J.Mol.Evol.6,61-76

(1975) 1975

© by Springer-Verlag

The Architecture of 5S rRNA and Its Relation to Function GEORGE E. FOX and CARL R. WOESE Department

of G e n e t i c s

Received November

25,

and D e v e l o p m e n t ,

U n i v e r s i t y of Illinois

at U r b a n a

1974

Summary. An e x t e n s i v e c o m p a r a t i v e a n a l y s i s of the available p r i m a r y seq u e n c e data on 5S r R N A has b e e n made. presented

for p r o c a r y o t i c

Eucaryotic

A universal

5S rRNA w h i c h c o n t a i n s architecture.

In addition,

s e r v e d s e g m e n t of more than thirty n u c l e o t i d e s half of the p r o c a r y o t i c molecule. tide -CGAAC- w h i c h p r e s u m a b l y this region,

role for the p r o c a r y o t i c

-CGAAC-

is d i s c u s s e d

change,

i.e.,

the n a t u r a l

ing and u n c o i l i n g of n u c l e i c

coil-

w h i c h can r e s u l t in a cyclic

of the 5S rRNA m o l e c u l e w i t h two t - R N A molecules.

also emerges:

nature

in this segment.

5S rRNA m o l e c u l e

conformational

ing and u n c o i l i n g of one of the helices, interaction

sequence

the p l a n t s d i s p l a y a p r o c a r y o t i c

in w h i c h it is e n v i s i o n e d to u n d e r g o

principle

is i d e n t i f i e d in the 5'

This s e g m e n t includes the o l i g o n u c l e o -

b u t no e u c a r y o t e has the sequence

A functional

and

a h i g h l y con-

binds to the t - R N A "common"

A m o n g the eucaryotes,

is

regions.

5S rRNAs are f o u n d to have only three of these h e l i c e s

thus have a s o m e w h a t d i f f e r e n t

-GT~CG-.

s e c o n d a r y structure four h e l i c a l

rotational motion

acid h e l i c e s

A general

i n h e r e n t in coil-

can be c o n v e r t e d quite

simply

to linear m e c h a n i c a l motion.

Key words: 5S r i b o s o m a l R N A - T r a n s l a t i o n - E v o l u t i o n - M o l e c u l a r Architecture

I.

- Conformational

Changes

INTRODUCTION

It

is

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relatively

A preliminary

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evolutionary This

1970a)

