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Circular dichroism of Escherichia coli ribosomes and tobacco mosaic virus
SHORT COMMUNICATIONS"
711
BBA 93359
Circular dichroism of Escherichia coli ribosomes and tobacco mosaic virus There have been several studies on the optical rotatory dispersion (ORD) of Escherichia coli ribosomes and their subunits z-3. None of these have, however, been concerned specifically with the interaction between the subunits in the 7o-S ribosomes, although several groups have suggested that there is no change in ORD on dissociating 7o-S ribosomes into their subunits z,3. We wish to report a difference in the circular dichroic (CD) spectra of 7o-S ribosomes and the dissociated subunits. Ribosomes from E. coli, MRE 600, a ribonuclease-free strain, were prepared and characterised as described previouslyt. Ribosomal RNA (I6-S+23-S) was prepared by the method of SPITNIK-ELSON5. 7 o - S ribosomes were suspended in 20 mM magnesium acetate, IO mM sodium phosphate, p H 7.0, under which conditions the ribosomes were 20 % dissociated. 3o-S and 5o-S ribosomes, and ribosomal RNA were suspended in I mM magnesium acetate, IO mM sodium phosphate, pH 7.0. Light scattering of such solutions was estimated from the absorbance in the long wavelength region adjacent to the absorption band and was found to be negligible. Tobacco mosaic virus (TMV) was prepared as described b y BOEDKTER AND SIMMONSe and TMV protein b y the method of FRAENKEL-CONRAT7. All preparations were examined spectrally and in a Beckman, Model E, analytical ultracentrifuge at 20 °. Ribosome concentrations were estimated fcom the absorbance per mole of phosphorus at 260 m/4 determined previously4; TMV was estimated using g210 -~265mg/ml m, -~ 30.6 (see ref. 6) and TMV protein using J-'282 ~1 mg/~__ m~ - - 1.27 (see ref. 7). CD spectra were measured at 18-2o ° in a Roussel-Jouan circular dichrographe, Model CD 185, using cells of 1.o- and o.z-cm pathlength. All spectra were reproducible within the noise level of the signal. The signal-to-noise ratio was 25 : 1 at the maxima. Results were expressed in terms of A E, the difference in extinction coefficient between left and right circularly-polarised light per mole of RNA phosphorus. Light scattering in solutions of TMV and TMV protein was considerable. However, A E was proportional to concentration and (]
J
i
i
20 15
f~ .c
o
E
5~ <1
2~o 2~o 36o
2~o
2~o
~oo
z (mY)
Fig. I. a. T h e CD s p e c t r a of R N A a n d r i b o s o m e s . I, R N A ; 2, 3o-S a n d 5o-S r i b o s o m e s ; 3, 7 o-S r i b o s o m e s ; 4, - difference s p e c t r a of 7o-S r i b o s o m e s a n d s u b u n i t s , b. CD s p e c t r u m of T M V In HaO ( ) a n d T M V p r o t e i n in o.oi M N a H I P O 4, p H 5.6 (- - -). A b b r e v i a t i o n s : O R D , optical r o t a t o r y dispersion, CD, circular d i c h r o i s m ; TMV, t o b a c c o mosaic virus.
Biochim. Biophys. Acta, 166 (I968) 711-713
712
SHORT COMMUNICATIONS
pathlength at all wavelengths, which indicated that there were no artifacts due to scattering. The CD spectra of ribosomal R1VA, 7o-S, 5o-S and 3o-S ribosomes, shown in Fig. I, were similar in shape with a large, positive, dichroic band centred at 265 m y and a small, negative band at 300 m y (c/. ref. 8). A E was zero at 247 and 294 m y for RN'A and 247 and 293 m y for ribosomes. The band at 265 m y was not symmetrical. The spectral characteristics are summarised in Table I. TABLE
I
CHARACTERISTICS OF THE DICHROIC SPECTRA OF R N A , RIBOSOMES AND T~c~V
System
2m.x
LIE,~a.
Rotational strength X I040
2,ni.
d Emi.
