Initiation of Transcription - a General Tool for Affinity Labeling of RNA Polymerase« by Autocatalysis

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B i o l . C h e m . Hoppe-Seyler

V o l . 369, pp. 7 7 5 - 7 8 8 , September 1988

R E V I E W

Twelfth Fritz Lipmann Lecture

d e l i v e r e d at t h e J o i n t A n n u a l M e e t i n g o f t h e G e s e l l s c h a f t für B i o l o g i s c h e C h e m i e a n d t h e Ö s t e r r e i c h i s c h e B i o c h e m i - s c h e G e s e l l s c h a f t o n S e p t e m b e r 1 2 , 1 9 8 8 , I n n s b r u c k , A u s t r i a b y Guido Hart mann.

Initiation of Transcription - a General Tool for Affinity Labeling of RNA Polymerase« by Autocatalysis

Guido R . H A R T M A N N³, Christof B I E B R I C H E R⁰, Stephan J . G L A S E R³, Frank G R O S S E^C, Michael J . K A T Z A M E Y E R³, Anton J . L I N D N E R³, Helmut M O S I G³, Heinz-Peter N A S H E U E R^C,

Lucia B . R O T H M A N - D E N E S0'6, Anton R . S C H A F F N E R3, George J . S C H N E I D E R6, Karl-Otto S T E T T E RF

and Michael T H O M M^F

a I n s t i t u t f ü r B i o c h e m i e , L u d w i g - M a x i m i l i a n s - U n i v e r s i t ä t , D - 8 0 0 0 M ü n c h e n 2 .

b M a x - P l a n c k - I n s t i t u t f ü r B i o p h y s i k a l i s c h e C h e m i e , D - 3 4 0 0 G ö t t i n g e n - N i k o l a u s b e r g .

c M a x - P l a n c k - I n s t i t u t f ü r E x p e r i m e n t e l l e M e d i z i n , D - 3 4 0 0 G ö t t i n g e n .

d D e p a r t m e n t o f M o l e c u l a r G e n e t i c s a n d C e l l B i o l o g y , T h e U n i v e r s i t y o f C h i c a g o , I L 6 0 6 3 7 , U . S . A .

e D e p a r t m e n t o f B i o c h e m i s t r y a n d M o l e c u l a r B i o l o g y , T h e U n i v e r s i t y o f C h i c a g o , C h i c a g o , I L 6 0 6 3 7 , U . S . A

f L e h r s t u h l f ü r M i k r o b i o l o g i e , U n i v e r s i t ä t R e g e n s b u r g , D - 8 4 0 0 R e g e n s b u r g .

( R e c e i v e d 2 4 J u n e 1 9 8 8 )

It is well known that the transcription of struc- tural genes in eukaryotic cells is a highly regu- lated process. For this regulation, the D N A template contains numerous specific sequences which control the frequency and the precise start of initiation of transcription. There is ample evidence that these regulatory nucleotide sequences are recognized by a multitude of protein factors. In the presence of these factors, specific transcripts can be synthesized in vitro from suitable templates with highly purified R N A Polymerase B (II)^{1 1}'. In the absence of these factors very few free R N A chains of de- fined length are made. One possible explana- tion for this Observation is that purified eu- karyotic R N A Polymerase B (II) - as opposed to the eubacterial enzyme — does not bind to specific D N A sequences directly. Instead, it is believed that the enzyme recognizes a specific

complex of factors with their cognate pro- motor elements for initiation of transcrip- t i o n ^ .

Start of RNA synthesis in vitro

We were quite surprised when we found excep- tions to the aforementioned rule. In the follow- ing experiment, we used as template a blunt- ended D N A fragment of 1303 bp length (Fig. 1) which contains the promoter region and R N A initiation site followed by 128 bp of the coding region of one of the zein genes in maize^{1 3 1}. Upon incubation with highly purified R N A Polymerase B (II) from wheat g e r m^{l 4 ]} together with radio- actively labeled Substrate, R N A is formed which migrates during electrophoresis in the form of two closely spaced, discrete bands with apparent lengths of 750 and 650 nucleotides (Fig. la).

Enzymes:

D N A - d i r e c t e d R N A P o l y m e r a s e , n u c l e o s i d e - t r i p h o s p h a t e : R N A n u c l e o t i d y l t r a n s f e r a s e ( D N A - d i r e c t e d ) ( E C 2 . 7 . 7 . 6 ) ;

D N A - d i r e c t e d D N A P o l y m e r a s e , d e o x y n u c l e o s i d e - t r i p h o s p h a t e : D N A d e o x y n u c l e o t i d y l t r a n s f e r a s e ( D N A - d i r e c t e d ) ( E C 2 . 7 . 7 . 7 ) ;

a l k a l i n e P h o s p h a t a s e , o r t h o p h o s p h o r i c - m o n o e s t e r p h o s p h o h y d r o l a s e ( a l k a l i n e o p t i m u m ) ( E C 3 . 1 . 3 . 1 ) ; n u c l e o t i d e p y r o p h o s p h a t a s e , d i n u c l e o t i d e n u c l e o t i d o h y d r o l a s e ( E C 3 . 6 . 1 . 9 ) ;

d e o x y r i b o n u c l e a s e I, ( E C 3 . 1 . 2 1 . 1 ) ; r i b o n u c l e a s e T² ( E C 3 . 1 . 2 7 . 1 ) ;

r i b o n u c l e a s e ( p a n c r e a t i c ) ( E C 3 . 1 . 2 7 . 5 ) ;

T r i t i r a c h i u m a l k a l i n e P r o t e i n a s e ( E C 3 . 4 . 2 1 . 1 4 ) , a l s o n a m e d P r o t e i n a s e K . Abbreviation:

b p , base p a i r s .

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a b c d e f g h i k l m n

F i g . 1. I n f l u e n c e o f s p e c i f i c c l e a v a g e o f a p r o m o t e r - c o n t a i n i n g t e m p l a t e o n t r a n s c r i p t i o n b y h i g h l y p u r i f i e d R N A P o l y m e r a s e B ( I I ) f r o m w h e a t g e r m .

L a n e a: 0 . 2 5 p m o l o f a 1 3 0 3 b p D N A f r a g m e n t ( s h o w n s c h e m a t i c a l l y ) w h i c h w a s o b t a i n e d b y d i g e s t i o n o f a p U C 1 2 v e c t o r c o n t a i n i n g a z e i n g e n e ^³! w i t h PvuW, w a s i n c u b a t e d w i t h a l l f o u r r i b o n u c l e o s i d e t r i p h o s p h a t e s a n d 0 . 1 8 jug e n - z y m e l⁴! f o r 3 0 m i n at 3 0 ° C u n d e r c o n d i t i o n s s i m i l a r as d e s c r i b e d ^ J , b : as i n a, b u t t e m p l a t e c l e a v e d w i t h Hinfl;

c: as i n a, b u t w i t h t h e 3 4 1 b p Hinfl f r a g m e n t as t e m p l a t e ; d : as i n a , b u t w i t h t h e 9 6 2 b p Hinfl f r a g m e n t ; e: as i n b ; f: as i n e, b u t f o l l o w e d b y i n c u b a t i o n w i t h 2 jug d e o x y r i b o n u c l e a s e I; g : as i n e, b u t f o l l o w e d b y i n c u b a t i o n w i t h 2 jug r i b o n u c l e a s e A ; h : as i n e; i : as i n h , b u t w i t h o u t t e m p l a t e ; k : as i n h , b u t w i t h o u t e n z y m e ; 1: as i n h , b u t w i t h o u t M n C l²; m : as i n h , b u t i n p r e s e n c e o f 1 fJig/ml a- a m a n i t i n ; n : as i n h , b u t i n p r e s e n c e o f 2 Hg/ml h e p a r i n . A u t o r a d i o - g r a p h i c a n a l y s i s w a s p e r f o r m e d a f t e r e l e c t r o p h o r e s i s i n a 4 % P o l y a c r y l a m i d e g e l c o n t a i n i n g 6 M u r e a .