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product

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arose

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codon

account o f this work has a p p e a r e d in N a t u r e

61

in

a s s i g n m e n t s a s s u m e d t h e i r p r e s e n t form w i t h a c o n c o m i t a n t i n c r e a s e in the c o m p l e x i t y of the m e c h a n i s m , and a final stage in w h i c h the p r o c e s s b e c a m e e x t r e m e l y r a p i d and precise. It is i m p l i c i t in the n a t u r e of e v o l u t i o n that e s s e n t i a l f e a t u r e s of an a r c h e t y p a l p r o c e s s t e n d to p e r s i s t as that p r o c e s s evolves. In o t h e r w o r d s , c e n t r a l f e a t u r e s of p r i m i tive p r o c e s s e s r e m a i n c e n t r a l f e a t u r e s of t h e i r m o r e e v o l v e d c o u n t e r p a r t s . Hence, it is r e a s o n a b l e to e x p e c t e x t a n t versions of the t r a n s l a t i o n a p p a r a t u s to m a n i f e s t c h a r a c t e r i s tics of t h e i r e v o l u t i o n a r y p r e d e c e s s o r s . A s p e c i f i c m o l e c u l a r m o d e l for the e v o l u t i o n a r y o r i g i n of the t r a n s l a t i o n m e c h a n i s m was p r o p o s e d five y e a r s ago (Woese, 197Ob; 1973). The e s s e n t i a l and e x p e r i m e n t a l l y v e r i f i a b l e f e a t u r e of this m o d e l is an a l l o s t e r i c t r a n s i t i o n in the a n t i - c o d o n arm of t R N A or its a n c e s t r a l e q u i v a l e n t s . T h e s e t r a n s i t i o n s a l l o w m R N A to be r a t c h e t e d t h r o u g h the r i b o s o m e in t r i p l e t fashion. In its p r i m i t i v e stages, this t r a n s l a t i o n m a c h i n e c o u l d have b e e n d r i v e n by e n v i r o n m e n t a l p e r t u r b a tions alone. H o w e v e r , this w o u l d p r o v i d e n e i t h e r a r a p i d nor an a c c u r a t e m e a n s of p o l y p e p t i d e synthesis. The e v o l u t i o n of a f a s t e r and m o r e p r e c i s e v e r s i o n of the m e c h a n i s m w o u l d of n e c e s s i t y lead to m a r k e d i n c r e a s e s in c o m p l e x i t y and h e n c e e f f e c t i v e size, since a c c u r a c y and speed are in e s s e n c e mutually exclusive. The c r u c i a l t u r n i n g p o i n t in the e v o l u t i o n of such a p r i m i t i v e t r a n s l a t i o n s y s t e m r e s t s u p o n a c q u i s i t i o n of the cap a c i t y to d r i v e the m e c h a n i s m c h e m i c a l l y , i.e., to p r o v i d e it w i t h the a b i l i t y to t r a n s d u c e c h e m i c a l into m e c h a n i c a l energy. In this way, not o n l y w i l l speed be a c h i e v e d but t h o s e e r r o r s i n h e r e n t in a d e v i c e d r i v e n by p e r t u r b a t i o n s a l o n e can n o w in p r i n c i p l e be o v e r c o m e . 5S r R N A has b e e n s t r o n g l y i m p l i c a t e d in the e n e r g e t i c a s p e c t s of t r a n s l a t i o n ( H o r n e & E r d m a n n , 1973; G r u m m t et al., 1974; G r u m m t & Grummt, 1974). Hence, it m a y be a c e n t r a l c o m p o n e n t in such an energetic t r a n s d u c e r . H e r e i n we o f f e r a s p e c i f i c s u g g e s t i o n as to h o w the 5S r R N A m o l e c u l e m i g h t f u n c t i o n in this c a p a c i t y .

II.

THE A R C H I T E C T U R E

OF

5S RIBOSO~tAL

RNA

It is n e c e s s a r y to a p p r o a c h the o v e r a l l a r c h i t e c t u r e of 5S r R N A t a b u l a r a s a . The m o l e c u l e is not n e c e s s a r i l y b u i l t u p o n the same p r i n c i p l e s as is tRNA, w h i c h m a y e x p l a i n the failure of n u m e r o u s p h y s i c a l - c h e m i c a l t e c h n i q u e s and s o p h i s t i c a t e d a n a l y s e s to r e v e a l its nature. The b i o l o g i s t ' s s i m p l e s t and o f t e n m o s t e f f e c t i v e weapon, c o m p a r a t i v e a n a l y s i s , has