12.8 14.2
3oo 3oo
--o.2 -- o. 3
(e.~.s.) RNA 7o-S 5o-S } 3o-S TMV
265 265
6.25±o.1 7.o2 i o. 16
265
5.72±0.25
II. 4
3oo
--o.3
275
22.2±0.5o
52. 4
None
--
/
A comparison of the magnitudes and positions of the ribosomal bands with t h a t of RNA shows that the dichroism of the ribosomes is dominated b y the contribution arising from the secondary structure of the RNA. The similar rotational strengths and shapes of the bands of the 3o-S and 5o-S subunits are consistent with the similarities in their ORD curves ~,3 and are further evidence that their RNA components have very similar secondary structures. The difference in magnitude between the strength of this band in the subunits and in the RNA is only just significant. The dichroism of the 7o-S ribosomes is significantly greater than that of the subunits. The CD difference curve between 7o-S ribosomes and the averaged 3o-S and 5o-S curves is positive with a m a x i m u m at 265 m y (see Fig. I). This difference implies that there has been a change in conformation of the RNA nucleotides probably at the interface between subunits. However, the increase in the absorption maximum at 260 m y when the ribosomes dissociate into subunits is less than one per cent, and is only just greater than the error of the measurement. Preliminary observations using difference spectroscopy confirm that there are significant changes in absorbance of not more than one per cent over the wavelength range 240-300 In# (L. A. BALL ANn I. O. W A L K E R , unpublished results). These observations imply that there has been no gross change in hyperchromism and thus no great change in the internucleotide interactions either in double-helical regions or in single-stranded, "stacked" regions. There is considerable evidence that single-stranded, stacked, oligoand polynucleotides assume helical conformations 9,1°, which, when completely stacked, are fully hypochlomic. Theoretical studies n,l~ show that hypochromism in polynucleotides is primarily a result of nearest-neighbour electronic interactions between adjacent bases on the same chain. Contributions to hypochromism from complementary base pairs on the same or different chains are small. The CD difference spectrum between 7o-S ribosomes and that of the subunits shows that the increase in dichroism arises from a positive band centred at about 265 m/z. Several lines of evidence suggest that dichroism at this wavelength m a y be Biochim. Biophys. Acta, 166 (1968) 7 I I - 7 1 3
713
SHORT COMMUNICATIONS
associated with double-helical structure. First, the secondary structure of ribosomal RNA is thought to consist mainly of short, right-handed, double-helical regions 13. Thus dichroic bands centred at 265 m# m a y be attributed to the optical activity of this type of structure. The difference CD curve m a y therefore result from the net formation of similar structure. Secondly, the CD spectrum of TMV (Fig. I and Table I) is dominated b y the contribution from the RNA. This has a conformation within the virus particle of an extended, ordered, single strand 14 in which, however, the bases m a y not necessarily be stacked (but see ref. 3). The dichroism is located at longer wavelengths than t h a t of ribosomal RNA or the ribosomes and consists of a positive symmetrical band at 275 m#. Finally this difference in the position of the dichroic band between single- and double-stranded RNA parallels differences in the ORD curves of TMV-RNA and certain polynucleotides which have also been attributed to the difference in optical activity between single-stranded and double-helical conformations 15,3. The evidence from the CD studies presented here together with 0 R D studies cited above strongly support the possibility that th~ dichroism at 265 m/~ arises from the formation of right-handed, double helical structure. We suggest that the increased dichroism associated with the formation of 7o-S ribosomes from the subunits arises from the net formation of right-handed double helical structure from existing single-stranded helix located on the surface between the subunits. Such interactions can account for the increased optical activity and the absence of concomitant changes in hypochromism. Steric considerations show that these regions cannot consist of more than half a turn of helix, about 5 bases, otherwise the formation of complementary base pairs results in the two strands of RNA in the subunits becoming intertwined. The half turns of helical, stacked structure m a y be located in the closed loop of polynucleotide at the ends of the double helical regions. Thus the type of interaction envisaged here m a y be similar to the one suggested for the anticodon loop in transfer RN'A 16. The authors t h a n k the Jouan Instrument Company (Paris) for use of the Dichrographe. Department o[ Biochemistry, University o] Ox]ord, Ox/ord (Great Britain)
S . H . MIALL I. 0. WALKER
I P. McPHIE AND W. GRATZER, Biochemistry, 5 (1966) 131o. 2 P. SARKAR, J. T. YANG AND P. BOa'Y, Biopolymers, 5 (1967) I.
