Proc. Natl. Acad. Sci. USA
Vol. 75, No. 10, pp. 4901-4905, October 1978 Biophysics
Laser Raman evidence for new cloverleaf secondary structures for eukaryotic 5.8S RNA and prokaryotic 5S RNA (tRNA binding/Mg2+-dependence)
GREG A. LUOMA* AND ALAN G. MARSHALL* Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1W5, Canada
Communicated by Alexander Rich, June 27, 1978
ABSTRACT Neither of the two previously proposed secondary structures for eukaryotic 5.8S RNA is consistent with the present laser Raman results. A new, highly stable "cloverleaf" secondary structure not only fits the Raman data but also accounts for previously determined enzymatic partial cleavage patterns, base sequence and pairing homologies, and GC and A'U base pair numbers and ratios. The new cloverleaf model also conserves several structural features (constant loops, bulges, and stems) consistent with known 5.8S RNA functions. Finally, we propose a similar new cloverleaf secondary structure for Escherichia coli 5S RNA, consonant with many known properties of prokaryotic 5S RNA.
secondary and tertiary structures of RNA molecules in solution (13, 14), particularly for determining the types and extent of base stacking, the percentage of U residues in base-paired versus single-stranded regions, and the degree of order (rigidity) at the backbone phosphate linkages (13-16). Raman spectra can also reflect changes in RNA secondary or tertiary conformation (17). Because two markedly different yeast 5.8S RNA secondary structures have been proposed, Raman studies are expected to provide critical tests for these structures. Raman spectra are reported here for Saccharomyces cerevisiae (yeast) 5.8S RNA in the presence or absence of Mg2+ in H20 and in the presence of Mg2+ ions in 2H20. Data obtained from these experiments, combined with prior information, lead to a new proposed "cloverleaf" secondary structure for 5.8S RNA. The new structure is adaptable to 5.8S RNA from other species and to E. coil 5S RNA.
5S RNA and 5.8S RNA belong to a class of small RNA molecules (including tRNA) that function in protein synthesis. The structure and function of the smaller tRNA is now understood largely because (i) tRNAPhe has been successfully crystallized and its three-dimensional structure has been determined (1, 2), and (ii) tRNA function is present in part in the free cytoplasm. In contrast, 5S RNA and 5.8S RNA are intimately bound to the ribosome. Their three-dimensional crystal structures have not yet been reported, and conclusions based on low-field hydrogen-bonded proton nuclear magnetic resonance spectra have been severely limited by poor resolution (3, 4). Less-direct structural information from other techniques has been reviewed (5), and more recent results are now discussed. Pene et al. (6) demonstrated that 5.8S RNA is strongly hydrogen-bonded to the 28S RNA of the large ribosomal subunit of eukaryotes. This interaction is between the 3' end of the 5.8S RNA molecule and a complementary segment of the 288 RNA and is stabilized by the presence of a G-C-rich loop near the 3' end of the 5.8S RNA (7). Moreover, all 5.8S RNA nucleotide sequences determined to date contain a sequence of bases that is complementary to the T4/CG-loop of tRNA (8-10). These facts suggest that 5.8S RNA in eukaryotes binds tRNA at the ribosome during transcription. In addition, the following facts connect the structure and function of eukaryotic 5.8S RNA to prokaryotic 5S RNA. First, yeast 5.8S RNA can bind the same ribosomal proteins (EL-18 and EL-25) that Escherichia coli 5S RNA binds most strongly, and this 5.8S RNA-E. colh protein complex exhibits GTPase and ATPase activities similar to those for the homologous 5S RNA-protein complex (11). Second, all prokaryotic 5S RNA molecules contain the complement of the TXCG-loop of tRNA and can bind the tetranucleotide UUCG (5). Third, both the prokaryotic 5S RNA sequence and the eukaryotic 5.8S RNA sequence are contained in the large ribosomal RNA transcription units (12). Therefore, because eukaryotic 5.8S RNA and prokaryotic 5S RNA appear to have similar origin and function, their secondary structures should be similar. Raman spectroscopy has proved to be a sensitive probe of