82

b e e n s p a r i n g l y e m p l o y e d . C o n s e q u e n t l y , an e x t e n s i v e r e - e x a m i n a t i o n of the a v a i l a b l e c o m p a r a t i v e data on 5S r R N A has b e e n m a d e and is r e p o r t e d here. This a n a l y s i s p r o v i d e s c o n s i d e r able i n s i g h t to the p r i m a r y , s e c o n d a r y and t e r t i a r y structural c o n s t r a i n t s that u n d e r l i e the a r c h i t e c t u r e of this molecule. To date, six d i s t i n c t p r o c a r y o t i c s e q u e n c e s have b e e n de(Brownlee et al., 1967), Pseudomonas termined: Escherichia coli fluorescens (Dubuy & W e i s s m a n , 1971 ) , Bacillus stearothermophilus (Marotta et al., 1973), Bacillus megaterium (Pribula et al., (Corry et al. , 1974a) and Photobacter, 1974) , Anacystis nidulans s t r a i n 8265 (Woese et al., 1975). Four d i s t i n c t e u c a r y o t i c s e q u e n c e s have also been d e t e r m i n e d : KB cell (Forget & W e i s s man, 1967), Xenopus laevis - k i d n e y cell (Brownlee et al., (Nishikawa & T a k e m u r a , 1974) and 1972), Torulopsis utilis Chlorella pyrenoidosa (Jordan et al., 1974). In addition, sev e r a l s e q u e n c e s of c l o s e l y r e l a t e d o r g a n i s m s are k n o w n or can be inferred: Aerobacter aerogenes, Salmonella typhimurium, Erwinia aroideae, Proteus mirabilis (Sogin et al., 1972), Xenopus laevis o v a r y cell (Ford & S o u t h e r n , 1973), Saccharomyces carlsbergensis (Hindley & Page, 1972), Gallus gallus (chicken) (Pace et al., 1974), Yersinia pestis (Zablen et al., 1975). These s e q u e n c e s are a u g m e n t e d by the e x i s t e n c e of c a t a l o g s of v a r i o u s ribon u c l e a s e d i g e s t i o n p r o d u c t s from a b r o a d range of a d d i t i o n a l o r g a n i s m s : Serratia marcescens, Pseudomonas aeruginosa, Alcaligenes faecalis, Clostridium perfringens (Sogin et al., 1972), Vicia faba (broad bean) , Lycopersicum esculentum (tomato) , Helianthus anuus (sunflower) , Secale cereale (rye grain) , Phaseolus vulgaris (dwarf bean) (Payne et al. , 1 973) , and Oscillatoria tenuis C o r r y et al., 1974b) and u n p u b l i s h e d r e s u l t s in this l a b o r a t o r y for Acinetobacter calcoaceticus, Serratia marinoruba, Photobacter s t r a i n M A V , Clostridium thermosaccharolyticum and Bacillus subtilis. This f r a g m e n t a r y s e q u e n c e i n f o r m a t i o n a l l o w s large p o r t i o n s of a d d i t i o n a l s e q u e n c e s to be deduced. Even t h o u g h the e x i s t i n g d a t a are not yet f u l l y r e p r e s e n t a t i v e of the 5S r R N A spectrum, it does p r o v i d e s u f f i c i e n t range that i m p o r t a n t archit e c t u r a l f e a t u r e s s h o u l d be r e c o g n i z a b l e . E a r l i e r p r o p o s a l s for s e c o n d a r y s t r u c t u r e have been r e v i e w e d by S i d d i q u i (1973). Two r e c e n t m o d e l s (Nishikawa & T a k e m u r a , 1974; J o r d a n et al., 1974) have b e e n p r o p o s e d exc l u s i v e l y for the e u c a r y o t e s . K e a r n s & W o n g (1974) have a t t e m p t e d to infer the s e c o n d a r y s t r u c t u r e of E.Coli 58 r R N A from t h e i r N M R data. T h e r e is no g e n e r a l a g r e e m e n t as to the n a t u r e of the s e c o n d a r y s t r u c t u r e of this m o l e c u l e . H o w e v e r , there m u s t be h i g h l y c o n s e r v e d , if not t o t a l l y u n i v e r s a l , s t r u c t u r a l f e a t u r e s to the m o l e c u l e . This is s u g g e s t e d by the facts: (I) that 5S r R N A m u s t be in a p a r t i c u l a r c o n f o r m a -

63

i i !~ii i i i:~::~ ....

D,c'C'C.A U I G/ 'A'A- G" C ~

Fig.l. Schematic representation of 5S RNA secondary structure Normal base pairs are connected by vertical lines while G-U pairs are indicated by dots. The shaded portion of the molecule is that which has been identified as being involved in interaction with specific 5OS subunit proteins (Gray et al., 1973). The structure is drawn with helices aligned along the long axis in accord with the suggestion of Connors and Beeman (1972)