H o w e v e r , w h e n the template is first cleaved at its Single Hinfl site t w o a d d i t i o n a l very intense radioactive bands w i t h length o f a p p r o x i m a t e l y 3 3 0 and 3 4 0 nucleotides appear ( F i g . l b , e, h).

A l l four labeled bands consist o f R N A since they can be h y d r o l y s e d w i t h ribonuclease ( F i g . l g ) . H o w e v e r , the r a d i o a c t i v i t y o f the upper t w o bands, consists o f labeled r i b o n u c l e o t i d e s c o - valently b o u n d to D N A since the r a d i o a c t i v i t y migrates o u t o f the gel u p o n i n c u b a t i o n w i t h d e o x y r i b o n u c l e a s e ( F i g . l f ) . O b v i o u s l y , w i t h - out the specific cleavage o f this template, R N A Polymerase B (II) does not initiate de n o v o but o n l y elongates at n i c k s or ends o f the template.

T h i s is i n agreement w i t h earlier o b s e r v a t i o n s '^{5 1}. A l l four radioactive bands are not f o r m e d i n the absence o f e n z y m e ( F i g . l k ) n o r i n the absence o f D N A ( F i g . 1 i) or M n^{2 e} ( F i g . 11) o r i n the presence o f either 1 jug/m/ a - a m a n i t i n ( F i g . I m ) or 2 /xg/m/ heparin ( F i g . I n ) . The bands are synthesized w h e n 0. 8 m M M n^{2 @} is replaced b y 8mM M g² (data not s h o w n ) . T h e t w o R N A

bands i n i t i a t e d de n o v o are transcribed f r o m the same Hinfl cleavage fragment o f a p p r o x i - mately 3 4 0 bp i n length as evident f r o m e x p e r i - ments where the separated Hinfl fragments were used as template ( F i g . l c , d). The s t i c k y ends o f the Hinfl cleavage site, present o n b o t h fragments, are alone not sufficient to i n d u c e the de novo i n i t i a t i o n o f R N A synthesis. T h i s c o n - c l u s i o n is s u p p o r t e d b y experiments w i t h an isolated D N A fragment 3 1 6 bp l o n g o b t a i n e d b y cleavage o f p U C 1 2 w i t h Pvull and w i t h another fragment o f 521 bp o b t a i n e d b y cleavage o f p U C 1 2 w i t h i 4 / w l . O n l y w i t h the 316 bp fragment are large a m o u n t s o f free R N A synthesized after cleavage o f the template w i t h Hinfl (data not s h o w n ) ( H . M o s i g , P h . D . thesis, F a k u l t ä t für C h e m i e u n d Pharmazie der L u d w i g - M a x i m i l i a n s - U n i v e r s i t ä t M ü n c h e n , i n preparation). P r o b a b l y some a d d i t i o n a l sequence requirements, missing in the latter fragment, must be met. H o w e v e r , the p r o t r u d i n g 5^;-end o f the template is essential for the de n o v o synthesis. A f t e r e x t e n s i o n o f the

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recessive 3'-ends with D N A Polymerase I, the de novo R N A product is no longer formed (Fig. 2a). Both newly initated R N A chains start with A T P since they become labeled only when [7-^{3 2}P]ATP is used as Substrate (Fig. 2b).

To determine the start site, the 5'-terminal 35 nucleotides of the R N A of 330 nucleotides length were sequenced.

DNA 5 ' - A A T C A A A A T A G A T G T A T A C C T A A C A T C A G C A A A T G G A A A T A A A A . . . - 3 * 3 ' - G T T T T A T C T A C A T A T G G A T T G T A G T C G T T T A C C T T T A T T T T . . . - 5 '

RNA p p p A G A U G U A U A C C U A A C A U C A G C A A A U G G A A A U A A A A . . .

Comparison of this sequence with that of the template^{1 3 1} reveals that the start site of R N A synthesis is at the 7th base pair downstream of the double stranded part of the Hinfl cleavage site. This start is not the one selected in vivo and in vitro R N A synthesis proceeds opposite to the in vivo direction (scheme in Fig. 1).

Abortive initiation

The start of the R N A Polymerase reaction in vitro can also be studied by abortive initia-

a) fl b)

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F i g . 2 . I n f l u e n c e o f t h e p r o t r u d i n g e n d s o f t h e t e m p l a t e o n t r a n s c r i p t i o n a n d d e t e r m i n a t i o n o f t h e 5 ' - n u c l e o t i d e o f t h e t r a n s c r i p t ( d e n s i t o m e t r i c t r a c i n g o f a u t o r a d i o - g r a p h y ) .

a) E x p e r i m e n t A : t r a n s c r i p t i o n o f t h e / f t w f l - c l e a v e d t e m p l a t e w a s d o n e as d e s c r i b e d i n F i g . 1, l a n e b ; e x p e r i - m e n t B : t h e p r o t r u d i n g e n d s o f t h e / / z w f l - c l e a v e d f r a g - m e n t s w e r e f i l l e d b y i n c u b a t i o n w i t h D N A P o l y m e r a s e I ( K l e n o w f r a g m e n t ) a n d t h e t e m p l a t e w a s r e i s o l a t e d b y p h e n o l t r e a t m e n t a n d t r a n s c r i b e d as b e f o r e . b ) T r a c i n g A : t r a n s c r i p t i o n i n t h e p r e s e n c e o f [ a-^{3 2}P ] U T P ; t r a c i n g B : i n t h e p r e s e n c e o f [ 7 -^{3 2}P ] A T P ; t r a c i n g C : i n t h e p r e s c e n c e o f [ 7 -^{3 2}P ] G T P as l a b e l e d s u b s t r a t e .

t i o n^{| 6 , 7 ]}. Here, R N A Polymerase is incubated with template and an incomplete set of ribo- nucleoside triphosphates as Substrate. Under these conditions mainly di- or trinucleotides are formed which are readily released from the transcription complex.

P P P NI + P P p N² P P p N i p N² + p p

P P p N i p N² + P P p N³ p p p N ! p N²p N³ + p p

The product contains at its 5'-terminus the intact triphosphate group of the starting nu- cleotide. Under abortive initiation conditions the first step of the R N A Polymerase reaction is continuously repeated. This reaction is catalysed by eubacterial R N A polymerases^{[ 6 1} as well as by eukaryotic R N A Polymerase B ( I I )^{I 7 , 8 ]}. In the case of the highly purified eukaryotic enzyme, however, it is not known if abortive initiation requires the same condi- tions as the initiation of unprimed synthesis of R N A . In vivo the start site of eukaryotic m R N A synthesis is much less precisely defined than in bacteria and may start at several nucleotides within a small sequence^{[ 9 )}. There- fore a direct comparison of the sequence of the product of abortive initiation with the 5'- terminus of the R N A formed in vivo is incon- clusive. If under our experimental conditions (Fig. 1) abortive initiation really represents the initial step of de novo R N A synthesis in vitro, one would expect to observe significant dinucleoside tetraphosphate synthesis only after cleavage of the template with Hinfl and with the shorter of the two Hinfl cleavage frag- ments. Indeed, only the shorter fragment leads to the formation of large amounts of p p p A p U (Fig. 3B). To a small extent pppApA can be synthesized on both fragments (Fig. 3 A , E).

