Effect of secondary structure on the degradation of ribosomal RNA

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CASAS et al. s t h a t Sr 2+ can s u b s t i t u t e for Ca 2+ in a c t i v a t i o n of d e o x y r i b o n u c l e a s e , b u t not ribonuclease, activity. The possible reason for this d i s c r e p a n c y m a y be due to c o n t a m i n a t i o n of r e a g e n t s (used in e x p e r i m e n t s i n v o l v i n g Sr 2+) with traces of Ca 2+ sufficient to p r o m o t e h y d r o l y s i s of the polymer. This w o r k was s u p p o r t e d b y C o n t r a c t A T (3o-1)363o of the A t o m i c E n e r g y Commission, b y G r a n t s P R P - 3 o a n d E - I 5 7 of t h e American Cancer Society a n d b y G r a n t GB-6o58 of t h e N a t i o n a l Science F o u n d a t i o n .

Laboratory o] Enzymology, Roswell P a r k Memorial Institute, Bu]]alo, N . Y . I42o 3 (U.S.A.)

E. SULKOWSKI M.LAsKOWSKI, SR.

I IV[.LASKOWSKI, SR., Advan. Enzymol., 29 (1967) 165. 2 A. J. MIKULSKI, E. SULKOWSKI, L. STASIUK, AND M. LASKOWSKI,SR., J. Biol. Chem., 244 (1969) 6559. 3 E. SULKOWSKI, A . M. ODLYZKO AND M. LASKOWSK1, SR., Anal. Biochem., in the press. 4 J. N. TUAL, in H. A. SOBER, Handbook o] Biochemistry, Chemical Rubber Co., Cleveland, Ohio 1968, p. G-94. 5 F. FELIX, J. L. POTTER AND M. LASKOWSKI,SR., J. Biol. Chem., 235 (196o) 115o. 6 N. S. KONDO, ]f~. N. FANG, P. S. MILLER AND P. O. P. TS'O, Abstr. z6oth Meeting Am. Chem. Soe., Chicago, Sept. 197o. 7 E . SULKOWSKI AND M. LASKOWSKI, SR., J. Biol. Chem., 244 (1969) 3818. 8 P. CUATRECASAS,S. FUCHS AND C. B. ANFINSEN, J. Biol. Chem., 242 (1967) 1541. R e c e i v e d J u n e 4th, 197o Biochim. Biophys. Acta, 217 (197o) 538-54 °

BBA 93544 Effect of secondary structure on the degradotion of ribosomal R N A L o o p e d configurations (of unspecified detail) have been suggested for s o m e high m o l e c u l a r weight R N A ' s on the basis of E D T A t r e a t m e n t 1, d e n a t u r a t i o n 2,3, or chain l e n g t h m e a s u r e m e n t 4, a n d a m u l t i l o o p e d a r r a n g e m e n t is implicit in the configuration p r o p o s e d for r i b o s o m a l R N A (rRNA) b y SPIRIN 5. This p a p e r considers t h e r e a l i t y of these looped s t r u c t u r e s b y following changes in the m o l e c u l a r weight a n d v o l u m e d u r i n g e n d o n u c l e o l y t i c d e g r a d a t i o n of r R N A u n d e r conditions of stabilisation a n d d e s t a b i l i s a t i o n of t h e s e c o n d a r y structure. E x p e r i m e n t s w i t h b a c t e r i o p h a g e ~ X I 7 4 a n d fd D N A ' s 6,v show t h a t these changes are characteristic if the molecule is circular. The results show t h a t such looped s t r u c t u r e s p r o b a b l y occur u n d e r some p h y s i c a l c o n d i t i o n s b u t t h e y are easily d e s t a b i l i s e d in lower ionic s t r e n g t h solutions when r R N A b e h a v e s as a linear polymer. Escherichia coli W was grown in a s y n t h e t i c g l u c o s e - s a l t s m e d i u m at 37 ° with forced a e r a t i o n a n d ribosomes isolated 3. r R N A was p r e p a r e d b y the phenol m e t h o d using five e x t r a c t i o n s a t 4 ° a n d was p r e c i p i t a t e d from 0.025 M E D T A , where a p p r o priate, to r e m o v e d i v a l e n t (and other) cations. The R N A was s u s p e n d e d in various buffers a t a c o n c e n t r a t i o n of 2" lO .4 g / m l m e a s u r e d b y a b s o r b a n c e a t 260 n m \(E - - I~% Gill = 24o), in 2. 5 m M Mg*+-Tris buffer (pH 7.6). Solutions for light s c a t t e r i n g were m a d e u p in double glass-distilled water, a n d d e d u s t e d b y c e n t r i f u g a t i o n a t 30 ooo × g for Biochim. Biophys. Acta, 217 (I97 o) 54o-543