MATERIALS AND METHODS Isolation and Purification of 5.8S RNA and tRNA. S. cerevssiae cultures were grown to midlogarithmic phase, harvested by centrifugation, and stored at -20°. RNA was extracted with phenol and precipitated at -200 with ethanol. 28S RNA was separated from 5.8S RNA by heat denaturation in 1.2 M NaCl at 65° for 5 min, and the 28S RNA (insoluble) was removed from the 5.8S RNA (soluble) by centrifugation at 0°. Chromatography on DEAE-cellulose (DE-32) removed traces of non-RNA material, and the RNA was precipitated with ethanol containing 0.05 M MgCl2. Highly purified 5.8S RNA was obtained by two successive gel filtrations on Sephadex G-100 (10 X 130-cm and 2 X 190-cm columns) in the presence of 10 mM MgCl2. The purified 5.8S RNA had the same gel electrophoretic mobility relative to tRNA as previously reported for this species (8). The purified 5.8S RNA was precipitated, desalted on Sephadex G-25, and stored as a lyophilized powder. tRNA was prepared similarly from the first Sephadex G-100 fractionation. Preparation of Raman Samples. tRNA or 5.8S RNA was dissolved in H20 or 2H20 containing 10 mM phosphate (pH 7), 10 mM MgCl2, and 100 mM NaCl. Samples were 4% RNA (wt/vol), except for the low-Mg2+ sample (2%), and all the 5.8S RNA samples were renatured at 650 for 5 min before spectra were recorded. Low-Mg2+ 5.8S RNA was prepared as by Chen et al. (17). Raman spectra were recorded on a Spex Ramalog 4 laser Raman system equipped with a Spectra-Physics 265 exciter argon laser tuned to the 5148 A excitation line. Sample tubes (0.8-mm inside diameter glass capillaries) were irradiated with 600 mW of laser power (8 cm-I slit width). Scan speed was 1 cm1 /sec at a period of 5 sec for 4% samples and 0.5 cm-1 /sec at a 10-sec period for the 2% sample. Each reported spectrum was recorded at least three (and usually six or more) times.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
*
4901
To whom reprint requests should be addressed.
4902
Proc. Natl. Acad. Sci. USA 75 (1978)
Biophysics: Luoma and Marshall
RESULTS Comparison of 5.8S and tRNA Raman Spectra. The Raman spectra of 5.8S RNA and tRNA are shown in Fig. 1. The positions and intensities (peak heights) of prominent lines are listed in Table 1. Line intensities were normalized with respect to the intensity of the P02- line at 1100 cm-1 (15), measured as the peak height above a base line drawn tangent at 1065 and 1130 cm 1 (17). That line exhibits constant halfwidth and integrated area in spectra from all Mg2+-containing samples (13-16) and changes only slightly after 100-fold changes in Mg2f concentration (18). The normalized intensity at 814 cm-1 [measured from baseline tangent at 740 and 840 cm-' (15)] is identical for 5.8S RNA and tRNA (Table 1). This line is assigned to a C-O-P-O-C stretching vibration of the phosphodiester group (13-16) and is known to be very sensitive to "order" in the backbone structure of RNA. Because previous studies have shown that all tRNA species are 85% "ordered" (at the phosphodiester linkage) relative to completely "ordered" polyribonucleotides such as poly(rA)-poly(rU), 5.8S RNA must also be 85% "ordered" in solutions containing Mg2+. The Raman line at 670 cm-1 exhibits.reverse hypochromism-i.e., line intensity increases with increased G-base stacking at a given G-base concentration. After correction for difference in G-base content between tRNA (-30%) and 5.8S RNA (23%), the 670 cm-1 intensity4or 5.8S RNA is greater by 35%. In fact, when compared with that of tRNAPhe (17), the 5.8S RNA line is greater by 60%. Therefore, 5.8S RNA has a higher proportion of stacked G bases than does tRNA. The G-C-rich arm (see Fig. 3) containing a segment of five consecutive G bases produces this large stacking effect when these bases are helically
paired. The Raman line at 725 cm-1 is due to A bases and is hypochromic (13-16). After correction for the different A-base content of 5.8S RNA and tRNA, their 725 cm-1 intensities are approximately equal, suggesting similar A-base stacking in both cases.