If abortive initiation occurs exactly at the same site of the template as the synthesis of R N A with the sequence p p p A p G p A at its 5'-terminus Starts, then formation of p p p A p G would be expected, but is not observed (Fig. 3C, G). Also a combination of A T P and CTP is inactive (Fig. 3D, H). In agreement with earlier studies^{l 7 ]} these observations indicate that p p p A p U is the preferred product if allowed by the sequence of the template. A distinct Stimulation of abor- tive initiation, similar to the increase in synthesis of free R N A when the template is cleaved with Hinfl, is also observed with the D N A fragment

of 316 bp length obtained by cleavage of p U C l 2 with Pvull (data not shown).

It was shown above that the protruding 5'-termi- nus of the Hinfl cleavage site is necessary for the induction of de novo R N A synthesis in vitro (Fig. 2a). The same is found for p p p A p U syn- thesis. Repairing the gap of tlie Hinfl cleavage

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F

c E

. —Ju

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F i g . 3 . I n f l u e n c e o f s p e c i f i c c l e a v a g e o f a p r o m o t e r - c o n t a i n i n g t e m p l a t e o n t h e a b o r t i v e i n i t i a t i o n b y w h e a t g e r m R N A P o l y m e r a s e B ( I I ) .

T h e e x p e r i m e n t s w e r e c a r r i e d o u t u n d e r i d e n t i c a l c o n - d i t i o n s as u s e d f o r t r a n s c r i p t i o n ( F i g . 1) e x c e p t t h a t o n l y o n e o r t w o Substrates w e r e a d d e d . T h e d i n u c l e o s i d e t e t r a p h o s p h a t e s w e r e s e p a r a t e d b y e l e c t r o p h o r e s i s i n a 2 5 % P o l y a c r y l a m i d e g e l c o n t a i n i n g 6 M u r e a . T h e d e n s i t o m e t r i c t r a c i n g s o f t h e a u t o r a d i o g r a p h y are s h o w n . T e m p l a t e : t h e 3 4 1 b p ( A - D ) o r 9 6 2 b p ( E - H ) f r a g m e n t o b t a i n e d b y Hinfl c l e a v a g e o f t h e 1 3 0 3 b p f r a g m e n t d e s c r i b e d i n F i g . 1. S u b s t r a t e : [ a -3 2P ] A T P ( A , E ) ; [ a -^{3 2}P ] A T P + U T P ( B , G ) ; [ a -^{3 2}P ] A T P + G T P ( C , G ) ; [ a -3 2P ] A T P + C T P ( D , H ) .

site leads to the loss of template activity also in abortive initiation (Fig. 4).

The following conclusions may be drawn from these experiments:

1) In the absence of additional protein factors, eukaryotic R N A Polymerase B (II) from plants can initiate selectively the synthesis of free R N A of defined length. This is in agreement with

observations made with R N A Polymerase B (II) from calf t h y m u s^{1 1 0 1}.

2) Efficient abortive initiation by purified eukaryotic R N A Polymerase B (II) requires properties of the template similar to those re- quired for the unprimed synthesis of R N A of specific length.

Labeling of the active site by autocatalysis The findings described above suggest an applica- tion of an elegant method developed for labeling the region which contains the active center of Escherichia coli R N A Polymerase1 1 1 , 1 2 1 to eukaryotic R N A polymerases. The essential features of this method, as it is used in most of our experiments, consists of incubating the R N A Polymerase with a chemically reactive nucleotide derivative in the absence of template and Substrate^{1 1 3 1}. Numerous different derivati- ves have been synthesized^{1 1 4}'^{1 5 1}. The chemical structure of only one of them is shown.

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F i g . 4 . I n f l u e n c e o f t h e p r o t r u d i n g e n d s o f t h e t e m p l a t e o n a b o r t i v e i n i t i a t i o n .

T h e d e n s i t o m e t r i c t r a c i n g s o f t h e a u t o r a d i o g r a p h y o f the electrophoretic Separation are shown. U p p e r t r a c i n g : a b o r t i v e i n i t i a t i o n w i t h t h e / / / « f l - c l e a v e d t e m p l a t e ( F i g . 1 ) w i t h A T P a n d [ a -^{3 2}P ] U T P as s u b s t r a t e as d e - s c r i b e d i n F i g . 3 . L o w e r t r a c i n g : T h e s a m e e x p e r i m e n t w i t h t h e / / / w f l - c l e a v e d t e m p l a t e w h o s e p r o t r u d i n g e n d s h a d b e e n f i l l e d b y i n c u b a t i o n w i t h D N A P o l y m e r a s e I ( K l e n o w f r a g m e n t ) .

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O H

4-Hydroxybenzaldehyde ester of ATP

If the c h e m i c a l l y reactive group in the nucleotide derivative is benzaldehyde, the reactive group may f o r m a Schiff-base w i t h the e-amino group o f lysine residues i n the P o l y p e p t i d e chain (equation 1).

P o l y p e p t i d e - N H² + 0 = C H - C⁶H⁴- 0 - p p p A -+ ( 1 ) p o l y p e p t i d e - N = C H — C6H4 - O - p p p A

P o l y p e p t i d e - N = C H - C⁶H⁴- 0 - p p p A ^N^a^B ^H⁴ > ( 2 ) p o l y p e p t i d e - N H - C H2- C6H4- 0 - p p p A

D N A

P o l y p e p t i d e - N H - C H2- C6H4- 0 - p p p A + p p p * U • p o l y p e p t i d e - N H - C H²- C⁶H⁴- 0 - p p p A p * U + p p

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These derivatives w i l l react preferentially w i t h amino-acid residues in the v i c i n i t y o f nucleotide- b i n d i n g sites on the e n z y m e such as the b i n d i n g site w i t h i n the active center o f R N A P o l y - merase. However, a strict selectivity o f b i n d i n g is not essential for the success o f this m e t h o d . In a second reaction the unstable Schiff-base is converted i n t o a stable secondary amine by r e d u c t i o n under m i l d c o n d i t i o n s w i t h s o d i u m b o r o h y d r i d e (equation 2). S i m u l t a n e o u s l y , any excess o f the aldehyde derivative is reduced to the non-reactive a l c o h o l . So far the m o d i f i e d en-

z y m e is not labeled radioactively. T o avoid un- specific l a b e l i n g^{1 1 6 1} r a d i o a c t i v i t y is i n t r o d u c e d in the next step (equation 3) where the m o d i f i e d enzyme is incubated w i t h template and a Single radioactive nucleoside triphosphate. O f rele- vance are enzyme molecules w h i c h contain the derivative covalently b o u n d such that its nucleotide m o i e t y is b o u n d to the active site in the same c o n f i g u r a t i o n as the p r i m i n g nucleotide during abortive i n i t i a t i o n . If such a m o d i f i e d en- z y m e is still c a t a l y t i c a l l y active in a template- dependent m o d e , then a phosphodiester b o n d w i l l be formed by autocatalysis between the covalently attached nucleotide and the added radioactive second Substrate. The result is a covalently b o u n d d i n u c l e o t i d e w h i c h by its radioactivity f i r m l y labels the P o l y p e p t i d e to w h i c h it is attached. T h e distance o f the active center to the site o f attachment cannot be larger than the length o f the spacer between the nucleotide and the C-a-atom o f the a m i n o acid to w h i c h the nucleotide is b o u n d (about 1.5 — 2.5 n m ) . W i t h o u t covalent m o d i f i c a t i o n or re- d u c t i o n by b o r o h y d r i d e , or in the absence o f template no radioactive labeling should be observed. F u r t h e r m o r e , the o l i g o n u c l e o t i d e label should disappear after digestion w i t h proteases but not u p o n i n c u b a t i o n w i t h d e o x y - ribonuclease or ribonuclease.

