20°C and Table 1 lists the
Raman spectroscopic studies of ribosomal 5S RNAs: H. Fabian et al.
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Figure 2 Raman spectra in the region 500-1750 cm- 1of H20 solutions of 5S RNAs (40-50 #g//~l)in 0.004 M MgClz and 0.01 MNacacodylate at pH 7.0. The sample tamperature is 20_+1°C for all spectra. (A) B. subtilis, (B) E. coil (A-form), (C) T. thermophilus, (D) yeast, (E) rat liver. Abbreviations:A, adenine; U, uracil; G, guanine; C, cytosine;-O PO-, OPO diestergroup; POf, PO~ dioxy group
normalized peak intensities and origins of the various resolved Raman lines. The comparative discussion of the spectra is based mainly on known Raman studies of model compounds and tRNAs. Lines in Raman spectra between approximately 780-880cm-1 generally reflect changes in the ribophosphate backbone conformation9'21'22. Base stacking is monitored by lines between about 600-800 cm - ~ and 1200-1600 cm - ~ (Refs 6-9). Carbonyl bands located roughly between 16001750cm-1 are sensitive primarily to base pairing z3. Backbone conformation It is well established that the ratio of intensities of lines near 812cm -1 and 1100crn -1 (Islz/Illoo) in Raman spectra of ribopolymers indicates the degree of order in the ribophosphat¢ backbone 13. The very stable line near 1100cm-1 (POf symmetric stretching vibration) serves as an internal intensity standard while the line near 812cm -1 (diester OPO stretching vibration) is very sensitive in intensity and position. Highly ordered ribopolymers with uniform backbone geometry exhibit an intense and sharp line near 812cm -1 in the Raman spectrum. In completely disordered structures the Raman intensity at 812cm -1 falls virtually to zero. From the Raman intensity quotient (ls12/IHoo) observed for a natural RNA the percentage of nucleotides in ordered regions is calculated through division by the quotient
Is12/I11oo measured for A-helix structures of uniform geometry22. I s ~2/I 110o = 1.78 _ 0.03 has been measured under our experimental conditions for poly(rI), poly(rC) in Mg 2+containing buffer as a model substance for a completely ordered double-stranded RNA (spectra not shown). A value of 1.67_+0.03 for Is12/I~1oo has been measured in all five 5S RNAs (Table 1). The coincidence of I s 1z / I 11oo in all 5S RNAs studied is important, because it shows that the regularity in the ribophosphate backbone is the same in all samples. Furthermore, 1812/I110o = 1.67 suggests a rather homogeneous backbone geometry in 5S RNA for about 94 ~ of the whole molecule. This number is much higher than an expected value corresponding simply to the double helical regions in 5S RNAs. In 5S RNAs nearly two-thirds of the bases are base paired as demonstrated by different biophysical studies, e.g. Refs 14-16, 19, 20, 24-27. Therefore, the Raman data show that not only nucleotides in base-paired regions, but also the majority of unpaired bases should exist in the same highly specific backbone geometry that gives rise to the 812cm -1 Raman line. The few remaining 5S RNA nucleotides (about 6 ~ ) may exist in random-chain or highly constrained segments of the 5S RNA backbone. The regularity in the sugar-phosphate backbone is approximately 10~ higher than in tRNAs ~4,1s. Deviations from a uniform A-form configuration in
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Raman spectroscopic studies of ribosomal 5S RNAs: H. Fabian et al. Table 1 Frequencies, relative intensities and assignments of some Raman lines of five 5S RNAs Frequency (cm- 1)
Origin of line
669 724 785 812 1100 1237 1250 1300 1320 1337 1375 1484 1575
G A C,U -OPOPO 2 U C,A C,A G A G,A G,A A,G
Composition
A U G C
B. subtilis
E. coli
0.68 0.72 2.32 1.67 1.00 1.44 1.47 1.12 1.34 1.41 0.94 2.16 1.34
0.74 0.67 2.30 1.64 1.00 sh 1.58 1.17 1.51 1.30 0.93 2.30 1.46
25 23 36 32
23 20 41 36
Others
T. thermophilus 0.84 0.64 2.23 1.68 i .00 sh 1.48 1.15 1.55 1.26 0.94 2.57 1.48 23 16 44 38
Yeast 0.61 0.78 2.39 1.69 1.00 1.76 1.65 1.42 1.60 1.97 1.16 2.28 1.50 30 27 33 30 1
Rat liver 0.70 0.61 2.38 1.68 1.00 1.47 1.51 1.13 1.42 1.32 0,95 2.29 1.39 22 27 39 33
Intensities are peak intensities at the indicated frequencyrelative to the PO2 line at 1100cm 1. All values are averages of at least five spectra; each individual normalizedintensity differsby less than + 3 % for intense lines and + 5 % for weak lines, respectively,from the average,sh denotes shoulder tRNAs are found at positions where chain foldings switch abruptly from helical to looped ones and where chains are stretched. These special configurations arise because the molecule is folded into a compact structure 2 s. The smaller deviations from a uniform A-form structure found for 5S RNAs indicate, so far as one can conclude from R a m a n spectroscopy, a sugar-phosphate backbone in 5S RNAs probably less constrained by the spatial folding of the molecules than in tRNAs. In agreement with these data are results obtained for 5S RNA using 31p n.m.r. spectroscopy2 4.