The 785 cm-' Raman line is also hypochromic, so its intensity increases with increasing proportion of unstacked C and U bases, for fixed C-base and U-base composition (13-16). Because
A G
A G
A
Table 1. Comparison of Raman lines of tRNA, 5.8S RNA, and low-Mg2+ 5.8S RNA Frequency, cm-1
Origin of line
670 725 785 814 1100 1234 1251 1300 1321 1338 1375
G A C,U -OPOPO2 U C,A C,A G A G,A
0.53 0.58 2.24 1.61
0.56 0.75 2.28 1.61
0.48 0.61 2.31 1.41
1.00
1.00
1.00
1.05 1.10 0.90 1.15 1.22 1.88
1.21 1.18 0.93 1.19 1.65 1.91
1.35 1.41 0.90 1.36 1.67 1.90
* Each value represents an average from at least six spectra; each individual normalized intensity differs by less than ±5% from the average. t As in (*); averages from at least three spectra.
5.8S RNA has about 20% more C and U residues than does tRNA, the 785 cm-1 intensities (Table 1) indicate that 5.8S RNA has more C and U stacking than does tRNA. A large stacking contribution is expected for the four C bases paired to G bases in the G'C-rich arm (see Fig. 3); also, there are several instances of three consecutive U bases, with potentially large contribution to U-base stacking. Furthermore, the hypochromic Raman line due to U residues at 1234 cm-1 has greater relative intensity for tRNA than for 5.8S RNA, when U-base contents are considered. In 2H20, the carbonyl stretch at 1660 cm-1 arises from base-paired U residues, whereas the one at 1688 cm-' arises from unpaired U residues (16). The ratio of these two intensities is thus a direct measure of the percentage of base-paired U residues. Fig. 2 indicates that 5.8S RNA has proportionately more base-paired U residues than does mixed tRNA. In particular, because the 1660/1688 intensity ratio is larger for 5.8S RNA than for tRNAPhe (16) and because two-thirds of the U
AA
G AGC CU
tRNA*
Relative intensity Low-Mg2+ 5.8S RNAt 5.8S RNA*
Po-
U OPOC
A
G
60-0 800 1000 cm-1 FIG. 1. Tracings of the original Raman spectra of mixed tRNA (a), normal 5.8S RNA (b), and low-Mg2+ 5.8S RNA (c). Samples for a and b were dissolved in H20 containing 10 mM phosphate (pH 7), 10 mM MgCl2, and 100 mM NaCl. Low-Mg2+ 5.8S RNA was prepared as described bv Chen et al. (17). No lines except the H20 line in the spectra are attributed to the buffer. 1800
1600
1400
1 200
Biophysics:
Luoma and Marshall
Proc. Natl. Acad. Sci. USA 75 (1978)
4903
Table 2. Properties of various models for eukaryotic 5.8S RNA Property
1700
1600
cm-1
FIG. 2. Superposition of the carbonyl stretch regions of the spectra of mixed tRNA (-) and normal 5.8S RNA (--- -) to compare the percentage of base-paired U residues. Samples were dissolved in 2H20 containing 10 mM phosphate pH 7, 10 mM MgCl2, and 100 mM NaCl. No lines are attributed to the buffer.
It
Yeast IIt III§
A-U base pairs 15 20 G-C base pairs 21 16 Total A.U and G-C pairs 31 41 G.Ubasepairs 4 4 A-*basepairs 1 1 Total base pairs 36 46 % nucleotides in A.U and G-C pairs 52 39 % U in base pairs 44 56 % nucleotides in all types of pairing 46 58 Sum of stability numbers 16 16 * From Novikoff hepatoma cells. t I, model as proposed by Rubin (8). 1 II, model as proposed by Nazar et al. (9). § III, cloverleaf model proposed here.
Rat* III§
IF
23 23 46 8 1 55
12 33 45 3 2 50
12 32 44 6 1 51
58 72
57 55
56 64
70 22
63 28
65 21
residues of tRNAPhe are base-paired (1, 2), 5.8S RNA must have more than 70% of its U residues in base-paired configurations.