The l o c a t i o n o f the labeled dinucleotide can be a p p r o x i m a t e l y determined by an elegant M a x a m - G i l b e r t type o f amino-acid specific l i m i t e d p r o t e o l y s i s1 1 7 - 1 9 1 provided the enzyme contains o n l y one molecule o f covalently b o u n d labeled dinucleotide per P o l y p e p t i d e chain.

8 8

5' 2'

H l

ii Ii t i i m i l IUI I H ,

^c

— I C

F i g . 5. K i n e t i c s o f the l i m i t e d cleavage b y C N B r o f the a f f i n i t y - l a b e l e d T 7 R N A P o l y m e r a s e .

0 . 2 jug T 7 R N A P o l y m e r a s e l a b e l e d s i m i l a r l y as d e s c r i b e d1 2 1 1 was t r e a t e d w i t h 5 0 m M C N B r i n 5 0 m M H C l i n the p r e s e n c e o f 0 . 0 5 % d o d e c y l s u l f a t e at 3 7 ° C . C l e a v a g e was t e r m i n a t e d b y n e u t r a l i s a t i o n . T h e p r o d u c t s w e r e s e p a r a t e d b y e l e c t r o p h o r e s i s i n the p r e s e n c e o f 0.1 % d o d e c y l s u l f a t e i n a gel c o n t a i n i n g a g r a d i e n t f r o m 7 . 5 - 2 0 % P o l y a c r y l - a m i d e . L a n e s a, b , c: 1, 2 , o r 5 m i n i n c u b a t i o n w i t h C N B r . T h e m i g r a t i o n o f t h e f r a g m e n t s is f r o m left t o r i g h t (the P o s i t i o n o f m a r k e r p r o t e i n s is i n d i c a t e d ) . T h e m o b i l i t y o f the m o s t r a p i d l y m i g r a t i n g s p o t c o r r e s p o n d s t o M^T ~ 2 0 0 0 0 . T h e d i s t r i b u t i o n o f the M e t - r e s i d u e s i n the P o l y p e p t i d e c h a i n is s h o w n s c h e m a t i c a l l y b e l o w the a u t o r a d i o g r a p h y .

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a b c d e f g h i k l m n

F i g . 6. A f f i n i t y l a b e l i n g b y a u t o c a t a l y s i s o f the R N A p o l y m e r a s e s f r o m b a c t e r i o p h a g e T 3 a n d N 4 .

Experiments with T3 RNA Polymerase /lanes a-f): a: the i n c u b a t i o n was c a r r i e d o u t s i m i l a r l y as d e s c r i b e d '^{2 1}' w i t h 0 . 5 / i g T 3 R N A P o l y m e r a s e , T 3 D N A a n d the 4 - h y d r o x y b e n z a l d e h y d e e s t e r o f G T P ; b : as i n a, b u t w i t h o u t d e r i v a t i v e : c: as i n a, b u t w i t h o u t D N A ; d : as i n a, b u t w i t h o u t r e d u e t i o n ; e: as i n b , b u t w i t h a d d e d G T P ; f: as i n a. but w i t h s u b - s e q u e n t i n c u b a t i o n w i t h P r o t e i n a s e K .

Experiments with N4 virion RNA Polymerase^^23;'²⁴¹ (lanes g and h): g : 0 . 6 6 fig e n z y m e was m o d i f i e d w i t h 4 - h y d r o x y - b e n z a l d e h y d e e s t e r o f G T P s i m i l a r l y as i n r e f . '2 1 1 b u t i n p r e s e n c e o f 2 ßg h e a t - d e n a t u r e d N 4 D N A . A f t e r r e d u e t i o n D N A was a d d e d a g a i n . I n c u b a t i o n was c a r r i e d o u t w i t h [ a -3 2 P ] A T P ; h : as i n g , b u t w i t h o u t d e r i v a t i v e .

Experiments with N4 RNA Polymerase / / '2 5'2 61 (lanes i-n): 5 / i g o f a p 4 p 7 p r e p a r a t i o n was m o d i f i e d w i t h 4 - h y d r o x y - b e n z a l d e h y d e e s t e r o f A T P s i m i l a r l y as i n r e f . '2 1 1 b u t i n p r e s e n c e o f h e a t - d e n a t u r e d N 4 D N A . T h e S u b s t r a t e f o r the e n z y m a t i c r e a c t i o n was [ a -^{3 2}P ] U T P ; k : as i n i , b u t w i t h o u t d e r i v a t i v e ; 1: as i n i , b u t w i t h o u t D N A ; m : as i n i . b u t w i t h o u t r e d u e t i o n ; n : as i n i , b u t f o l l o w e d b y i n c u b a t i o n w i t h P r o t e i n a s e K .

E x p e r i m e n t s performed during recent years have s h o w n that this affinity labeling o f R N A p o l y - merases b y autocatalysis can be applied rather generally to m a n y enzymes o f this type w i t h interesting results c o n c e r n i n g the region w h i c h contains the active site.

DNA-Directed RNA polymerases from phages W i t h T 7 R N A Polymerase, w h i c h contains o n l y a Single P o l y p e p t i d e c h a i n , strong labeling was o b s e r v e d^{1 2 0}'^{2 1 1}. It was also s h o w n that this labeling is not o n l y D N A - d e p e n d e n t but s t r i c t l y p r o m o t e r - c o n t r o l l e d '^{2 1}' . T o o b t a i n labeling, the reactive nucleotide derivative can be attached to the R N A Polymerase p r i o r to the a d d i t i o n o f the template. It follows that the active site is accessible for the derivative i n the absence o f template and, furthermore, that the correct b i n d i n g o f the template to the e n z y m e is not prevented b y the covalent attachment o f the nucleotide derivative.