Base stacking The intensities of base specific R a m a n lines in a given spectrum depend on the relative abundances of the bases in the RNA, as well on their degree of stacking. Neglecting different contributions depending on the base stacking, an expected intensity can be calculated (I=lod) for a R a m a n line in the spectrum of one RNA compared to that observed (Iob~) in the spectrum of another RNA if the base composition of both RNAs is known. Icalcd(RNA2)=
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(1) where NB is the number of bases in RNA~ and RNA 2 and NR is the total number of nucleotide residues in each RNA (Ref. 10). Differences between Ical~(RNA2) and Iobsd(RNA2) reflect differences in the degree of base stacking in RNA 1 and RNA 2. In this way the efficiency of base stacking between different RNAs can be compared. Especially the separated lines at 669 c m - ~ and 724 c m - 1 for G and A residues, respectively, and (to some extent) at 785 c m - 1 for pyrimidines (C + U) can be used for this purpose. The effect of stacking on these R a m a n lines, that means the quantitative sensitivity of the spectra to conformational changes, is very high as can be estimated by recording the R a m a n spectra of RNA solutions in
352
Int. J. Biol. Macromol., 1987, Vol 9, December
dependence on temperature. Unfortunately, at temperatures above about 65°C, Mg 2+-containing 5S RNA solutions became turbid and therefore acceptable Raman spectra could not be obtained. In Mg 2+-free solutions, however, R a m a n spectra could be measured even near 80°C. F r o m measurements in Mg 2 +-free solutions it was estimated that, e.g. in Raman spectra of B. subtilis 5S RNA and rat liver 5S RNA (spectra not shown) the intensity of the 669 c m - 1 line decreases by a factor of about 2, while the intensity of the lines at 724 cm - 1 and 785 c m - 1 increases by about 25 and 30~o, respectively, on the transition from the stacked or at least predominantly stacked state (at 20°C) to the unstacked state (near 80°C). These changes cannot simply be transferred quantitatively to measurements in Mg2+-containing buffers. Nevertheless, they show a very pronounced influence of stacking of the G, A and C + U bases on these intensities. Expected intensities of Raman lines have been calculated by equation (1) for all 5S RNAs from the data of Table 1, and the results are given in Table 2. The observed intensities in the Raman spectrum of B. subtilis 5S RNA have been used as a reference for comparison of different 5S RNAs. After correction for the different guanine contents of the 5S RNAs, the 6 6 9 c m - 1 line intensities become equal within experimental uncertainty. The same holds for the 7 2 4 c m - 1 and 785cm -1 line intensities. Since, as discussed above, the effect of stacking of the bases on these intensities is considerable, this coincidence indicates with high sensitivity a comparable degree of G-base stacking and a rather similar overall Abase stacking and pyrimidine-base stacking for the 5S RNAs studied. R a m a n lines above 1200cm-1 cannot be used readily for quantitative comparison of base stacking because they are rather overlapping. However, for 5S RNAs with similar base composition, like for 5S RNAs from B. subtilis and rat liver, the spectral characteristics of the whole R a m a n spectrum should be similar if the structures of the native 5S RNAs are comparable. Really, the
Raman spectroscopic studies of ribosomal 5S RNAs: H. Fabian et al. Table
2 Calculatedand observed relative intensities of G, A and C + U lines Line at 669 cm- 1 (G)
Line at 724 cm- 1 (A)
Line at 785 era(C + U)
5S RNA
Calcd
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Calcd
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Calcd
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B. subtilis E. coil T. thermophilus Yeast Rat liver
(0.68) 0.74 0.80 0.60 0.71
0.68 0.74 0.84 0.61 0.70
(0.72) 0.64 0.64 0.83 0.61
0.72 0.67 0.64 0.78 0.61
(2.32) 2.28 2.18 2.31 2.43
2.32 2.30 2.23 2.39 2.38
similarity of the Raman spectra of B. subtilis and rat fiver 5S RNA is very high (Table 1). Taking into account minor differences of the relative abundances of the bases the intensities for all Raman lines of both 5S RNAs practically coincide. This agreement gives further support for a high structural similarity between B. subtilis and rat fiver 5S RNA. Further information on the structure and dynamics of 5S RNAs has been obtained by comparing the Raman spectra measured at 20°C (Figure 2) and 50°C (Figure 3) in the presence of Mg 2 +-ions. Table 3 lists the relative intensities of some separated lines in the Raman spectra of 5S RNAs measured at 50°C. The Raman data for yeast 5S RNA at 50°C are not included in Table 3 for a quantitative comparison because the signal-to-noise ratio of the available spectra is worse. Qualitatively, yeast 5S RNA has shown the most pronounced spectral changes between 20 and 50°C among the 5S RNAs studied. Interestingly, the intensity of some Raman lines (at 669 cm- 1 and 812 cm- 1) remains nearly constant while the intensity of some other lines (at 725 cm -1 and 785 cm-1) increases when the temperature is raised from 20 to 50°C. The lines at 669cm -1 and 812cm -I are connected with G stacking and conformational transitions in the ribophosphate backbone, respectively. As mentioned before, it is just these lines that show the most pronounced changes in intensity among the Raman lines between 600-1200cm-1 on denaturation of RNAs. Their constancy in the spectra in the premelting temperature range indicates that neither significant unstacking of G bases nor alterations in the ribophosphate backbone occur in 5S RNAs in the presence of Mg 2+-ions between 20 and 50°C. However, intensity changes of the Raman lines at 724 cm-1 and 785 cm- 1 which can be correlated with the unstacking of adenines and pyrimidines, respectively, were observed in the premelting range. The extent of intensity changes differs from species to species. From the data obtained for T. thermophilus 5S RNA and B. subtilis 5S RNA conclusions concerning the localization of structural changes within the molecule during the premelting can be obtained. In T. thermophilus 5S RNA, a pronounced change of the A fine at 724 cm- 1 (+ 15 %) but only a small intensity increase of + 5 % of the C + U line at 785 cm- 1 was observed. In B. subtilis 5S RNA, on the contrary, a small change of + 7 % of the A line at 724 cm-1 but a pronounced change of +13~o of the C + U line was found. Therefore, it can be concluded that the induced conformational change should be localized in pyrimidine rich part(s) of B. subtilis 5S RNA and in adenine rich part(s) of T. thermophilus 5S RNA. Potential candidates for premelting transitions are less
stable loop regions of the molecules. Inspection of the models (Figure 1) reveals that only the base composition of loop II is compatible with the Raman data. Loop II contains four pyrimidines and three adenines in B. subtilis 5S RNA, but only one cytosine and five adenines in T. thermophilus 5S RNA. Furthermore, loop II is poor in guanines. Although minor changes in other parts of the molecules cannot be excluded, it is tempting to correlate temperature induced premelting transitions in 5S RNA with structural changes of loop II. This proposal is derived from the Raman data and the specific nucleotide sequences of B. subtilis and T. thermophilus 5S RNA but can be extended probably also to other 5S RNAs. Recently it was shown by infrared spectroscopy that conformational changes for T. thermophilus 5S RNA in the presence of Mg 2 +-ions between 20 and 60°C cannot be correlated with the melting of canonical base pairs 2°. Striking was the reduction of A stacking in this temperature interval. Similar spectral changes, though less pronounced for the adenine band, have been observed for E. coli 5S RNA 19, B. subtilis 5S RNA and rat liver 5S RNA TM. B. subtilis 5S RNA has shown the smallest unstacking of adenines between 20 and 50°C. These results strongly support the Raman data discussed above.