Comparison of High- and Low-Mg2+ 5.8S RNA Raman Spectra. The Raman spectra of normal and lowMg2+ 5.8S RNA (Fig. 1) and the normalized peak intensities (Table 1) indicate an important role for Mg2+ in 5.8S RNA structure. Because some lines (1234, 1251, and 1321 cm-') increase and others (670 and 814 cm-') decrease on removal of Mg2+, the observed intensity changes are not principally due to a change in intensity of the 1100 cm-1 normalization peak. First, the 12% decrease in intensity of the 814 cm-1 line suggests a decrease in order of the phosphodiester backbone on removal of Mg2+. Second, the 15% decrease in intensity of the 670 cm-l line indicates a small decrease in G-base stacking in 5.8S RNA lacking Mg2+, whereas the decrease in intensity of the 725 cm- line suggests an increase in A-base stacking. These observations are confirmed by the negligible change in the intensities at 1375 and 1485 cm-l, because the decreased G-stacking intensity contribution to these lines is largely compensated for by the increased A-stacking contribution. Finally, the slight increase in intensity of the 1234 cm-l line suggests a small decrease in U stacking when Mg2+ is absent. These results indicate that the removal of Mg2+ from 5.8S RNA causes a slight disordering of the backbone and a rearrangement of base-stacking interactions. These changes are identical in type but less in degree than for tRNAPhe (17), which has three strong Mg2+ binding sites (19, 20). The structure of 5.8S RNA is therefore expected to be less dependent on Mg2+ than is the structure of tRNA, although Mg2+ is required to maintain native conformation in both cases.
DISCUSSION Raman Data Conflict with Previously Proposed Structures. The above Raman data suggest that yeast 5.8S RNA has the following properties: (i) a highly ordered backbone structure similar to that of tRNA and indicative of a high degree of base pairing, (ii) a G-C-rich arm giving rise to extensive G (and C) stacking, (iii) secondary and tertiary structures containing >70% base-paired U residues involving significant U stacking, (iv) a smaller structural requirement for Mg2+ than tRNA, suggesting either less extensive tertiary folding or less dependence on Mg2+ for this folding, and (v) only moderate A stacking, which increases when Mg2+ is absent. Neither of the previously proposed structures- (8, 9) has all of the above properties. As shown in Table 2, Rubin's model (8)
has only 44% of its U residues in base-paired regions and is insufficiently "ordered" (too much single-strandedness) to fit the Raman data. Although the alternative model of Nazar et al. (9) has a less-open structure than Rubin's model, it contains neither a high enough percentage of base-paired U residues nor an unpaired GAAC region with which to bind tRNA. Moreover, when adapted to different 5.8S RNA species, the Nazar-type structure lacks consistency in conserved base-paired regions and lacks the stability expected for a sequence that shows a very high degree of homology between different plant and animal species (10, 22). Such homologous sequences imply a highly conserved pattern of base pairs, but the Nazar et al. model contains only minor base-pair homologies. Also, the stability number (22) for the Nazar et al. structure for hepatoma 5.8S RNA is 75% larger than that for yeast 5.8S RNA, which seems highly unlikely for a largely conserved sequence. New Cloverleaf Model Fits Raman and Other Data. Based on the Raman data for yeast 5.8S RNA, we have developed a new cloverleaf secondary structure (Fig. 3 left) which exhibits all the above properties, accounts for several independent previous observations, and is adaptable to Novikoff hepatoma 5.8S RNA (Fig. 3 right) and E. colt SS RNA (Fig. 4). The new model has numerous advantages over previous structures. First, the cloverleaf model accounts for all the listed Raman data. The structural similarity of the 5.8S RNA and tRNA cloverleafs is consistent with their similar backbone order, and the percentage of base-paired U residues (72% for the cloverleaf structure) agrees with the Raman determination. Furthermore, the cloverleaf contains a GC-rich arm and accounts for the low level of A stacking by forcing many A residues into hairpin loops, bulges, or interior loops. Also, the removal of Mg2+ from a yeast 5.8S RNA solution produces effects very similar to those for tRNAPhe, again supporting the proposed secondary structure. Second, the new cloverleaf secondary structure is conserved among different 5.8S RNA species, as suggested by the basesequence homology. Not only are the stability numbers for yeast and hepatoma 5.8S RNA very similar, but the total number of base pairs also remains constant (Table 2). The cloverleaf structure allows many conserved regions to be base-paired in the same manner among different species, thereby stabilizing each of the three arms similarly among various types of 5.8S RNA (Fig. 3). Third, the new cloverleaf accommodates a wide range of
4904
Proc. Natl. Acad. Sci. USA 75 (1978)
Biophysics: Luoma and Marshall
A -UA
C
pC-G C
G-C A-U
~~~~~~~~~~~C-G
U-A
C~ U~~~~~~~~~~C-G
C-G
AC
GA
UC GU A-U
t
A
CA~U U
-G XCG GU-DXCG-C U
2O~~~~C~
\ i ar GCrU 20U
Vol. 75, No. 10, pp. 4901-4905, October 1978 Biophysics
Laser Raman evidence for new cloverleaf secondary structures for eukaryotic 5.8S RNA and prokaryotic 5S RNA (tRNA binding/Mg2+-dependence)
GREG A. LUOMA* AND ALAN G. MARSHALL* Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1W5, Canada
Communicated by Alexander Rich, June 27, 1978
ABSTRACT Neither of the two previously proposed secondary structures for eukaryotic 5.8S RNA is consistent with the present laser Raman results. A new, highly stable "cloverleaf" secondary structure not only fits the Raman data but also accounts for previously determined enzymatic partial cleavage patterns, base sequence and pairing homologies, and GC and A'U base pair numbers and ratios. The new cloverleaf model also conserves several structural features (constant loops, bulges, and stems) consistent with known 5.8S RNA functions. Finally, we propose a similar new cloverleaf secondary structure for Escherichia coli 5S RNA, consonant with many known properties of prokaryotic 5S RNA.