What do we k n o w about the p o s i t i o n o f the labeled site? T h e T 7 R N A Polymerase, w i t h a length o f 883 a m i n o acids contains a very trypsin-sensitive site about 170 a m i n o acids away f r o m the N - t e r m i n u s '^{2 1}' . When the native e n z y m e labeled w i t h the 4 - h y d r o x y b e n z a l - dehyde ester o f G T P is digested for a very short time w i t h t r y p s i n , this small fragment is cleaved o f f w i t h o u t loss o f r a d i o a c t i v i t y i n the remain- ing large fragment. Therefore the label cannot be attached to the N - t e r m i n a l part o f the en- z y m e^{1 2 1 1}. O n the other h a n d , a very short i n - c u b a t i o n o f labeled intact T 7 R N A Polymerase w i t h eyanogen b r o m i d e^{1 1 8 1}, w h i c h cleaves next to m e t h i o n i n e residues, results in the f o r m a t i o n o f a small labeled P o l y p e p t i d e (M^r ~ 20 k) ( F i g . 5). T h e e n z y m e contains 26 m e t h i o n i n e residues scattered more or less evenly over the whole P o l y p e p t i d e c h a i n . Therefore, the appearance o f the small radioactively labeled P o l y - peptide w i t h i n a very short time o f i n c u b a t i o n indicates that the site labeled w h e n the 4 h y -

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d r o x y b e n z a l d e h y d e ester o f G T P is used as the m o d i f y i n g nucleotide derivative is located close to one o f the t w o t e r m i n i . Since the N-terminus has been previously e x c l u d e d '^{2 1 1} the short labeled B r C N - c l e a v e d fragment must be derived from the C-terminus ( F i g . 5). T h i s c o n c l u s i o n is supported by a d d i t i o n a l k i n e t i c experiments based o n the rapid cleavage o f the t w o A s n - G l y bonds in T 7 R N A Polymerase by h y d r o x y l a m i n e (data not s h o w n ) .

T h e R N A Polymerase c o d e d for by the genome of the bacteriophage T 3 is closely related to the T 7 R N A P o l y m e r a s e '^{2 2 1}. Therefore it is not surprising that affinity labeling o f this enzyme by autocatalysis w i t h T 3 D N A as template is also achieved ( F i g . 6a).

The R N A Polymerase encapsulated i n the v i r i o n o f the bacteriophage N 4 is very different from the T 3 or T 7 R N A p o l y m e r a s e s^{1 2 3 1}. The N 4 v i r i o n R N A Polymerase consists o f a very long P o l y p e p t i d e chain (M^T ~~ 3 2 0 k) and recognizes Promoters w i t h G p A or G p G R N A starting S i t e s^{1 2 4 1}. The genome o f bacteriophage N 4 codes for a second R N A Polymerase, w h i c h transcribes N 4 m i d d l e R N A s during the N 4 life c y c l e . The core f o r m o f this e n z y m e consists o f t w o subunits w i t h Af^r o f about 30 k (p7) and 4 0 k ( p 4 )^{1 2 5 1}. Here, the transcribed R N A s start w i t h A T P1 2 5 , 2 6 1. In each case a specific labeling o f the e n z y m e by autocatalysis is observed ( F i g . 6g and F i g . 6i—n). O f the t w o subunits, w h i c h constistute N 4 R N A Polymerase II, the p o l y

a b c d e f g h i k l m n o p q r

F i g . 7. A f f i n i t y l a b e l i n g b y a u t o c a t a l y s i s o f the R N A P o l y m e r a s e f r o m the G r a m - n e g a t i v e Thermotoga maritima s p . n o v . , Anabaena 11 2 0 a n d the G r a m - p o s i t i v e Lactobacillus curvatus.

Lanes a-i: E x p e r i m e n t s w i t h t h e e n z y m e f r o m Anabaena^29). a: 2 / i g e n z y m e w a s m o d i f i e d w i t h 0 . 3 m M 4 - h y d r o x y - b e n z a l d e h y d e ester o f A T P i n the p r e s e n c e o f 1 / i g p l a s m i d p T E 5 5 ( E l l i o t , T . , K a s s a v e t i s , G . A . & G e i d u s c h e k , E . P . ( 1 9 8 4 ) , c i t e d i n r e f . '2 9 1) c o n t a i n i n g a s t r o n g p r o m o t e r f o r t h i s R N A P o l y m e r a s e . A f t e r r e d u e t i o n the e n z y m a t i c l a b e l i n g w a s c a r r i e d o u t w i t h 0 . 1 6 / i M [ o >^{3 2}P ] U T P ; b : as i n a, b u t w i t h o u t d e r i v a t i v e ; e: as i n a, b u t w i t h o u t D N A ; d : as i n a, b u t w i t h o u t r e d u e t i o n ; e : as i n a, b u t f o l l o w e d b y i n c u b a t i o n w i t h P r o t e i n a s e K ; f: as i n a, b u t f o l l o w e d b y i n c u b a t i o n w i t h r i b o n u c l e a s e ; g: as i n a, b u t f o l l o w e d b y i n c u b a t i o n w i t h d e o x y r i b o n u c l e a s e ; h : as i n a, b u t w i t h 4 - [ A ^ - ( 2 ' - h y d r o x y e t h y l ) - A ^ - m e t h y l ] a m i n o b e n z a l d e h y d e e s t e r o f A D P ; i : as i n h , b u t w i t h the ester o f A M P as the d e - r i v a t i v e .

Lanes k-n: E x p e r i m e n t s w i t h the e n z y m e f r o m Thermotoga^2^ w e r e c a r r i e d o u t w i t h 4 - h y d r o x y b e n z a l d e h y d e ester o f A T P as the d e r i v a t i v e as d e s c r i b e d '3 7! , b u t w i t h 4 0 m M M g C l2; the e n z y m a t i c l a b e l i n g w a s p e r f o r m e d at 55 C k : c o m p l e t e m i x t u r e ; 1: as i n k , b u t w i t h o u t D N A ; m : as i n k , b u t w i t h o u t r e d u e t i o n ; n : as i n k , b u t i n the presenc-e o f 1 0 0 / i g / m / h e p a r i n .

Lanes o—r: E x p e r i m e n t s w i t h the e n z y m e f r o m / , . curvatus^2^. T h e e x p e r i m e n t s w e r e c a r r i e d o u t as d e s c r i b e d '3 7' w i t h the 4 - h y d r o x y b e n z a l d e h y d e e s t e r o f A T P b u t w i t h 2 . 5 m M M n C l² a n d l O O m M K C l i n s t e a d o f 1 0 m M M g C l².

L a b e l i n g w a s c a r r i e d o u t at 3 7 ° C . o : c o m p l e t e m i x t u r e ; p : as i n o , b u t w i t h 2 / i g / m / r i f a m p i c i n ; q : as i n o , b u t w i t h o u t D N A ; r: as i n o , b u t w i t h o u t r e d u e t i o n .

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peptide with7lf^r ~ 30 k becomes labeled. In each case all appropriate controls confirm the specific labeling.

DNA-Directed RNA polymerases from Eubacteria

Eubacteria contain a Single DNA-directed R N A Polymerase (M^T approximately 500 k) usually consisting of 4 to 5 Polypeptide chains^{1 2 7 1}. The first enzyme investigated with the method of affinity labeling by autocatalysis was from Escherichia c o / /^{[ 1 1}'^{1 2 1}. Depending on the nature of the reactive nucleotide used, either the se- cond largest subunit ß (M^r ~~ 150 k) or both ß and o subunits become labeled^{1 1 5 1}.

We have investigated the highly purified R N A Polymerase of the extremely thermophilic Gram-negative Eubacterium Thermotoga maritima^ which consists of only the core subunits ß' (184 k), ß (141 k) and oc (45 k).

As in E. coli the second largest subunit becomes distinctly labeled (Fig. 7k—n).