Base pairing The interaction of polynucleotides to form helical structures results in characteristic changes in the carbonyl region of the vibrational spectrum. Although the interpretation of these changes is complex, e.g. Refs 29 and 30, these spectral changes are used for determination for the base pairing content. In the Raman spectrum of an RNA in D20 solution, the intensity ratio of the bands near 1684 cm- 1 and near 1655 cm- 1 reflects primarily the ratio of paired and unpaired U residues in the RNA. For tRNAs, the Raman data have provided percentages of paired uracils which were in satisfactory agreement with values obtained from the cloverleaf models 8. In the present work this procedure has been applied to 5S RNAs. To compare the different spectra with one another, the peak intensities were determined from a baseline drawn between about 1440 and 1740 c m - t in each spectrum. For unpaired U residues a quotient 11684/11655=0.56 was taken from published spectra23,29. Intensities in the Raman spectrum of yeast 5S RNA (11684//1655 = 1.28) have been used as a second reference. The 5S RNA has approximately two-thirds of its U residues in base paired regions 15. The results are given in Table 4.
Int. J. Biol. Macromol., 1987, Vol 9, December
353
Raman spectroscopic studies o f ribosomal 5S RNAs: H. Fabian et al.
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Table
3
Frequencies and relative intensities of some Raman lines of 5S RNAs at 50°C
B. subtilis
E. coli
T. thermophilus
Rat liver
Frequency (cm -1)
Origin of line
I
AI (%)
I
AI (%)
I
AI (%)
I
AI (%)
669 724 785 812
G A C+ U -OPO-
0.66 0.77 2.62 1.67
- 3 +7 + 13 0
0.77 0.73 2.48 1.66
+4 +9 + 8 + 1
0.80 0.74 2.34 1.64
- 5 + 15 +5 - 2
0.69 0.68 2.59 1.69
- 1 + 12 + 10 + 1
Al indicates the change in intensity over the range 50-20°C in each of these lines in percent of the 20°C value
354
I n t . J. Biol. M a c r o m o l . , 1987, V o l 9, D e c e m b e r
Raman spectroscopic studies of ribosomal 5S RNAs: H. Fabian et al.
Table 4 U residues in base-paired regions of 5S RNAs
11684//1655 Base-paired U residues (%) Number of paired U residues Number of A.U base pairsa Number of G.U base pairsb Model Figure 1)
B. subtilis
E. coil
T. thermophilus
Yeast
Rat liver
1.15 55 13 10 3 8 A.U 5 G-U
1.22 60 12 9 3 7 A.U 6 G'U
1.03 43 7 4 3 4 A.U 6 G'U
1.28 66 18 13 5 13 A.U 4 G'U
1.20 58 16 9 7 6 A.U 5 G'U
°Number of A'U pairs estimatedby infraredspectroscopyaccordingto Refs 14, 20, 26, and unpublisheddata bNumber of G'U pairs calculatedby subtractionof the numberof A-U pairs fromthe total numberof paired U residues
I
~
1700 L 1101 FREQUENCY (¢m-1} Figure 4 Raman spectra in the region 14(~--1750cm-~ of D20-buffer solutions of 5S RNAs (40-50/zg/#l) at experimental conditions indicated in the legend of Figure 2. (A) B. subtilis, (B) E. coil, (C) T. thermophilus, (D) yeast, (E) rat liver
agreement between our data for B. subtilis 5S RNA and E. coli 5S RNA (3 G.U pairs in both molecules) and the number of G.U pairs (3 G.U pairs in B. subtilis 5S RNA 27 and at least 3 G'U pairs in E. coli 5S RNA 31'a2) identified by n.m.r, spectroscopy very recently. Therefore, the combination of infrared and Raman spectroscopy offers a possibility for the rough estimation of G.U pairs in RNAs. The total number of paired U residues estimated by Raman spectroscopy agrees more or less with the number of base-paired U residues found in the secondary structure models. An exception seems to be rat liver 5S RNA. This could be explained by the existence of U.U mispairs which have been suggested as evolutionary alternatives to Watson42rick or G.U pairs in 5S RNAs a3. In two positions U bases are juxtaposed (U73-U102 and Uso-U96 ) in the model (Figure 1) as potential candidates for U.U pairs in rat liver 5S RNA. Recently small but significant differences between the infrared spectra of E. coli 5S RNA and rat liver 5S RNA were found ~4 which may be correlated with U bases, possibly with the presence of U'U pairs in rat liver 5S RNA at low temperatures. Experiments are in preparation to provide a quantitative proof of this suggestion and, therefore, the results for rat liver 5S RNA summarized in Table 4 should be considered as preliminary.