secondary and tertiary structures of RNA molecules in solution (13, 14), particularly for determining the types and extent of base stacking, the percentage of U residues in base-paired versus single-stranded regions, and the degree of order (rigidity) at the backbone phosphate linkages (13-16). Raman spectra can also reflect changes in RNA secondary or tertiary conformation (17). Because two markedly different yeast 5.8S RNA secondary structures have been proposed, Raman studies are expected to provide critical tests for these structures. Raman spectra are reported here for Saccharomyces cerevisiae (yeast) 5.8S RNA in the presence or absence of Mg2+ in H20 and in the presence of Mg2+ ions in 2H20. Data obtained from these experiments, combined with prior information, lead to a new proposed "cloverleaf" secondary structure for 5.8S RNA. The new structure is adaptable to 5.8S RNA from other species and to E. coil 5S RNA.
5S RNA and 5.8S RNA belong to a class of small RNA molecules (including tRNA) that function in protein synthesis. The structure and function of the smaller tRNA is now understood largely because (i) tRNAPhe has been successfully crystallized and its three-dimensional structure has been determined (1, 2), and (ii) tRNA function is present in part in the free cytoplasm. In contrast, 5S RNA and 5.8S RNA are intimately bound to the ribosome. Their three-dimensional crystal structures have not yet been reported, and conclusions based on low-field hydrogen-bonded proton nuclear magnetic resonance spectra have been severely limited by poor resolution (3, 4). Less-direct structural information from other techniques has been reviewed (5), and more recent results are now discussed. Pene et al. (6) demonstrated that 5.8S RNA is strongly hydrogen-bonded to the 28S RNA of the large ribosomal subunit of eukaryotes. This interaction is between the 3' end of the 5.8S RNA molecule and a complementary segment of the 288 RNA and is stabilized by the presence of a G-C-rich loop near the 3' end of the 5.8S RNA (7). Moreover, all 5.8S RNA nucleotide sequences determined to date contain a sequence of bases that is complementary to the T4/CG-loop of tRNA (8-10). These facts suggest that 5.8S RNA in eukaryotes binds tRNA at the ribosome during transcription. In addition, the following facts connect the structure and function of eukaryotic 5.8S RNA to prokaryotic 5S RNA. First, yeast 5.8S RNA can bind the same ribosomal proteins (EL-18 and EL-25) that Escherichia coli 5S RNA binds most strongly, and this 5.8S RNA-E. colh protein complex exhibits GTPase and ATPase activities similar to those for the homologous 5S RNA-protein complex (11). Second, all prokaryotic 5S RNA molecules contain the complement of the TXCG-loop of tRNA and can bind the tetranucleotide UUCG (5). Third, both the prokaryotic 5S RNA sequence and the eukaryotic 5.8S RNA sequence are contained in the large ribosomal RNA transcription units (12). Therefore, because eukaryotic 5.8S RNA and prokaryotic 5S RNA appear to have similar origin and function, their secondary structures should be similar. Raman spectroscopy has proved to be a sensitive probe of