t i o n in o r d e r to b i n d to t h e 5OS p a r t i c l e ( A u b e r t et al., 1973) a n d (2) t h a t p r o c a r y o t i c (but n o t e u c a r y o t i c vs. p r o caryotic) 5S r R N A m o l e c u l e s are functionally interchangeable (Wrede & E r d m a n n , 1973; B e l l e m a r e et al., 1973). The present comparative a n a l y s i s s h o w s t h a t all k n o w n procaryotic s e q u e n c e s c a n be w r i t t e n in a g e n e r a l f o r m c o n taining four helices (Fig. 1 a n d T a b l e I). T h e e u c a r y o t i c seq u e n c e s c o n f o r m to m u c h t h e s a m e g e n e r a l s t r u c t u r e e x c e p t t h a t o n l y t h r e e of t h e f o u r h e l i c e s a r e p r e s e n t . T h e s e t h r e e h e l i ces h a v e b e e n s u g g e s t e d i n d e p e n d e n t l y by J o r d a n et al. (1974) in C h l o r e l l a on the b a s i s of n u c l e a s e a t t a c k , b u t t h e y f a i l e d to r e c o g n i z e t h e i r u n i v e r s a l i t y . T h e f i r s t h e l i c a l r e g i o n is t h e " m o l e c u l a r stalk", a s t r e t c h of a b o u t t e n b a s e p a i r s w h i c h c o n n e c t s t h e t w o t e r m i n i of t h e m o l e c u l e . Chlorella a p p e a r s to be an e x c e p t i o n in t h a t t h e t h r e e t e r m i n a l b a s e s at the 5' e n d a r e n o t P a i r e d . The molecular s t a l k is g e n e r a l l y q u i t e s t a b l e a n d o f t e n c o n tains G-U pairs. The second base-paired r e g i o n is t h e " p r o c a r y o t i c loop", a hair-pin comprising 4-5 b a s e p a i r s in p o s i t i o n 8 2 - 8 6 a n d 9 0 - 9 4 (all p o s i t i o n s throughout this paper are numbered T h i s h e l i x is o f t e n rea c c o r d i n g to t h e E.coli c o n v e n t i o n ) . m a r k a b l y r e s i s t a n t to e n z y m a t i c a t t a c k ( B r o w n l e e et al.,

64

V

•

*

•

•

¸

•

|

¥

Fig.2. Summary of experimental results relating to 5S rRNA secondary and tertiary structure The E.coli 5S rRNA sequence is shown. Brackets indicate regions of base-pairing and dots above individual nucleotides specify positions involved in G-U base pairs. The various experimental results are indicated as follows: + = the point of unambiguous carbodiimide substitution (Lee and Ingram, 1968); = regions (T 1 oligomers) attacked by nitrous acid (Bellemare et al., 1972); . . . . nucleotides which appear to be available for binding with external tri- and tetranucleotides (Lewis and Doty, 1970); • = guanosine residues readily substituted by kethoxal and glyoxal (Bellemare et al., 1972); and V = cytosine residues possibly attacked by methoxyamine (Bellemare et al., 1972). Where these points of attack are to some extent ambiguous, there is in all cases an interpretation consistent with the proposed structure. IParticularly pertinent is the oligomer -UCUCCCCAUG- attacked by nitrous acid and methoxyamine. The results of pancreatic nuclease digestion studies on 5S rRNA - protein complexes indicate that the accessible cytosine is in position 88 (Gray et al., 1972). This is completely consistent with the proposed structure. I

1 9 6 8 ) . It is f r e q u e n t l y b r i d g e d b y o n l y t h r e e b a s e s , t h e m i nimum sterically possible. One unresolved question concerns a possible alternate pairing (even p o t e n t i a l t r i p l e h e l i x f o r m a t i o n ) of p o s i t i o n s 8 2 - 8 8 in t h i s r e g i o n w i t h p o s i t i o n s 3 3 - 3 9 . H o w e v e r , t h i s " o t h e r " p a i r i n g is n o t u n i v e r s a l in t h a t both its precise location and its length vary somewhat. This pairing may be fortuitous, r e f l e c t i n g t h e l a c k of v a r i a b i l i t y so f a r e n c o u n t e r e d in t h e p r i m a r y s t r u c t u r e of t h e p o t e n tially-involved p o r t i o n s of t h e m o l e c u l e . T h e t h i r d h e l i c a l r e g i o n ( p o s i t i o n s 1 8 - 2 3 a n d 60-65) is especially interesting. Its l e n g t h o f s i x b a s e p a i r s is essentially universal a l t h o u g h i t s p r i m a r y s t r u c t u r e is h i g h ly v a r i a b l e (except for a terminal G-C pair, positions 23-60, w h i c h is u n i v e r s a l ) . As discussed previously (Woese et al., 1974), this helix seems to be under energetic constraints a n d f o r t h i s r e a s o n w i l l h e r e i n be r e f e r r e d t o as " t u n e d " .