Conclusions The data indicate a high percentage of base-paired U residues between 43 and 66 %. A slight decrease in the order yeast 5S RNA>E. coil 5S RNA,~rat liver 5S RNA > B. subtilis 5S RNA > T. thermophilus 5S RNA is observed. The number of base-paired U residues estimated by Raman spectroscopy is larger than the number of U residues in A.U base pairs as estimated by infrared spectroscopy. This indicates the presence of U residues in non-Watson--Crick base pairs (e.g.G.U pairs) in 5S RNAs. We have calculated the number of G.U pairs as the difference of the total number of base-paired U residues determined from the Raman spectra and the number of A.U pairs estimated previously by infrared spectroscopy. The data are given in Figure 4. The G.U estimate is likely to be highly imprecise, since the procedure is based firstly on the assumption of comparable spectral characteristics of Raman spectra for U residues in A.U pairs and G.U pairs, and secondly the number of A.U pairs determined by infrared spectroscopy also involves relatively large errors 2°'26. This approach to estimate G.U pairs is supported, however, by the close
The spectroscopic results reported in this paper corroborate the conclusion that eubacterial and eukaryotic 5S RNAs are very similar with respect to their fundamental structural features. This high similarity concerning base stacking in single-stranded and basepaired regions and the comparable backbone geometry in the 5S RNAs studied is valid for the shape parameters as well. Very recently it was shown by X-ray scattering that the shape and dimensions of E. coli 5S RNA and rat liver 5S RNA are very similar too 34. Nevertheless, differences evolved between eubacterial and eukaryotic 5S RNAs render them non-exchangeable in reconstitution experiments with ribosomal proteins 17'1s. This could be due to subtle site or sequence specific structural differences which cannot be identified by Raman spectroscopy. Comparison of Raman spectra of 5S RNAs with different primary sequences and measured at different temperatures can aid in identifying changes in local parts of the molecule. The experiments carried out for B. subtilis 5S RNA, E. coli 5S RNA, T. thermophilus 5S RNA
Int. J. Biol. Macromol., 1987, Vol 9, December
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Raman spectroscopic studies of ribosomal 5S RNAs: H. Fabian et al. and rat liver 5S RNA suggest conformational alterations mainly in loop II during the first melting steps of some higher-order structure of 5S RNAs between 20 and 50°C. Despite manifold approaches, the higher-order structure of 5S RNAs remains unresolved until now. A fiat shape with a compact central region and two protruding arms was derived from X-ray scattering experiments 35, while rather elongated Y-shape structures are disproved by these data 36. The compacted model seems to require tertiary interactions for stabilization of the complicated folding of the nucleotide chain a7. Several tertiary interactions have been suggested in the last few years 2'3s 40. Although these structures differ significantly in detail and final proof for any of them is lacking, all models are characterized by a kinking of the nucleotide chain in the internal loop II. N.m.r. spectroscopic evidence for a dynamic secondary structure in the loop II region of a 5S RNA fragment of E. coli was described very recently 31 . Structural changes in loop II should occur before or be connected with the melting of tertiary interactions suggested and with the unfolding of the molecule. These suggestions are supported by the Raman data discussed before.
Acknowledgement We thank Mrs B. Kannen for skilful technical assistance.
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 28a 29 30
References 1 2 3 4 5 6 7 8 9 l0 11
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31 32 33 34 35 36 37 38 39 40
Giege, R., Huong, P. V. and Moras, D. Spectrochim. Aeta 1986, 42A, 387 Thomas, G. J. and Kyogoku, Y. in "Infrared and Raman Spectroscopy in Biological Science', (Eds E. G. Brame and J. G. Graselli), Dekker, New York, 1977, 1C, p. 775 Fabian, H., BiShm, S., Filimonov, V. V., Venyaminov, S. Y., Carius, W., HiSlzer, W., Misselwitz, R. and Welfle, H. studia biophysica 1985, 108, 187 Luoma, G. A. and Marshall, A. G. J. Mol. Biol. 1978, 125, 95 Fabian, H., BiShm, S., Welfle, H., Reich, P. and Bielka, H. FEBS Lett. 1981, 123, 19 Wrede, P. and Erdmann, V. A. FEBS Lett. 1973, 33, 315 Nazar, R. N. and Wildeman, A. G. Nucleic. Acids Res. 1983, 11, 3155 BiAhm,S., Fabian, H., Venyaminov, S. Y., Matveev, S. V., Lucius, H., Welfle, H. and Filimonov, V. V. FEBS Lett. 1981, 132, 357 B6hm, S., Venyaminov, S. Y., Fabian, H., Filimonov, V. V. and Welfle, H. Eur. J. Biochem. 1985, 147, 503 Brown, K. G., Kiser, E. J. and Peticolas, W. L. Biopolymers 1972, 11, 1855 Thomas, G. J. and Hartmann, K. A. Biochim. Biophys. Acta 1973, 312, 311 Lafleur, L., Rice, J. and Thomas, G. J. Biopolymers 1972, 11, 2423 Salemik, P. J. M., Raue, H. A., Heerschap, A., Planta, R. J. and Hilbers, C. W. Biochemistry 1981, 20, 265 Kime, M. J. and Moore, P. B. FEBS Lett. 1983, 153, 199 Burkey, K. O., Marshall, A. G. and Alben, J. O. Biochemistry 1983, 22, 4223 Chang, L.-H. and Marshall, A. G. Biochemistry 1986, 25, 3056 Saenger, W. in 'Principles of Nucleic Acid Structure', (Ed. C. R. Cantor), Springer-Verlag, Berlin, 1984, p. 338 Fabian, H. et al., in preparation Morikawa, K., Tsuboi, M., Takehashi, S., Kyogoku, Y., Mitsui, Y., Iltaka, Y. and Thomas, G. J. Biopolymers 1973, 12, 799 Tsuboi, M. and Nishimura, Y. in 'Raman Spectroscopy Linear and Nonlinear', (Eds J. Lascombe and P. V. Huong), John Wiley, Chichester, 1982, p. 683 Leontis, N. B. and Moore, P. B. Biochemistry 1986, 25, 3916 Leontis, N. B., Ghosh, P. and Moore, P. G. Biochemistry 1986, 25, 7386 Walker, W. F. and Doolittle, W. F. Nucleic Acids Res. 1983, I 1, 5159 Miiller, J. J., Zalkova, T. N., Zirwer, D., Misselwitz, R., Gast, K., Serdyuk, I. N., Welfle, H. and Damaschun, G. Eur. Biophys. J. 1986, 13, 301 Miiller, J. J., Damaschun, G., Wilhelm, P., Welfle, H. and Pilz, I. Int. J. Biol. Macromol. 1982, 4, 289 Welfle, H., BiShm,S., Damaschun, G., Fabian, H., Filimonov, V. V., Gast, K., Misselwitz, R., Miiller, J. J., Venyaminov, S. Y., Zalkova, T. N. and Zirwer, D. studia biophysica 1986, 114, 103 Miiller, J. J., Damaschun, G., BiShm, S., Fabian, H. and Welfle, H. studia biophysica 1982, 87, 11 Pieler, T. and Erdmann, V. A. Proc. Natl. Acad. Sci., USA 1982, 79, 4599 Hancock, J. and Wagner, R. Nucleic Acids Res. 1982, 10, 1257 Gbringer, H. U. and Wagner, R. Nucleic Acids Res. 1986, 14, 7473