When highly purified R N A Polymerase from the filamentous cyanobacterium Anabaena¹²⁹^ is used for affinity labeling by autocatalysis, again intensive labeling of the second largest subunit with 7V/^r ~ 124 k is observed with the 4-hydroxybenzaldehyde ester of A T P as the reactive nucleotide derivative (Fig. 7a—g). More- over, when the A D P ester of 4-[Af-(2'-hydroxy- ethylHV-methyl]amino benzaldehyde is used, an additional weak labeling of the sigma sub- unit (M^T ^ 52 k) is observed (Fig. 7h, i).

In Gram-positive Eubacteria the largest subunit of the R N A Polymerase seems to share some properties with the second largest subunit of the enzyme from gram-negative Eubacteria^. It was therefore of particular interest to investigate which subunit would become labeled in enzymes from these organisms. In fact, the enzyme from Gram-positive Eubacteria such as Micrococcus

luteus^[3l] can also be labeled by this method.

After electrophoretic Separation the label ap- pears in the region of the two largest subunits (data not shown). Since the large subunits are of very similar size they are difficult to separate by gel electrophoresis in presence of dodecyl sulfate.

Separation is easier in the case of the R N A Polymerase from Gram-positive Lactobacillus curvatus where the largest subunit (M^r — 151k) is 4% larger than the second largest subunit (M^T ~ 145 k )^{1 3 2 1}. After incubation under condi- tion of affinity labeling, autoradiography clearly revealed that the largest subunit becomes ex- clusively labeled (Fig. 7o—r). This result Sup- ports the hypothesis^{1 3 0 1} that in the R N A Poly- merase from Gram-positive Eubacteria the largest subunit fulfills the function of the se- cond largest subunit of the enzyme from Gram- negative Eubacteria. Rifampicin does not block dinucleotide synthesis in Eubacteria^. In agreement, the labeling is not inhibited (Fig. 7p).

DNA-Directed RNA polymerases from Archaebacteria

The DNA-directed R N A Polymerase from Archaebacteria is very different from the cor- responding enzyme in Eubacteria and consist of 8 to 10 Polypeptides^{1 3 4 1}. Is the active center of these enzymes also susceptible to affinity labeling by autocatalysis? The subunits of the R N A polymerases of Archaebacteria may be classified according to serological cross-reac- t i v i t y^{1 3 5}'^{3 6 1} (Table 1).

The application of affinity labeling by auto- catalysis to the DNA-directed R N A Polymerase from six different Archaebacteria clearly sup- ported and even refined the serological Classi- fication (Table 1). In each enzyme only one of the numerous subunits became labeled^{1 3 7 1}. In the enzyme from methanogenic or halophilic

Table 1. Affinity labeling of R N A Polymerase from Archaebacteria by autocatalysis!^{3 7}1.

A rchaebacterium Subunit Labeled subunit M^T

composition^a (x K T3)

Mc.^b vaniellii A B' B" C B' 79

Mc.^b thermolithotrophicus A B' B" C B' 79

Mb.c thermoautotrophicum A B' B" C B' 78

Halobacterium halobium A B' B" C B' 86

Archaeoglobus fulgidus (A + C) B' B" B' 80

Sulfolobus acidocaldarius B A C B 127

Sulfolobus B12 B A C B 127

a Ordered according to molecular size. Only the largest subunits are shown. The same capital letter is used for subunits which show serological cross-reactivity.

k Mc.: Methanococcus.

c Mb.: Methanobacterium.

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Archaebacteria exclusively the second largest subunit B ' was labeled (Table 1). In the e n z y m e from the sulfate-reducing Archaeoglobus fulgidus^ again o n l y the second largest subunit

B ' was labeled. In contrast, w h e n the e n z y m e from t w o different strains o f the non-methanogenic sulfur-dependent Sulfolobus (type B A C ) was tested the largest subunit B was labeled.

T h i s clearly shows that affinity labeling by autocatalysis and i m m u n o l o g i c a l cross reac- t i v i t y are m u c h better evidence for f u n c t i o n a l equivalence than the m o l e c u l a r size o f the subunits. It w i l l be o f interest to compare the sequence a r o u n d the b i n d i n g site o f the affinity label i n these various enzymes.

Eukaryotic DNA-directed RNA polymerases E u k a r y o t i c cells contain three different R N A polymerases designated as A (I), B (II) and C (III) w h i c h catalyse the synthesis o f the large r i b o s o m a l R N A s , messenger R N A s and small R N A s such as t R N A , respectively. E a c h o f these enzymes contains 10 to 14 P o l y p e p t i d e s o f different size.

Is it possible to also label these enzymes b y affinity labeling? We have answered this ques- t i o n b y a p p l y i n g this m e t h o d to the purified enzymes A (I), B (II) and C (III) f r o m yeast. In each case exclusively the second largest subunit became labeled, i.e., subunit A 1 3 5 , B 1 5 0 o r C 1 2 8^{1 1 3 1}.

These results suggest that the presence o f the active site o n the second largest subunit o f these polymerases w i t h different specialised functions has been preserved during e v o l u t i o n and differ- e n t i a t i o n .

A f f i n i t y labeling by autocatalysis is also possible w i t h the R N A Polymerase f r o m other e u k a r y o t i c sources. W h e n R N A Polymerase B (II) from wheat germ is used, again o n l y the second largest subunit W 1 4 0 becomes labeled. T h i s is prevented by l o w concentrations o f a-amanitin just as is the R N A synthesis catalysed b y this en- z y m e^{1 3 9 1}. Indeed, affinity labeling b y autocatalysis requires the same c a t a l y t i c a c t i v i t y as R N A synthesis.

T o analyse the p r o d u c t o f this affinity labeling in more detail the subunit W 1 4 0 labeled w i t h the 4-[N-(2'-hydroxyethyl)-A^-methyl]amino- benzaldehyde ester o f A D P and [ a -^{3 2}P ] U T P i n the presence o f p l a s m i d D N A was isolated after i n c u b a t i o n w i t h ribonuclease and was then digested w i t h Proteinase K . The radioactive p r o d u c t was cleaved o f f w i t h acid p y r o p h o s - phatase. T e r m i n a l phosphate groups were re- m o v e d by treatment w i t h Phosphatase as de- s c r i b e d^{1 1 3 1}. C h r o m a t o g r a p h i e analysis d e m o n -

strated u n e q u i v o c a l l y that the d i n u c l e o t i d e A p U and the t r i n u c l e o t i d e A p A p U had been formed during affinity labeling. W h e n the 4- h y d r o x y b e n z a l d e h y d e ester o f A T P was used, and w i t h o u t ribonuclease A treatment, the trinucleotides A p U p U and A p U p A were f o u n d . F o r the f o r m a t i o n o f A p A p U and A p U p A one w o u l d expect the presence o f A T P i n the reac- t i o n m i x t u r e ; however, A T P had not been

a b c d e

F i g . 8. A f f i n i t y l a b e l i n g b y a u t o c a t a l y s i s w i t h a c r u d e n u c l e a r e x t r a c t f r o m t o b a e c o c e l l s .