3 C. A. BUSH AND H. A. SCHERAGA,Biochemistry, 6 (1967) 3036. 4 S. n . MIALL AND I. O. WALKER, Biochim.
Biophys. Acta, 145 (1967) 82.
5 P. SPITZqlK-ELSON,Biochem. Biophys. Res. Commun., 18 (1965) 5576 H. BOEDKTER AND N. S. SIMMONS, J. Am. Chem. Soc., 80 (1958) 255 o.
7 H. FRAENKEL-CONRAT,Virology, 4 (1957) I. 8 P. SARKAR, B. WELLS AND J. T. YANG, J. Mol.
Biol., 25 (1967) 563 .
9 J. BRAHMS,Nature, 202 (1964) 797. IO J. BRAHMS AND W. F. H. ~¢~. MOMMAERa,S, J. Mol.
II 12 13 14
Biol., IO (1964) 73-
H. DEVOEAND I. TINOCO.J. Mol. Biol., 4 (1962) 5oo. H. OEVOEAND I. TINOCO,J. Mol. Biol., 4 (1962) 518. R. A. COX, Biochem. J., 98 (1966) 841. A. KLUGAND D. L. D. CASPAR,Advan. Virus Res., 7 (196o) 225.
15 C. R. CANTOR, S. R. JASKUNAS AND I. TINOCO, JR.,
J. Mol. Biol., 2o (1966) 39.
16 W. FULLERAND A. HODGSON,Nature, 215 (1967) 817. Received May 24th, 1968 Revised manuscript received J u l y 3Ist, 1968
Biochim. Biophys. Aaa, 166 (1968) 711-713
711
BBA 93359
Circular dichroism of Escherichia coli ribosomes and tobacco mosaic virus There have been several studies on the optical rotatory dispersion (ORD) of Escherichia coli ribosomes and their subunits z-3. None of these have, however, been concerned specifically with the interaction between the subunits in the 7o-S ribosomes, although several groups have suggested that there is no change in ORD on dissociating 7o-S ribosomes into their subunits z,3. We wish to report a difference in the circular dichroic (CD) spectra of 7o-S ribosomes and the dissociated subunits. Ribosomes from E. coli, MRE 600, a ribonuclease-free strain, were prepared and characterised as described previouslyt. Ribosomal RNA (I6-S+23-S) was prepared by the method of SPITNIK-ELSON5. 7 o - S ribosomes were suspended in 20 mM magnesium acetate, IO mM sodium phosphate, p H 7.0, under which conditions the ribosomes were 20 % dissociated. 3o-S and 5o-S ribosomes, and ribosomal RNA were suspended in I mM magnesium acetate, IO mM sodium phosphate, pH 7.0. Light scattering of such solutions was estimated from the absorbance in the long wavelength region adjacent to the absorption band and was found to be negligible. Tobacco mosaic virus (TMV) was prepared as described b y BOEDKTER AND SIMMONSe and TMV protein b y the method of FRAENKEL-CONRAT7. All preparations were examined spectrally and in a Beckman, Model E, analytical ultracentrifuge at 20 °. Ribosome concentrations were estimated fcom the absorbance per mole of phosphorus at 260 m/4 determined previously4; TMV was estimated using g210 -~265mg/ml m, -~ 30.6 (see ref. 6) and TMV protein using J-'282 ~1 mg/~__ m~ - - 1.27 (see ref. 7). CD spectra were measured at 18-2o ° in a Roussel-Jouan circular dichrographe, Model CD 185, using cells of 1.o- and o.z-cm pathlength. All spectra were reproducible within the noise level of the signal. The signal-to-noise ratio was 25 : 1 at the maxima. Results were expressed in terms of A E, the difference in extinction coefficient between left and right circularly-polarised light per mole of RNA phosphorus. Light scattering in solutions of TMV and TMV protein was considerable. However, A E was proportional to concentration and (]
J
i
i
20 15
f~ .c
o
E
5~ <1
2~o 2~o 36o
2~o
2~o
~oo
z (mY)
Fig. I. a. T h e CD s p e c t r a of R N A a n d r i b o s o m e s . I, R N A ; 2, 3o-S a n d 5o-S r i b o s o m e s ; 3, 7 o-S r i b o s o m e s ; 4, - difference s p e c t r a of 7o-S r i b o s o m e s a n d s u b u n i t s , b. CD s p e c t r u m of T M V In HaO ( ) a n d T M V p r o t e i n in o.oi M N a H I P O 4, p H 5.6 (- - -). A b b r e v i a t i o n s : O R D , optical r o t a t o r y dispersion, CD, circular d i c h r o i s m ; TMV, t o b a c c o mosaic virus.