W. Hiilzer and W. Carius Pedagogic University 'Dr Th. Neubauer', 5010 Erfurt, GDR
and V. V. Filimonov Institute of Protein Research, Academy of Sciences of the USSR, 142 292 Pushchino, USSR (Received 1 April 1987) Raman spectra of eubacterial ribosomal 5S RNAs of Escherichia coli, Bacillus subtilis and Thermus thermophilis and of eukaryotic 5S RNAs of yeast and rat liver have been compared. The spectra show a very high and comparable regularity in the ribophosphate backbone as indicated by the ratio 1.67+__0.03 for Is12/I1 loo in all samples. The 5S RNAs studied have a similar degree of stacking of the G, A and pyrimidine bases. A high percentage of base-paired U residues between 43 and 66 % is indicated. Conformational alterations occurring in 5S RNAs in the presence of Mg 2+ ions between 20 and 50"C are localized mainly in the region of loop II of the molecule. The implications of these results for the 5S RNA structure are discussed. Keywords: Ribosomal 5S RNA; conformation; Raman spectroscopy
Introduction The large subunits of eubacterial and eukaryotic ribosomes contain a small ribosomal RNA designated as 5S RNA. One 5S RNA molecule is a constituent required for protein synthesis in virtually any ribosome. The chain length of 5S RNA is usually close to 120 nucleotides and, at present, the nucleotide sequence is known for the 5S RNAs of about 350 species ~. In 1982, several research groups independently arrived at the conclusion that the 5S RNA secondary structure is universal and contains five helices in all species2-4. This general secondary structure model was derived from a comparative sequence analysis and is a modification of the model proposed by Nishikawa and Takemura in 19745. Such models have to be verified experimentally by biochemical and biophysical methods. Detailed knowledge of the threedimensional structure of RNAs may be derived from Xray diffraction studies on single crystals, such as for tRNAs. However, no 5S RNAs have been obtained up to now in crystalline forms that give suitable diffraction patterns. Thus, the investigation of the structure of 5S RNA by other physical techniques is important. Among these, high-resolution n.m.r, spectroscopy is a promising approach for determining the solution structure of small RNAs. Other powerful techniques are Raman and infrared spectroscopy. Studies on tRNAs have shown that the Raman spectra provide convenient means of identifying differences in conformation between related molecules, or on molecules in different environments 6-~2. 0141-8130/87/060349-08503.00 © 1987 Butterworth & Co. (Publishers) Ltd
Several Raman lines can serve that purpose, giving information on the types and extent of base stacking, on the percentage of U residues in base-paired versus singlestranded regions, and the regularity in the ribophosphate backbone (reviewed, e.g. in Ref. 13). In contrast to numerous measurements on tRNAs, Raman spectra of only one eubacterial 5S RNA1 o,14 and two eukaryotic 5S RNAs 14"-16have been published. The present work extends these earlier studies by a detailed comparative structural analysis of three eubacterial 5S RNAs and two eukaryotic 5S RNAs. Figure I shows the proposed secondary structure models of these 5S RNAs, namely for the eubacterial 5S RNAs of Bacillus subtilis, Escherichia coil and Thermus thermophilus and for the eukaryotic 5S RNAs of yeast and rat liver. The Raman studies of these 5S RNAs were undertaken to test the generalized secondary structure model and to search for significant conformational differences between 5S RNAs of organisms from different kingdoms that may account for the non-exchangeability of eubacterial and eukaryotic 5S RNAs in reconstitution experiments with ribosomal proteins ~7,~8. The results obtained from experiments reported in this communication point to rather high structural similarities between 5S RNAs. Furthermore, the data suggest that conformational alterations in loop II of the molecule are related to the first step of the melting of some higher-order structures of 5S RNAs.
Int. J. Biol. Macromol., 1987, Vol 9, December
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Experimental Sample preparation 5S RNAs were prepared from ribosomes of E. coli 19, B. subtilis, T. thermophilus 2°, yeast (Saccharomyces cerevisiae) and rat liver 16 using standard procedures. Purified 5S RNAs were dialysed against diluted buffers, lyophilized, and dissolved in the appropriate volumes of H 2 0 or D 2 0 , respectively, in order to yield the buffer conditions of 0.004 M MgCI 2 and 0.01 M Na-cacodylate at pH7.0. The purity of 5S RNAs was checked by polyacrylamide gel electrophoresis. Raman scattering Raman spectra excited at 488 nm (200-300 mW of laser power) with an argon-ion laser (ILA-120, VEB Carl Zeiss, Jena) were recorded on a Raman spectrometer consisting of a double monochromator G D M 1000 (VEB Carl Zeiss, Jena), a photomultiplier with $20-
350
Int. J. Biol. Macromol., 1987, Vol 9, December
photocathode and direct current amplification. Sample tubes were glass microcells kept at the indicated temperature by a circulating water bath. A 90 ° geometry was used, with a slit width of 6 cm-1. Scan speed was 0.2cm-~/s at a time constant of 3 s. Spectra of high signal-to-noise ratio were obtained under these conditions without data smoothing. The intensity measurements for all resolved Raman lines are peak heights above background measured with respect to the 1100 cm - x line, since this line is insensitive to conformation and indicates the total concentration of phosphate groups 13. The peak height was determined from a baseline drawn tangent to the spectrum as for tRNAs ~o.
Results and discussion Figure 2 shows the Raman spectra for the 5S RNAs studied in H 2 0 - b u f f e r at 20°C and Table 1 lists the
Raman spectroscopic studies of ribosomal 5S RNAs: H. Fabian et al.