MATERIALS AND METHODS Isolation and Purification of 5.8S RNA and tRNA. S. cerevssiae cultures were grown to midlogarithmic phase, harvested by centrifugation, and stored at -20°. RNA was extracted with phenol and precipitated at -200 with ethanol. 28S RNA was separated from 5.8S RNA by heat denaturation in 1.2 M NaCl at 65° for 5 min, and the 28S RNA (insoluble) was removed from the 5.8S RNA (soluble) by centrifugation at 0°. Chromatography on DEAE-cellulose (DE-32) removed traces of non-RNA material, and the RNA was precipitated with ethanol containing 0.05 M MgCl2. Highly purified 5.8S RNA was obtained by two successive gel filtrations on Sephadex G-100 (10 X 130-cm and 2 X 190-cm columns) in the presence of 10 mM MgCl2. The purified 5.8S RNA had the same gel electrophoretic mobility relative to tRNA as previously reported for this species (8). The purified 5.8S RNA was precipitated, desalted on Sephadex G-25, and stored as a lyophilized powder. tRNA was prepared similarly from the first Sephadex G-100 fractionation. Preparation of Raman Samples. tRNA or 5.8S RNA was dissolved in H20 or 2H20 containing 10 mM phosphate (pH 7), 10 mM MgCl2, and 100 mM NaCl. Samples were 4% RNA (wt/vol), except for the low-Mg2+ sample (2%), and all the 5.8S RNA samples were renatured at 650 for 5 min before spectra were recorded. Low-Mg2+ 5.8S RNA was prepared as by Chen et al. (17). Raman spectra were recorded on a Spex Ramalog 4 laser Raman system equipped with a Spectra-Physics 265 exciter argon laser tuned to the 5148 A excitation line. Sample tubes (0.8-mm inside diameter glass capillaries) were irradiated with 600 mW of laser power (8 cm-I slit width). Scan speed was 1 cm1 /sec at a period of 5 sec for 4% samples and 0.5 cm-1 /sec at a 10-sec period for the 2% sample. Each reported spectrum was recorded at least three (and usually six or more) times.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
*
4901
To whom reprint requests should be addressed.
4902
Proc. Natl. Acad. Sci. USA 75 (1978)
Biophysics: Luoma and Marshall
RESULTS Comparison of 5.8S and tRNA Raman Spectra. The Raman spectra of 5.8S RNA and tRNA are shown in Fig. 1. The positions and intensities (peak heights) of prominent lines are listed in Table 1. Line intensities were normalized with respect to the intensity of the P02- line at 1100 cm-1 (15), measured as the peak height above a base line drawn tangent at 1065 and 1130 cm 1 (17). That line exhibits constant halfwidth and integrated area in spectra from all Mg2+-containing samples (13-16) and changes only slightly after 100-fold changes in Mg2f concentration (18). The normalized intensity at 814 cm-1 [measured from baseline tangent at 740 and 840 cm-' (15)] is identical for 5.8S RNA and tRNA (Table 1). This line is assigned to a C-O-P-O-C stretching vibration of the phosphodiester group (13-16) and is known to be very sensitive to "order" in the backbone structure of RNA. Because previous studies have shown that all tRNA species are 85% "ordered" (at the phosphodiester linkage) relative to completely "ordered" polyribonucleotides such as poly(rA)-poly(rU), 5.8S RNA must also be 85% "ordered" in solutions containing Mg2+. The Raman line at 670 cm-1 exhibits.reverse hypochromism-i.e., line intensity increases with increased G-base stacking at a given G-base concentration. After correction for difference in G-base content between tRNA (-30%) and 5.8S RNA (23%), the 670 cm-1 intensity4or 5.8S RNA is greater by 35%. In fact, when compared with that of tRNAPhe (17), the 5.8S RNA line is greater by 60%. Therefore, 5.8S RNA has a higher proportion of stacked G bases than does tRNA. The G-C-rich arm (see Fig. 3) containing a segment of five consecutive G bases produces this large stacking effect when these bases are helically
paired. The Raman line at 725 cm-1 is due to A bases and is hypochromic (13-16). After correction for the different A-base content of 5.8S RNA and tRNA, their 725 cm-1 intensities are approximately equal, suggesting similar A-base stacking in both cases.