Biochim. Biophys. Acta, 166 (I968) 711-713
712
SHORT COMMUNICATIONS
pathlength at all wavelengths, which indicated that there were no artifacts due to scattering. The CD spectra of ribosomal R1VA, 7o-S, 5o-S and 3o-S ribosomes, shown in Fig. I, were similar in shape with a large, positive, dichroic band centred at 265 m y and a small, negative band at 300 m y (c/. ref. 8). A E was zero at 247 and 294 m y for RN'A and 247 and 293 m y for ribosomes. The band at 265 m y was not symmetrical. The spectral characteristics are summarised in Table I. TABLE
I
CHARACTERISTICS OF THE DICHROIC SPECTRA OF R N A , RIBOSOMES AND T~c~V
System
2m.x
LIE,~a.
Rotational strength X I040
2,ni.
d Emi.
12.8 14.2
3oo 3oo
--o.2 -- o. 3
(e.~.s.) RNA 7o-S 5o-S } 3o-S TMV
265 265
6.25±o.1 7.o2 i o. 16
265
5.72±0.25
II. 4
3oo
--o.3
275
22.2±0.5o
52. 4
None
--
/
A comparison of the magnitudes and positions of the ribosomal bands with t h a t of RNA shows that the dichroism of the ribosomes is dominated b y the contribution arising from the secondary structure of the RNA. The similar rotational strengths and shapes of the bands of the 3o-S and 5o-S subunits are consistent with the similarities in their ORD curves ~,3 and are further evidence that their RNA components have very similar secondary structures. The difference in magnitude between the strength of this band in the subunits and in the RNA is only just significant. The dichroism of the 7o-S ribosomes is significantly greater than that of the subunits. The CD difference curve between 7o-S ribosomes and the averaged 3o-S and 5o-S curves is positive with a m a x i m u m at 265 m y (see Fig. I). This difference implies that there has been a change in conformation of the RNA nucleotides probably at the interface between subunits. However, the increase in the absorption maximum at 260 m y when the ribosomes dissociate into subunits is less than one per cent, and is only just greater than the error of the measurement. Preliminary observations using difference spectroscopy confirm that there are significant changes in absorbance of not more than one per cent over the wavelength range 240-300 In# (L. A. BALL ANn I. O. W A L K E R , unpublished results). These observations imply that there has been no gross change in hyperchromism and thus no great change in the internucleotide interactions either in double-helical regions or in single-stranded, "stacked" regions. There is considerable evidence that single-stranded, stacked, oligoand polynucleotides assume helical conformations 9,1°, which, when completely stacked, are fully hypochlomic. Theoretical studies n,l~ show that hypochromism in polynucleotides is primarily a result of nearest-neighbour electronic interactions between adjacent bases on the same chain. Contributions to hypochromism from complementary base pairs on the same or different chains are small. The CD difference spectrum between 7o-S ribosomes and that of the subunits shows that the increase in dichroism arises from a positive band centred at about 265 m/z. Several lines of evidence suggest that dichroism at this wavelength m a y be Biochim. Biophys. Acta, 166 (1968) 7 I I - 7 1 3
713
SHORT COMMUNICATIONS