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5 ° min. The upper half of the centrifuged sample was removed and filtered directly into the light-scattering cell through a Millipore filter (0.3/~). Light-scattering measurements were made in a S.O.F.I.C.A. (Paris) instrument in I5-ml cells at 546 nm and 2o °, at angles between 30 and 15o°. (dn/dc) of rRNA was measured at 546 nm in a Briee-Phoenix differential refractometer at 25 ° and gave a value of o.174 ml/g -1 (0.04 M N a C l + o . o I M acetate buffer, pH 4.6). The absolute Rayleigh ratio of dustfree benzene was taken as 15.8" lO-6 cm -1, and a secondary glass standard, supplied by S.O.F.I.C.A., was used for calibration purposes. RNA degradation by pancreatic ribonuclease (Worthington Biochemicals) was followed in three buffer systems. These buffers given below were chosen, since earlier work 8 had shown that rRNA in o.oi M NaC1 was either on the point of denaturation or slightly denatured at 20 °, whereas in the other buffers there was a substantial amount of secondary structure (based on thermal denaturation experiments). (A) o.ooi M magnesium aeetate+o.oo5 M Tris+o.oo2 M succinic acid, pH 7.6. (B) 0.05 M N a C l + o . o I M Tris+o.oo 4 M succinic acid, pH 7.6. (C) o.oi M NaCl+o.ooo 5 M T l i s + o.0002 M succinic acid, pH 7.6). Qualitatively, if a population of linear molecules receives one random hit per molecule, the molecular weight and dimensions fall. A hit on a circular polymer, however, results in no change in molecular weight and an increase in dimensions. This is expressed in the following equations 9. For a linear molecule: ~V/w 2 M0 x~ (e-~--I +x) (I) R~

R~=3

{2

e-'+I

~+e_x~+ x

}

(2)

For a circular molecule: MW ] I2] { I ] e - ' ( I 21//0 x

+~) }

R~

6 { i--e-'(i + x + 5]l,x') }

R0~

x

1 --e-®(i + ' I s )

(3 )

(4)

where Mw is the (weight-average) molecular weight and Rg the square of the (z-average) radius of gyration after x scissions. M 0 and R0z are the same quantities when x = o. Zimm plots of rRNA in the three buffers showed reasonable linearity of Kc/Ro from 3 °0 to about lO5-115 ° but then became curved up to 15 °0 (see also ref. IO). Consequently Mw and RG were evaluated from (least squares) plots of Kc/Ro vs. sin 2 0/2 in the range 3O-lO5 °. Fig. IA shows that the change in M w / M o of rRNA during degradation in magnesium buffer (upper curve) is close to that expected for the degradation of a circular molecule (Eqn. 3). including the lag period at the start of the reaction. The arrow shows the value of M w / M o corresponding to an average of 1. 9 scissions per molecule. Similarly M w / M o is well represented by expression I for the breakdown of a linear moecule (lower curve). The arrow in this case corresponds to I.O scissions per molecule. The modes of degradation are distinct, and Expression 3, for instance, cannot be reasonably fitted to the experimental points in low-sodium buffers. The initial lag in magnesium buffers is not due to a slow rate of reaction, since this is about twice as fast as in low-sodium buffer which shows an immediate fall in M w / M o.

Biochim. Biophys. Acta, 217 (197 o) 540-543

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The data for RG/R o (Fig. IB) are not as well fitted by Expressions 2 and 4 as the molecular weight data by i and 3.The data from experiments in low-sodium buffers are low compared with the theoretical. The reasons for this are not clear, although there is less accuracy in measuring R G than Mw (since R e is the ratio slope/intercept, each with its standard error). Also, error in the measurement of R 0 leads to a systematic error in RG/Ro; this may partly explain to low values in low sodium buffers. The increase in RG/R o in magnesium buffers compared with the immediate and continuous fall in low-sodium buffers is quite clear however. 1,0( ~

~

0.9

0 0.001M Mg 2+ @0.01M Na"1"

°'ox

1.0

" ""o.,~....^

0.8

0.9 Mw

Mw 0,7

Mo 0.6

0.8

"~'-O

/Wo

0.5 0.4

0/7

0.3 I

I

I

I

I

I

I

I

I

1.4

I

I

A

0 0£)01M Mg 2"i" O

1,3 RG Ro

I

O0£)1 M Na÷

1.2 1.1' 1.0'

~----1~~

o.g "~

•

•

0.8

• I

I

I

10 20 20 40

l

,~ 1,3p n O0 1

I

L

I

I

I

I

I

I

B

30 40 50 60 70 80 9 0 100 110 120 60 80 100 120 1,40 160 180 200 220 240 Time ( mln )

.