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normalized peak intensities and origins of the various resolved Raman lines. The comparative discussion of the spectra is based mainly on known Raman studies of model compounds and tRNAs. Lines in Raman spectra between approximately 780-880cm-1 generally reflect changes in the ribophosphate backbone conformation9'21'22. Base stacking is monitored by lines between about 600-800 cm - ~ and 1200-1600 cm - ~ (Refs 6-9). Carbonyl bands located roughly between 16001750cm-1 are sensitive primarily to base pairing z3. Backbone conformation It is well established that the ratio of intensities of lines near 812cm -1 and 1100crn -1 (Islz/Illoo) in Raman spectra of ribopolymers indicates the degree of order in the ribophosphat¢ backbone 13. The very stable line near 1100cm-1 (POf symmetric stretching vibration) serves as an internal intensity standard while the line near 812cm -1 (diester OPO stretching vibration) is very sensitive in intensity and position. Highly ordered ribopolymers with uniform backbone geometry exhibit an intense and sharp line near 812cm -1 in the Raman spectrum. In completely disordered structures the Raman intensity at 812cm -1 falls virtually to zero. From the Raman intensity quotient (ls12/IHoo) observed for a natural RNA the percentage of nucleotides in ordered regions is calculated through division by the quotient
Is12/I11oo measured for A-helix structures of uniform geometry22. I s ~2/I 110o = 1.78 _ 0.03 has been measured under our experimental conditions for poly(rI), poly(rC) in Mg 2+containing buffer as a model substance for a completely ordered double-stranded RNA (spectra not shown). A value of 1.67_+0.03 for Is12/I~1oo has been measured in all five 5S RNAs (Table 1). The coincidence of I s 1z / I 11oo in all 5S RNAs studied is important, because it shows that the regularity in the ribophosphate backbone is the same in all samples. Furthermore, 1812/I110o = 1.67 suggests a rather homogeneous backbone geometry in 5S RNA for about 94 ~ of the whole molecule. This number is much higher than an expected value corresponding simply to the double helical regions in 5S RNAs. In 5S RNAs nearly two-thirds of the bases are base paired as demonstrated by different biophysical studies, e.g. Refs 14-16, 19, 20, 24-27. Therefore, the Raman data show that not only nucleotides in base-paired regions, but also the majority of unpaired bases should exist in the same highly specific backbone geometry that gives rise to the 812cm -1 Raman line. The few remaining 5S RNA nucleotides (about 6 ~ ) may exist in random-chain or highly constrained segments of the 5S RNA backbone. The regularity in the sugar-phosphate backbone is approximately 10~ higher than in tRNAs ~4,1s. Deviations from a uniform A-form configuration in
Int. J. Biol. Macromol., 1987, Vol 9, December
351
Raman spectroscopic studies of ribosomal 5S RNAs: H. Fabian et al. Table 1 Frequencies, relative intensities and assignments of some Raman lines of five 5S RNAs Frequency (cm- 1)
Origin of line
669 724 785 812 1100 1237 1250 1300 1320 1337 1375 1484 1575
G A C,U -OPOPO 2 U C,A C,A G A G,A G,A A,G
Composition
A U G C
B. subtilis
E. coli
0.68 0.72 2.32 1.67 1.00 1.44 1.47 1.12 1.34 1.41 0.94 2.16 1.34
0.74 0.67 2.30 1.64 1.00 sh 1.58 1.17 1.51 1.30 0.93 2.30 1.46
25 23 36 32
23 20 41 36
Others
T. thermophilus 0.84 0.64 2.23 1.68 i .00 sh 1.48 1.15 1.55 1.26 0.94 2.57 1.48 23 16 44 38
Yeast 0.61 0.78 2.39 1.69 1.00 1.76 1.65 1.42 1.60 1.97 1.16 2.28 1.50 30 27 33 30 1
Rat liver 0.70 0.61 2.38 1.68 1.00 1.47 1.51 1.13 1.42 1.32 0,95 2.29 1.39 22 27 39 33
Intensities are peak intensities at the indicated frequencyrelative to the PO2 line at 1100cm 1. All values are averages of at least five spectra; each individual normalizedintensity differsby less than + 3 % for intense lines and + 5 % for weak lines, respectively,from the average,sh denotes shoulder tRNAs are found at positions where chain foldings switch abruptly from helical to looped ones and where chains are stretched. These special configurations arise because the molecule is folded into a compact structure 2 s. The smaller deviations from a uniform A-form structure found for 5S RNAs indicate, so far as one can conclude from R a m a n spectroscopy, a sugar-phosphate backbone in 5S RNAs probably less constrained by the spatial folding of the molecules than in tRNAs. In agreement with these data are results obtained for 5S RNA using 31p n.m.r. spectroscopy2 4.
Base stacking The intensities of base specific R a m a n lines in a given spectrum depend on the relative abundances of the bases in the RNA, as well on their degree of stacking. Neglecting different contributions depending on the base stacking, an expected intensity can be calculated (I=lod) for a R a m a n line in the spectrum of one RNA compared to that observed (Iob~) in the spectrum of another RNA if the base composition of both RNAs is known. Icalcd(RNA2)=
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1)
NR(RNA,)] NR(RNA2)]
(1) where NB is the number of bases in RNA~ and RNA 2 and NR is the total number of nucleotide residues in each RNA (Ref. 10). Differences between Ical~(RNA2) and Iobsd(RNA2) reflect differences in the degree of base stacking in RNA 1 and RNA 2. In this way the efficiency of base stacking between different RNAs can be compared. Especially the separated lines at 669 c m - ~ and 724 c m - 1 for G and A residues, respectively, and (to some extent) at 785 c m - 1 for pyrimidines (C + U) can be used for this purpose. The effect of stacking on these R a m a n lines, that means the quantitative sensitivity of the spectra to conformational changes, is very high as can be estimated by recording the R a m a n spectra of RNA solutions in
352
Int. J. Biol. Macromol., 1987, Vol 9, December