associated with double-helical structure. First, the secondary structure of ribosomal RNA is thought to consist mainly of short, right-handed, double-helical regions 13. Thus dichroic bands centred at 265 m# m a y be attributed to the optical activity of this type of structure. The difference CD curve m a y therefore result from the net formation of similar structure. Secondly, the CD spectrum of TMV (Fig. I and Table I) is dominated b y the contribution from the RNA. This has a conformation within the virus particle of an extended, ordered, single strand 14 in which, however, the bases m a y not necessarily be stacked (but see ref. 3). The dichroism is located at longer wavelengths than t h a t of ribosomal RNA or the ribosomes and consists of a positive symmetrical band at 275 m#. Finally this difference in the position of the dichroic band between single- and double-stranded RNA parallels differences in the ORD curves of TMV-RNA and certain polynucleotides which have also been attributed to the difference in optical activity between single-stranded and double-helical conformations 15,3. The evidence from the CD studies presented here together with 0 R D studies cited above strongly support the possibility that th~ dichroism at 265 m/~ arises from the formation of right-handed, double helical structure. We suggest that the increased dichroism associated with the formation of 7o-S ribosomes from the subunits arises from the net formation of right-handed double helical structure from existing single-stranded helix located on the surface between the subunits. Such interactions can account for the increased optical activity and the absence of concomitant changes in hypochromism. Steric considerations show that these regions cannot consist of more than half a turn of helix, about 5 bases, otherwise the formation of complementary base pairs results in the two strands of RNA in the subunits becoming intertwined. The half turns of helical, stacked structure m a y be located in the closed loop of polynucleotide at the ends of the double helical regions. Thus the type of interaction envisaged here m a y be similar to the one suggested for the anticodon loop in transfer RN'A 16. The authors t h a n k the Jouan Instrument Company (Paris) for use of the Dichrographe. Department o[ Biochemistry, University o] Ox]ord, Ox/ord (Great Britain)
S . H . MIALL I. 0. WALKER
I P. McPHIE AND W. GRATZER, Biochemistry, 5 (1966) 131o. 2 P. SARKAR, J. T. YANG AND P. BOa'Y, Biopolymers, 5 (1967) I.
3 C. A. BUSH AND H. A. SCHERAGA,Biochemistry, 6 (1967) 3036. 4 S. n . MIALL AND I. O. WALKER, Biochim.
Biophys. Acta, 145 (1967) 82.
5 P. SPITZqlK-ELSON,Biochem. Biophys. Res. Commun., 18 (1965) 5576 H. BOEDKTER AND N. S. SIMMONS, J. Am. Chem. Soc., 80 (1958) 255 o.
7 H. FRAENKEL-CONRAT,Virology, 4 (1957) I. 8 P. SARKAR, B. WELLS AND J. T. YANG, J. Mol.
Biol., 25 (1967) 563 .
9 J. BRAHMS,Nature, 202 (1964) 797. IO J. BRAHMS AND W. F. H. ~¢~. MOMMAERa,S, J. Mol.
II 12 13 14
Biol., IO (1964) 73-
H. DEVOEAND I. TINOCO.J. Mol. Biol., 4 (1962) 5oo. H. OEVOEAND I. TINOCO,J. Mol. Biol., 4 (1962) 518. R. A. COX, Biochem. J., 98 (1966) 841. A. KLUGAND D. L. D. CASPAR,Advan. Virus Res., 7 (196o) 225.
15 C. R. CANTOR, S. R. JASKUNAS AND I. TINOCO, JR.,
J. Mol. Biol., 2o (1966) 39.
16 W. FULLERAND A. HODGSON,Nature, 215 (1967) 817. Received May 24th, 1968 Revised manuscript received J u l y 3Ist, 1968
Biochim. Biophys. Aaa, 166 (1968) 711-713