2

O0.O01M Mg 'z÷ &0.0~M Na"l"

0

~

1

,

1

A AQOO1M NEt+

R~_G

Ro

\, 0"9~

dl'

• A

o.7t°8 • i

~ ~..~.

" "~"

l

I

i

i

0,8

0.6

0,4

0. 2

C

Mw Mo Fig. i. Degradation of r R N A b y pancreatic ribonuclease. A. Variation in M w / M o with time in Buffers A ( O ) and C (O)- L e f t - h a n d ordinate a n d u p p e r - t i m e scale (O), 2ooffg r R N A per ml, 0.75 ng ribonuclease per ml. R i g h t - h a n d ordinate and lower-time scale ( O ) , 200 fig r R N A per ml, 2.2 ng ribonuclease per ml. U p p e r arrow 1.9, lower I.O scissions per molecule, average. Points are experimental data; lines c o m p u t e d from Expressions 3 ( - - ) and i ( - - - ) . B. Variation in RG/R o with time in Buffers A ( O ) and C ( 0 ) . U p p e r time scale ( O ) , lower ( 0 ) . Conditions as above. P o i n t s are experimental, lines c o m p u t e d from Expressions 4 ( ) and 2 (- - -). C. Variation in RG/R o with Mw/M0. P o i n t s are experimental; lines c o m p u t e d from E x pressions I, 2 (- - - ) and 3, 4 ( -). Biochim. 13iophys. Acta, 217 (197 o) 54 ° 543

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543

Several e x p e r i m e n t s are collected in Fig. IC, in which RG/R o is plotted a g a i n s t M w / M o, irrespective of the time-course of the d e g r a d a t i o n . The lines show the expected relation based on Expressions I a n d 2, a n d 3 a n d 4. I t is concluded: (a) W h e n the s e c o n d a r y structure of r R N A is s t r o n g l y stabilised (o.ooi M m a g n e s i u m or 0.05 M sodium buffer), e n d o n u c l e o l y t i c d e g r a d a t i o n follows a course characteristic of a circular molecule. (b) W h e n the secondary structure is destabilised b u t n o t lost (o.oi M sodium buffer), the kinetics of d e g r a d a t i o n are those of a linear polymer; therefore evidence for this (or a b r a n c h e d ) model is supported n. (e) r R N A degrades as a linear p o l y m e r when (assumed) h a i r p i n loops are present, suggesting t h a t early hits b y the e n z y m e are in the i n t e r h a i r p i n regions, a n d t h a t cross-chain interactions, present in o.ooi M m a g n e s i u m a n d 0.05 M sodium buffers, are lost at lower ionic strength. Such i n t e r a c t i o n s would lead to a structure like t h a t proposed b y SPIRIN 5.

S i r W i l l i a m D u n n School o] Pathology, South Parks Road, Ox/ord (Great Britain) I 2 3 4 5 6 7 8 9 io

A. RODGERS

L. MIRTH, P. HORN AND C. STRAZIELLE,J. Mol. Biol., 13 (1965) 735. M. L. FENWlCK, Biochem. J., lO7 (1968) 851. A. ]RODGERS, Biochem. J., IOO (1966) lO2. W. T. ]RILEY,Nature, 222 (1969) 446. A. S. SPIRIN, Macromolecular Structure o] Nucleic Acids, Reinhold, New York, I964, p. 37. W. FIERS AND ]R. L. SINSHEIMER, J. Mol. Biol., 5 (1962) 424. D. A. 1V[ARVINAND H. SCHALLER,J. Mol. Biol., 15 (1966) I. A. ]RODGERS,Biopolymers, 9 (197o) 843. C. STRAZlELLEAND H. BENOIT, J. Chim. Phys., 62 (1965) 986. S. N. TIMASHEFF, ]R. A. BROWN, J. S. COLTERAND ~[. DAVIES, Biochim. Biophys. Acta, 27

(1958) 662. I I H. BOEDTKER, W. M6LLER AND E. KLEMPERER,

Nature, 194 (1962) 444.

Received J u n e I 8 t h I97o,

Biochim. Biophys. Acta, 217 (197o) 54o-543