The 785 cm-' Raman line is also hypochromic, so its intensity increases with increasing proportion of unstacked C and U bases, for fixed C-base and U-base composition (13-16). Because
A G
A G
A
Table 1. Comparison of Raman lines of tRNA, 5.8S RNA, and low-Mg2+ 5.8S RNA Frequency, cm-1
Origin of line
670 725 785 814 1100 1234 1251 1300 1321 1338 1375
G A C,U -OPOPO2 U C,A C,A G A G,A
0.53 0.58 2.24 1.61
0.56 0.75 2.28 1.61
0.48 0.61 2.31 1.41
1.00
1.00
1.00
1.05 1.10 0.90 1.15 1.22 1.88
1.21 1.18 0.93 1.19 1.65 1.91
1.35 1.41 0.90 1.36 1.67 1.90
* Each value represents an average from at least six spectra; each individual normalized intensity differs by less than ±5% from the average. t As in (*); averages from at least three spectra.
5.8S RNA has about 20% more C and U residues than does tRNA, the 785 cm-1 intensities (Table 1) indicate that 5.8S RNA has more C and U stacking than does tRNA. A large stacking contribution is expected for the four C bases paired to G bases in the G'C-rich arm (see Fig. 3); also, there are several instances of three consecutive U bases, with potentially large contribution to U-base stacking. Furthermore, the hypochromic Raman line due to U residues at 1234 cm-1 has greater relative intensity for tRNA than for 5.8S RNA, when U-base contents are considered. In 2H20, the carbonyl stretch at 1660 cm-1 arises from base-paired U residues, whereas the one at 1688 cm-' arises from unpaired U residues (16). The ratio of these two intensities is thus a direct measure of the percentage of base-paired U residues. Fig. 2 indicates that 5.8S RNA has proportionately more base-paired U residues than does mixed tRNA. In particular, because the 1660/1688 intensity ratio is larger for 5.8S RNA than for tRNAPhe (16) and because two-thirds of the U
AA
G AGC CU
tRNA*
Relative intensity Low-Mg2+ 5.8S RNAt 5.8S RNA*
Po-
U OPOC
A
G
60-0 800 1000 cm-1 FIG. 1. Tracings of the original Raman spectra of mixed tRNA (a), normal 5.8S RNA (b), and low-Mg2+ 5.8S RNA (c). Samples for a and b were dissolved in H20 containing 10 mM phosphate (pH 7), 10 mM MgCl2, and 100 mM NaCl. Low-Mg2+ 5.8S RNA was prepared as described bv Chen et al. (17). No lines except the H20 line in the spectra are attributed to the buffer. 1800
1600
1400
1 200
Biophysics:
Luoma and Marshall
Proc. Natl. Acad. Sci. USA 75 (1978)
4903
Table 2. Properties of various models for eukaryotic 5.8S RNA Property
1700
1600
cm-1
FIG. 2. Superposition of the carbonyl stretch regions of the spectra of mixed tRNA (-) and normal 5.8S RNA (--- -) to compare the percentage of base-paired U residues. Samples were dissolved in 2H20 containing 10 mM phosphate pH 7, 10 mM MgCl2, and 100 mM NaCl. No lines are attributed to the buffer.
It
Yeast IIt III§
A-U base pairs 15 20 G-C base pairs 21 16 Total A.U and G-C pairs 31 41 G.Ubasepairs 4 4 A-*basepairs 1 1 Total base pairs 36 46 % nucleotides in A.U and G-C pairs 52 39 % U in base pairs 44 56 % nucleotides in all types of pairing 46 58 Sum of stability numbers 16 16 * From Novikoff hepatoma cells. t I, model as proposed by Rubin (8). 1 II, model as proposed by Nazar et al. (9). § III, cloverleaf model proposed here.
Rat* III§
IF
23 23 46 8 1 55
12 33 45 3 2 50
12 32 44 6 1 51
58 72
57 55
56 64
70 22
63 28
65 21
residues of tRNAPhe are base-paired (1, 2), 5.8S RNA must have more than 70% of its U residues in base-paired configurations.