dependence on temperature. Unfortunately, at temperatures above about 65°C, Mg 2+-containing 5S RNA solutions became turbid and therefore acceptable Raman spectra could not be obtained. In Mg 2+-free solutions, however, R a m a n spectra could be measured even near 80°C. F r o m measurements in Mg 2 +-free solutions it was estimated that, e.g. in Raman spectra of B. subtilis 5S RNA and rat liver 5S RNA (spectra not shown) the intensity of the 669 c m - 1 line decreases by a factor of about 2, while the intensity of the lines at 724 cm - 1 and 785 c m - 1 increases by about 25 and 30~o, respectively, on the transition from the stacked or at least predominantly stacked state (at 20°C) to the unstacked state (near 80°C). These changes cannot simply be transferred quantitatively to measurements in Mg2+-containing buffers. Nevertheless, they show a very pronounced influence of stacking of the G, A and C + U bases on these intensities. Expected intensities of Raman lines have been calculated by equation (1) for all 5S RNAs from the data of Table 1, and the results are given in Table 2. The observed intensities in the Raman spectrum of B. subtilis 5S RNA have been used as a reference for comparison of different 5S RNAs. After correction for the different guanine contents of the 5S RNAs, the 6 6 9 c m - 1 line intensities become equal within experimental uncertainty. The same holds for the 7 2 4 c m - 1 and 785cm -1 line intensities. Since, as discussed above, the effect of stacking of the bases on these intensities is considerable, this coincidence indicates with high sensitivity a comparable degree of G-base stacking and a rather similar overall Abase stacking and pyrimidine-base stacking for the 5S RNAs studied. R a m a n lines above 1200cm-1 cannot be used readily for quantitative comparison of base stacking because they are rather overlapping. However, for 5S RNAs with similar base composition, like for 5S RNAs from B. subtilis and rat liver, the spectral characteristics of the whole R a m a n spectrum should be similar if the structures of the native 5S RNAs are comparable. Really, the
Raman spectroscopic studies of ribosomal 5S RNAs: H. Fabian et al. Table
2 Calculatedand observed relative intensities of G, A and C + U lines Line at 669 cm- 1 (G)
Line at 724 cm- 1 (A)
Line at 785 era(C + U)
5S RNA
Calcd
Obsd
Calcd
Obsd
Calcd
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B. subtilis E. coil T. thermophilus Yeast Rat liver
(0.68) 0.74 0.80 0.60 0.71
0.68 0.74 0.84 0.61 0.70
(0.72) 0.64 0.64 0.83 0.61
0.72 0.67 0.64 0.78 0.61
(2.32) 2.28 2.18 2.31 2.43
2.32 2.30 2.23 2.39 2.38
similarity of the Raman spectra of B. subtilis and rat fiver 5S RNA is very high (Table 1). Taking into account minor differences of the relative abundances of the bases the intensities for all Raman lines of both 5S RNAs practically coincide. This agreement gives further support for a high structural similarity between B. subtilis and rat fiver 5S RNA. Further information on the structure and dynamics of 5S RNAs has been obtained by comparing the Raman spectra measured at 20°C (Figure 2) and 50°C (Figure 3) in the presence of Mg 2 +-ions. Table 3 lists the relative intensities of some separated lines in the Raman spectra of 5S RNAs measured at 50°C. The Raman data for yeast 5S RNA at 50°C are not included in Table 3 for a quantitative comparison because the signal-to-noise ratio of the available spectra is worse. Qualitatively, yeast 5S RNA has shown the most pronounced spectral changes between 20 and 50°C among the 5S RNAs studied. Interestingly, the intensity of some Raman lines (at 669 cm- 1 and 812 cm- 1) remains nearly constant while the intensity of some other lines (at 725 cm -1 and 785 cm-1) increases when the temperature is raised from 20 to 50°C. The lines at 669cm -1 and 812cm -I are connected with G stacking and conformational transitions in the ribophosphate backbone, respectively. As mentioned before, it is just these lines that show the most pronounced changes in intensity among the Raman lines between 600-1200cm-1 on denaturation of RNAs. Their constancy in the spectra in the premelting temperature range indicates that neither significant unstacking of G bases nor alterations in the ribophosphate backbone occur in 5S RNAs in the presence of Mg 2+-ions between 20 and 50°C. However, intensity changes of the Raman lines at 724 cm-1 and 785 cm- 1 which can be correlated with the unstacking of adenines and pyrimidines, respectively, were observed in the premelting range. The extent of intensity changes differs from species to species. From the data obtained for T. thermophilus 5S RNA and B. subtilis 5S RNA conclusions concerning the localization of structural changes within the molecule during the premelting can be obtained. In T. thermophilus 5S RNA, a pronounced change of the A fine at 724 cm- 1 (+ 15 %) but only a small intensity increase of + 5 % of the C + U line at 785 cm- 1 was observed. In B. subtilis 5S RNA, on the contrary, a small change of + 7 % of the A line at 724 cm-1 but a pronounced change of +13~o of the C + U line was found. Therefore, it can be concluded that the induced conformational change should be localized in pyrimidine rich part(s) of B. subtilis 5S RNA and in adenine rich part(s) of T. thermophilus 5S RNA. Potential candidates for premelting transitions are less
stable loop regions of the molecules. Inspection of the models (Figure 1) reveals that only the base composition of loop II is compatible with the Raman data. Loop II contains four pyrimidines and three adenines in B. subtilis 5S RNA, but only one cytosine and five adenines in T. thermophilus 5S RNA. Furthermore, loop II is poor in guanines. Although minor changes in other parts of the molecules cannot be excluded, it is tempting to correlate temperature induced premelting transitions in 5S RNA with structural changes of loop II. This proposal is derived from the Raman data and the specific nucleotide sequences of B. subtilis and T. thermophilus 5S RNA but can be extended probably also to other 5S RNAs. Recently it was shown by infrared spectroscopy that conformational changes for T. thermophilus 5S RNA in the presence of Mg 2 +-ions between 20 and 60°C cannot be correlated with the melting of canonical base pairs 2°. Striking was the reduction of A stacking in this temperature interval. Similar spectral changes, though less pronounced for the adenine band, have been observed for E. coli 5S RNA 19, B. subtilis 5S RNA and rat liver 5S RNA TM. B. subtilis 5S RNA has shown the smallest unstacking of adenines between 20 and 50°C. These results strongly support the Raman data discussed above.
Base pairing The interaction of polynucleotides to form helical structures results in characteristic changes in the carbonyl region of the vibrational spectrum. Although the interpretation of these changes is complex, e.g. Refs 29 and 30, these spectral changes are used for determination for the base pairing content. In the Raman spectrum of an RNA in D20 solution, the intensity ratio of the bands near 1684 cm- 1 and near 1655 cm- 1 reflects primarily the ratio of paired and unpaired U residues in the RNA. For tRNAs, the Raman data have provided percentages of paired uracils which were in satisfactory agreement with values obtained from the cloverleaf models 8. In the present work this procedure has been applied to 5S RNAs. To compare the different spectra with one another, the peak intensities were determined from a baseline drawn between about 1440 and 1740 c m - t in each spectrum. For unpaired U residues a quotient 11684/11655=0.56 was taken from published spectra23,29. Intensities in the Raman spectrum of yeast 5S RNA (11684//1655 = 1.28) have been used as a second reference. The 5S RNA has approximately two-thirds of its U residues in base paired regions 15. The results are given in Table 4.