65

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This t u n e d h e l i x is g e n e r a l l y w e a k e s t in m a r i n e p r o c a r y o t e s , p o s s i b l y r e f l e c t i n g the high ionic s t r e n g t h of their e n v i r o n ment. [ C o n s i s t e n t w i t h this, the i n t r a c e l l u l a r c a t i o n c o n c e n are signit r a t i o n s of the m a r i n e o r g a n i s m S e r r a t i a m a r i n o r u b a f i c a n t l y d i f f e r e n t from those of its close r e l a t i v e S e r r a t i a marcescens (Gale et al., 1970; C o l w e l l & Mandel, 1965)-I The p r o c a r y o t i c v e r s i o n s of the t u n e d h e l i x r e s o r t to the freq u e n t use of G-U and A - U pairs w h i l e the e u c a r y o t i c v e r s i o n s involve only "normal" p a i r i n g w i t h i n c r e a s e d usage of the s t r o n g e r G-C pairs. The 5' p r o x i m a l s t r a n d of the t u n e d h e l i x is s e p a r a t e d from the 3' p r o x i m a l s t r a n d by a s e g m e n t of 36 n u c l e o t i d e s . This segment, as d i s c u s s e d below, e n c o m p a s s e s a n o t h e r helix, r e s u l t i n g in two b u l g e loops. A l t h o u g h not c h a r a c t e r i s t i c of tRNA, this a r c h i t e c t u r a l p r i n c i p l e m a y in fact be c h a r a c t e r i s t i c of r i b o s o m a l RNAs. The final r e g i o n of b a s e - p a i r i n g is d e s i g n a t e d as the "common arm base" and i n v o l v e s p o s i t i o n s 31-34 and 48-51. This h e l i x c l o s e s a r a t h e r large loop and the d o u b l e - s t r a n d e d stalk is l o c a t e d i m m e d i a t e l y a d j a c e n t to the -CGAAC- s e q u e n c e w h i c h p r e s u m a b l y i n t e r a c t s w i t h the c o m m o n arm of tRNA (Erdm a n n et al., 1973). All p r o c a r y o t i c 5S rRNAs e x a m i n e d by p a n c r e a t i c n u c l e a s e d i g e s t i o n , i n c l u d i n g Sarcina, M i c r o coccus, A l c a l i g e n e s , L a c t o b a c i l l u s , S t r e p t o c o c c u s (Woese et al., u n p u b l i s h e d ) and O s c i l l a t o r i a (Corry et al., 1974) as well as those i n d i c a t e d in T a b l e 2 c o n t a i n the s e q u e n c e -pyrGAAC-. However, the e u c a r y o t e s do not c o n s e r v e this exact sequence. C h l o r e l l a and the f l o w e r i n g p l a n t s e x a m i n e d by Payne et al. (1973) have the s e q u e n c e - A G A A C - in this position, w h i l e the o t h e r e u c a r y o t e s have a s e q u e n c e w h i c h is s u g g e s t i v e of i n t e r a c t i o n w i t h the c o m m o n a r m of the i n i t i a tor (Dube, 1973) r a t h e r than the n o r m a l tRNAs. The c o m m o n arm base c o n t a i n s four pairs in all o r g a n i s m s so far e x a m i n e d e x c e p t T o r u l o p s i s , w h e r e it a p p e a r s to have three. Due to its small size and h i g h l y c o n s e r v e d p r i m a r y structure, o n l y four v e r s i o n s of this h e l i x h a v e b e e n found to date. In a d d i t i o n to these r e g i o n s of b a s e - p a i r i n g , s e v e r a l o t h e r a r c h i t e c t u r a l p r o p e r t i e s of p r o c a r y o t i c 5S rRNA a p p e a r in the p r e s e n t analysis. M o s t d i s t i n c t i v e is a r e m a r k a b l e s t r e t c h of m o r e than t h i r t y n u c l e o t i d e s that are very h i g h l y c o n s e r v e d (positions 28-59). The c o m m o n arm base and its loop are found herein. Due to the n u m b e r of large T I d i g e s tion p r o d u c t s u s u a l l y p r o d u c e d by this segment, it is o f t e n p o s s i b l e to d e d u c e its s e q u e n c e from p r e l i m i n a r y data alone. Table 2 shows the s e q u e n c e in this r e g i o n for all those organisms w h e r e it is k n o w n o u t r i g h t or can be deduced. Table 3 p r o v i d e s a d i s t a n c e m a t r i x of the v a r i o u s c o m p a r i s o n s t h a t can be made. A l t h o u g h this short s t r e t c h of bases is c l e a r l y

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