N u c l e i f r o m c u l t u r e d Nicotiana tabacum c e l l s w e r e i s o l a t e d '4 2! a n d b r o k e n i n t h e K o n t e s m i n i - b o m b c e l l d i s r u p t i o n C h a m b e r . A f t e r a d d i t i o n o f 0 . 2 5 m M p h e n y l - m e t h y l s u l f o n y l f l u o r i d e t h e e x t r a c t w a s c e n t r i f u g e d f o r 3 0 m i n at 1 1 0 0 0 0 x g. F o r m o d i f i c a t i o n t h e s u p e r n a t a n t (1 7 / i g p r o t e i n ) w a s i n e u b a t e d as d e s c r i b e d '1 3 1 b u t o n l y f o r 4 m i n w i t h 4 - h y d r o x y b e n z a l d e h y d e e s t e r o f A T P a n d , s u b s e q u e n t l y , w i t h N a B H4. T h e e n z y m i c r e a c t i o n w a s s t a r t e d w i t h 3.8 / i g d e n a t u r e d c a l f t h y m u s D N A a n d [ a -3 2P ] U T P f o r 4 m i n . L a n e a: s u b u n i t s o f w h e a t g e r m R N A P o l y m e r a s e as m a r k e r s ; b : s i l v e r s t a i n e d p r o t e i n s o f t h e s u p e r n a t a n t ; c : c o m p l e t e i n c u b a t i o n m i x t u r e ; d : as i n c, b u t w i t h o u t d e r i v a t i v e ; e: as i n c , b u t w i t h o u t r e d u e t i o n ( c - e : a u t o r a d i o g r a p h y ) .

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added. Maybe A T P existed as an i m p u r i t y in the Substrate o r was f o r m e d b y h y d r o l y s i s o f the A T P derivative. P o s s i b l y , the latter c o u l d also f u n c t i o n to a l i m i t e d extent as elongating Substrate^{1 4 0 1}.

F o r m a t i o n o f trinucleotides during affinity labeling by autocatalysis has also been observed w i t h R N A Polymerase B (II) and C (III) f r o m y e a s t^{1 1 3 1}.

T o e x p l a i n these results one has to assume a certain f l e x i b i l i t y o f the P o l y p e p t i d e chain to w h i c h the p r i m i n g n u c l e o t i d e is covalently b o u n d to a l l o w thereby a small m ovem ent o f the d i n u c l e o t i d e f o r m e d w i t h i n the p r o d u c t - b i n d i n g site. Otherwise R N A Polymerase must c o n t a i n t w o adjacent active sites for the forma- t i o n o f t w o phosphodiester b o n d s^{1 4 1 1}.

A f f i n i t y labeling by autocatalysis is a rather sensitive m e t h o d as was s h o w n b y the f o l l o w i n g e x p e r i m e n t . Isolated n u c l e i f r o m Nicotiana tabacum c e l l s^{1 4 2 1} were plasmolysed and the re- sulting crude 100 0 0 0 x g supernatant d i r e c t l y used w i t h o u t further p u r i f i c a t i o n for affinity labeling. Several radioactive bands appear i n the autoradiography o f the electrophoretic analysis ( F i g . 8) but the band w i t h the slowest m o b i l i t y

a b c d e f g h

is absent w h e n the nucleotide derivative o r w h e n the r e d u e t i o n step are o m i t t e d ( F i g . 8, d , e).

T h i s b a n d migrates at the p o s i t i o n o f the second largest subunit o f R N A Polymerase B (II) from Nicotiana tabacum R N A Polymerase

(M^T - 135 k )^{1 4 2 1}.

DNA Primase and Qß Replicase

The w i d e a p p l i c a b i l i t y o f affinity labeling by autocatalysis suggests that even more distantly related polymerases such as D N A primase m a y be susceptible to this procedure. T h i s e n z y m e is a D N A - d i r e c t e d R N A Polymerase w i t h a rather specialised f u n c t i o n . It synthesizes o l i g o r i b o - nucleotide primers required b y D N A Polymerase for i n i t i a t i o n o f D N A synthesis. T h e e n z y m e has been highly p u r i f i e d f r o m yeast c e l l s '^{4 4 1} as w e l l as from calf t h y m u s^{1 4 5 1} and is strongly associated w i t h D N A Polymerase. The c o m p l e x f r o m yeast contains 5 P o l y p e p t i d e chains. The P o l y p e p t i d e s w i t h M^r ^ 58 k and ^ 48 k are associated w i t h primase a c t i v i t y . When the m e t h o d o f affinity labeling b y autocatalysis is applied to the c o m p l e x f r o m yeast, b o t h subunits o f primase become labeled ( F o i a n i , M . , L i n d n e r , A . J . , H a r t m a n n , G . R . , L u c c h i n i , G . and Plevani, P., s u b m i t t e d for p u b l i c a t i o n ) . The same result

F i g . 9. A f f i n i t y l a b e l i n g b y a u t o c a t a l y s i s o f Q ß r e p l i c a s e a n d the c o m p l e x o f D N A p r i m a s e a n d D N A P o l y m e r a s e f r o m c a l f t h y m u s .

Lanes a-d: E x p e r i m e n t s w i t h Qß r e p l i c a s e ( h o l o e n z y m e w i t h the s u b u n i t s ot, ß, y, 5 ) , c a r r i e d o u t as d e s c r i b e d '^{3 7 1} w i t h 1 fdg e n z y m e '4 7 1 a n d 4 - h y d r o x y b e n z a l d e h y d e e s t e r o f G T P . 0 . 0 2 ßg/ßl M D V - I (+) R N A '⁴⁷¹ u s e d as t e m p l a t e , a n d [ ö >3 2P ] G T P u s e d as S u b s t r a t e , a : c o m p l e t e m i x t u r e ; b : as i n a, b u t w i t h o u t t e m p l a t e ; c : as i n a , b u t w i t h 1 0 0 jJtg/ml h e p a r i n ; d : as i n a, b u t w i t h o u t r e d u e - t i o n .

Lanes e-h: E x p e r i m e n t s w i t h t h e c o m p l e x o f D N A p r i m a s e a n d D N A P o l y m e r a s e , c a r r i e d o u t s i m i l a r as d e s c r i b e d '3 7' w i t h 2 / i g e n z y m e '4 5 1 i n 1 0 m M p h o s p h a t e b u f f e r p H 7.8 a n d 1 0 0 ßg/ml p o l y ( d C , d T ) as t e m p l a t e a n d [ a -3 2P ] G T P as S u b s t r a t e , e: w i t h 4 - [ 7 V - ( 2 ' - h y d r o x y - e t h y l ) - A f - m e t h y l ) a m i n o b e n z a l d e h y d e e s t e r o f A T P ; f: w i t h the e s t e r o f A D P ; g : w i t h the e s t e r o f A M P ; h : w i t h 4 - h y d r o x y b e n z a l d e h y d e ester o f A T P . T h e r a d i o a c t i v e b a n d i n the u p p e r p a r t o f t h e gel w a s n o t i d e n t i f i e d . T h i s b a n d is p a r t i c u l a r l y s t r o n g w i t h t h e ester o f A D P , is n o t s e n s i t i v e t o d i g e s t i o n w i t h P r o - t e i n a s e K a n d is t h e r e f o r e n o t b o u n d t o a p r o t e i n .

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is f o u n d for the c o m p l e x from calf t h y m u s ( F i g . 9e—h). However, the extent o f labeling o f the two subunits differs depending o n the particular c o n d i t i o n s a p p l i e d . F r e q u e n t l y , the larger subunit is more heavily labeled. These observations are reminiscent o f the Situation f o u n d i n several eubacterial R N A polymerases ( F i g . 7 and r e f . '^{1 5 1})

a b c d e

1 589

N | u ' " 1^{C 6 6}

I 1³⁴ I 1³¹ I 1²⁷ I 1¹⁷ I 1¹⁵

I I met-ser

F i g . 10. K i n e t i c s o f l i m i t e d cleavage b y C N B r o f t h e a f f i n i t y l a b e l e d Qß r e p l i c a s e ß s u b u n i t .