Int. J. Biol. Macromol., 1987, Vol 9, December
353
Raman spectroscopic studies o f ribosomal 5S RNAs: H. Fabian et al.
z z
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Table
3
Frequencies and relative intensities of some Raman lines of 5S RNAs at 50°C
B. subtilis
E. coli
T. thermophilus
Rat liver
Frequency (cm -1)
Origin of line
I
AI (%)
I
AI (%)
I
AI (%)
I
AI (%)
669 724 785 812
G A C+ U -OPO-
0.66 0.77 2.62 1.67
- 3 +7 + 13 0
0.77 0.73 2.48 1.66
+4 +9 + 8 + 1
0.80 0.74 2.34 1.64
- 5 + 15 +5 - 2
0.69 0.68 2.59 1.69
- 1 + 12 + 10 + 1
Al indicates the change in intensity over the range 50-20°C in each of these lines in percent of the 20°C value
354
I n t . J. Biol. M a c r o m o l . , 1987, V o l 9, D e c e m b e r
Raman spectroscopic studies of ribosomal 5S RNAs: H. Fabian et al.
Table 4 U residues in base-paired regions of 5S RNAs
11684//1655 Base-paired U residues (%) Number of paired U residues Number of A.U base pairsa Number of G.U base pairsb Model Figure 1)
B. subtilis
E. coil
T. thermophilus
Yeast
Rat liver
1.15 55 13 10 3 8 A.U 5 G-U
1.22 60 12 9 3 7 A.U 6 G'U
1.03 43 7 4 3 4 A.U 6 G'U
1.28 66 18 13 5 13 A.U 4 G'U
1.20 58 16 9 7 6 A.U 5 G'U
°Number of A'U pairs estimatedby infraredspectroscopyaccordingto Refs 14, 20, 26, and unpublisheddata bNumber of G'U pairs calculatedby subtractionof the numberof A-U pairs fromthe total numberof paired U residues
I
~
1700 L 1101 FREQUENCY (¢m-1} Figure 4 Raman spectra in the region 14(~--1750cm-~ of D20-buffer solutions of 5S RNAs (40-50/zg/#l) at experimental conditions indicated in the legend of Figure 2. (A) B. subtilis, (B) E. coil, (C) T. thermophilus, (D) yeast, (E) rat liver
agreement between our data for B. subtilis 5S RNA and E. coli 5S RNA (3 G.U pairs in both molecules) and the number of G.U pairs (3 G.U pairs in B. subtilis 5S RNA 27 and at least 3 G'U pairs in E. coli 5S RNA 31'a2) identified by n.m.r, spectroscopy very recently. Therefore, the combination of infrared and Raman spectroscopy offers a possibility for the rough estimation of G.U pairs in RNAs. The total number of paired U residues estimated by Raman spectroscopy agrees more or less with the number of base-paired U residues found in the secondary structure models. An exception seems to be rat liver 5S RNA. This could be explained by the existence of U.U mispairs which have been suggested as evolutionary alternatives to Watson42rick or G.U pairs in 5S RNAs a3. In two positions U bases are juxtaposed (U73-U102 and Uso-U96 ) in the model (Figure 1) as potential candidates for U.U pairs in rat liver 5S RNA. Recently small but significant differences between the infrared spectra of E. coli 5S RNA and rat liver 5S RNA were found ~4 which may be correlated with U bases, possibly with the presence of U'U pairs in rat liver 5S RNA at low temperatures. Experiments are in preparation to provide a quantitative proof of this suggestion and, therefore, the results for rat liver 5S RNA summarized in Table 4 should be considered as preliminary.
Conclusions The data indicate a high percentage of base-paired U residues between 43 and 66 %. A slight decrease in the order yeast 5S RNA>E. coil 5S RNA,~rat liver 5S RNA > B. subtilis 5S RNA > T. thermophilus 5S RNA is observed. The number of base-paired U residues estimated by Raman spectroscopy is larger than the number of U residues in A.U base pairs as estimated by infrared spectroscopy. This indicates the presence of U residues in non-Watson--Crick base pairs (e.g.G.U pairs) in 5S RNAs. We have calculated the number of G.U pairs as the difference of the total number of base-paired U residues determined from the Raman spectra and the number of A.U pairs estimated previously by infrared spectroscopy. The data are given in Figure 4. The G.U estimate is likely to be highly imprecise, since the procedure is based firstly on the assumption of comparable spectral characteristics of Raman spectra for U residues in A.U pairs and G.U pairs, and secondly the number of A.U pairs determined by infrared spectroscopy also involves relatively large errors 2°'26. This approach to estimate G.U pairs is supported, however, by the close
The spectroscopic results reported in this paper corroborate the conclusion that eubacterial and eukaryotic 5S RNAs are very similar with respect to their fundamental structural features. This high similarity concerning base stacking in single-stranded and basepaired regions and the comparable backbone geometry in the 5S RNAs studied is valid for the shape parameters as well. Very recently it was shown by X-ray scattering that the shape and dimensions of E. coli 5S RNA and rat liver 5S RNA are very similar too 34. Nevertheless, differences evolved between eubacterial and eukaryotic 5S RNAs render them non-exchangeable in reconstitution experiments with ribosomal proteins 17'1s. This could be due to subtle site or sequence specific structural differences which cannot be identified by Raman spectroscopy. Comparison of Raman spectra of 5S RNAs with different primary sequences and measured at different temperatures can aid in identifying changes in local parts of the molecule. The experiments carried out for B. subtilis 5S RNA, E. coli 5S RNA, T. thermophilus 5S RNA
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Raman spectroscopic studies of ribosomal 5S RNAs: H. Fabian et al. and rat liver 5S RNA suggest conformational alterations mainly in loop II during the first melting steps of some higher-order structure of 5S RNAs between 20 and 50°C. Despite manifold approaches, the higher-order structure of 5S RNAs remains unresolved until now. A fiat shape with a compact central region and two protruding arms was derived from X-ray scattering experiments 35, while rather elongated Y-shape structures are disproved by these data 36. The compacted model seems to require tertiary interactions for stabilization of the complicated folding of the nucleotide chain a7. Several tertiary interactions have been suggested in the last few years 2'3s 40. Although these structures differ significantly in detail and final proof for any of them is lacking, all models are characterized by a kinking of the nucleotide chain in the internal loop II. N.m.r. spectroscopic evidence for a dynamic secondary structure in the loop II region of a 5S RNA fragment of E. coli was described very recently 31 . Structural changes in loop II should occur before or be connected with the melting of tertiary interactions suggested and with the unfolding of the molecule. These suggestions are supported by the Raman data discussed before.
Acknowledgement We thank Mrs B. Kannen for skilful technical assistance.
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