Comparison of High- and Low-Mg2+ 5.8S RNA Raman Spectra. The Raman spectra of normal and lowMg2+ 5.8S RNA (Fig. 1) and the normalized peak intensities (Table 1) indicate an important role for Mg2+ in 5.8S RNA structure. Because some lines (1234, 1251, and 1321 cm-') increase and others (670 and 814 cm-') decrease on removal of Mg2+, the observed intensity changes are not principally due to a change in intensity of the 1100 cm-1 normalization peak. First, the 12% decrease in intensity of the 814 cm-1 line suggests a decrease in order of the phosphodiester backbone on removal of Mg2+. Second, the 15% decrease in intensity of the 670 cm-l line indicates a small decrease in G-base stacking in 5.8S RNA lacking Mg2+, whereas the decrease in intensity of the 725 cm- line suggests an increase in A-base stacking. These observations are confirmed by the negligible change in the intensities at 1375 and 1485 cm-l, because the decreased G-stacking intensity contribution to these lines is largely compensated for by the increased A-stacking contribution. Finally, the slight increase in intensity of the 1234 cm-l line suggests a small decrease in U stacking when Mg2+ is absent. These results indicate that the removal of Mg2+ from 5.8S RNA causes a slight disordering of the backbone and a rearrangement of base-stacking interactions. These changes are identical in type but less in degree than for tRNAPhe (17), which has three strong Mg2+ binding sites (19, 20). The structure of 5.8S RNA is therefore expected to be less dependent on Mg2+ than is the structure of tRNA, although Mg2+ is required to maintain native conformation in both cases.
DISCUSSION Raman Data Conflict with Previously Proposed Structures. The above Raman data suggest that yeast 5.8S RNA has the following properties: (i) a highly ordered backbone structure similar to that of tRNA and indicative of a high degree of base pairing, (ii) a G-C-rich arm giving rise to extensive G (and C) stacking, (iii) secondary and tertiary structures containing >70% base-paired U residues involving significant U stacking, (iv) a smaller structural requirement for Mg2+ than tRNA, suggesting either less extensive tertiary folding or less dependence on Mg2+ for this folding, and (v) only moderate A stacking, which increases when Mg2+ is absent. Neither of the previously proposed structures- (8, 9) has all of the above properties. As shown in Table 2, Rubin's model (8)
has only 44% of its U residues in base-paired regions and is insufficiently "ordered" (too much single-strandedness) to fit the Raman data. Although the alternative model of Nazar et al. (9) has a less-open structure than Rubin's model, it contains neither a high enough percentage of base-paired U residues nor an unpaired GAAC region with which to bind tRNA. Moreover, when adapted to different 5.8S RNA species, the Nazar-type structure lacks consistency in conserved base-paired regions and lacks the stability expected for a sequence that shows a very high degree of homology between different plant and animal species (10, 22). Such homologous sequences imply a highly conserved pattern of base pairs, but the Nazar et al. model contains only minor base-pair homologies. Also, the stability number (22) for the Nazar et al. structure for hepatoma 5.8S RNA is 75% larger than that for yeast 5.8S RNA, which seems highly unlikely for a largely conserved sequence. New Cloverleaf Model Fits Raman and Other Data. Based on the Raman data for yeast 5.8S RNA, we have developed a new cloverleaf secondary structure (Fig. 3 left) which exhibits all the above properties, accounts for several independent previous observations, and is adaptable to Novikoff hepatoma 5.8S RNA (Fig. 3 right) and E. colt SS RNA (Fig. 4). The new model has numerous advantages over previous structures. First, the cloverleaf model accounts for all the listed Raman data. The structural similarity of the 5.8S RNA and tRNA cloverleafs is consistent with their similar backbone order, and the percentage of base-paired U residues (72% for the cloverleaf structure) agrees with the Raman determination. Furthermore, the cloverleaf contains a GC-rich arm and accounts for the low level of A stacking by forcing many A residues into hairpin loops, bulges, or interior loops. Also, the removal of Mg2+ from a yeast 5.8S RNA solution produces effects very similar to those for tRNAPhe, again supporting the proposed secondary structure. Second, the new cloverleaf secondary structure is conserved among different 5.8S RNA species, as suggested by the basesequence homology. Not only are the stability numbers for yeast and hepatoma 5.8S RNA very similar, but the total number of base pairs also remains constant (Table 2). The cloverleaf structure allows many conserved regions to be base-paired in the same manner among different species, thereby stabilizing each of the three arms similarly among various types of 5.8S RNA (Fig. 3). Third, the new cloverleaf accommodates a wide range of
4904
Proc. Natl. Acad. Sci. USA 75 (1978)
Biophysics: Luoma and Marshall
A -UA
C
pC-G C
G-C A-U
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U-A
C~ U~~~~~~~~~~C-G
C-G
AC
GA
UC GU A-U
t
A
CA~U U
-G XCG GU-DXCG-C U
2O~~~~C~
\ i ar GCrU 20U