0 . 2 / i g e l e c t r o p h o r e t i c a l l y p u r i f i e d l a b e l e d ß s u b u n i t ( F i g . 9 ) w a s d i s s o l v e d i n 5 0 / i / H C l c o n t a i n i n g 1 % d o d e c y l s u l f a t e a n d t h e n t r e a t e d w i t h 6 6 m M C N B r at p H 1 - 2 . C l e a v a g e w a s s t o p p e d b y n e u t r a l i s a t i o n i n p r e s e n c e o f 2 % m e r c a p t o e t h a n o l . T h e p r o d u c t s o f t h e l i m i t e d cleavage w e r e s e p a r a t e d b y e l e c t r o p h o r e s i s i n a 1 5 % P o l y a c r y l a m i d e gel c o n t a i n i n g 0 . 1 % d o d e c y l s u l f a t e . L a n e a ( c o n t r o l ) : i n c u b a t i o n f o r 15 m i n at 2 0 ° C w i t h o u t C N B r ; l a b e l e d b a n d s are t h e p r o d u c t s o f cleavage at p a r t i c u l a r l y a c i d l a b i l e p e p t i d e b o n d s '4 9 1. b - e : i n c u b a t i o n f o r 2 , 5, 10 a n d 15 m i n w i t h C N B r . T h e p a t t e r n o f t h e l a b e l e d C N B r p r o d u c t s a n d t h e i r size ( s h o w n ) c o r r e s p o n d s e x a c t l y t o t h e N - t e r m i n a l P e p t i d e s f o r m e d b y " s i n g l e h i t " C N B r cleavage at t h e m e t r e s i d u e s 3 2 3 , 3 0 0 , 2 3 0 , 1 4 8 a n d 1 3 0 ( M e t7 6- S e r7 7

is n o t c l e a v a b l e u n d e r these c o n d i t i o n s '^{4 9 1}) as s h o w n

i n the s c h e m e '4 8 1. ,

where t w o subunits (ß and a) become labeled.

These results suggest that the t w o subunits o f primase are associated i n such a way that the covalently b o u n d derivative can reach i n t o the active site f r o m attachment sites o n b o t h subunits.

A n o t h e r rather specialised Polymerase is the R N A - d i r e c t e d R N A replicase from E. coli bacteriophage Q ß . This R N A replicase consists o f four different subunits. Three o f t h e m (a, 7 and 5 ) are host-coded and are i d e n t i c a l w i t h the r i b o s o m a l p r o t e i n S l and the p r o t e i n e l o n - gation factors E F T^U and E F T^S, respectively.

O n l y subunit ß, is coded for by the viral g e n o m e '^{4 6 1}. The e n z y m e selectively transcribes specific R N A templates w i t h a c y t i d i n e Cluster at the 3'-end w h i c h acts as the starting site for R N A synthesis. E v i d e n t l y Q ß replicase is not a general R N A - d i r e c t e d R N A Polymerase. W i t h midivariant R N A ( M D V - 1 , 221 nucleotide l e n g t h '^{4 7 1}) as template and [ a -^{3 2}P ] G T P as Sub- strate the viral coded subunit ß (M^T ~~ 66 k '^{4 8 1}) becomes exclusively labeled. A s i n a l l previous examples the labeling is dependent o n the presence o f the derivative as w e l l as o n the template. Labeling is prevented by 100 / i g / m / heparin and does not o c c u r when the redue- t i o n step is o m i t t e d ( F i g . 9 a - d ) .

Where is the label attached to the P o l y p e p t i d e chain? U p o n inspection o f the sequence o f the 589 a m i n o acids o f the ß subunit from the replicase it becomes evident that m e t h i o n i n e residues o c c u r o n l y i n the N - t e r m i n a l half o f the P o l y - peptide c h a i n '^{4 8 1}. Cleavage o f the labeled P o l y - peptide chain w i t h eyanogen b r o m i d e for a short time under c o n d i t i o n s where o n l y one cleavage per P o l y p e p t i d e chain s h o u l d o c c u r1 1 7 - 1 9 , 4 9 1

leads to the appearance o f t w o relatively small fragments w i t h A /^r o f about 17 k and 14 k, respectively ( F i g . 10). T h e labeled double band corresponds i n length to the expected cleavage products at positions 148 and 130 o f the 589 a m i n o acid long sequence. T h i s result suggests that the labeled lysine residue occurs w i t h i n the first 130 a m i n o acids near the N - t e r m i n u s o f the P o l y p e p t i d e c h a i n .

Sequence homologies at the binding site The surprising fact that such a large variety o f different R N A polymerases can be labeled by the same m e t h o d suggests that all labeled P o l y - peptides should c o n t a i n a similar amino-acid sequence at or close to the active site. The benzaldehyde derivatives used i n our experiments are most l i k e l y attached to the e-amino group o f a lysine residue as has been suggested for the R N A Polymerase f r o m E. colfi^15,181.

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Table 2. Comparison of the amino-acid sequence between L y s1 0 4 8 and A r g1 0 5 8 in the ß subunit of E. coli R N A Polymerase with a similar region in labeled Polypeptides from other R N A poly- merases.

The subunit, its approximate molecular mass, the position of the first amino acid of the given se- quence and the source of the sequence is shown.

Lit.

E. coli ß (150 k) 1048 K I V K V Y L A V K R (50)

Yeast B140 962 K F V K V R V R T T K (52)

Mb. thermo*B' (78 k) 345 R L A K I R V R E Q R d S. acido.b B (126 k) 859 K L V K V R V R D L R e

T3 (T7) (98 k) 704 K L L A A E V K D K K (22)

Qßß(66 k) 95 K F L A A E A _c E C A (48)

Yeast primase (48 k) 135 K F ₁ S L A M K ₁ T N (53)

a Methanobacterium thermoautotrophicum strain Winter.

b Sulfolobus acidocaldarius.

c Assumed deletion.

d Berghöfer, B., Schallenberg, J . & Klein, A . , Fachbereich Biologie-Molekulargenetik, Universität, D-3550 Marburg, personal communication.

e Pühler, G . & Zillig, W., Max-Planck-Institut für Biochemie, D-8033 Maxtinsried, personal com- munication.

Depending on the nature of the nucleotide deri- vative used, the labeled oligonucleotide is co- valently attached in this enzyme to either one of two adjacent regions of the ^-subum^^{5 0 1}. One is located between I l e^{1 0 3 6} and M e t1 0 6 6 ( 1 8 1, the other between M e t^{1 2 3 2} and M e t1 2 4 3 1 1 9 1. When searching for similar sequences among the labeled R N A Polymerase Polypeptides with known primary structure such regions are in- deed found (Tables 2 and 3). They differ mostly in conservative substitutions.

Particularly striking is the similarity of these regions between eukaryotic and archaebacterial R N A polymerases. It is also interesting to note that even so different polymerases as Qj3 repli- case and T7 or T3 R N A Polymerase contain similar sequences. Compared at the nucleotide

level the similarity of these regions in T7 Poly- merase and Qj3 replicase is even closer. 16 out of 19 nucleotides are identical.

Outlook

Affinity labeling of the active site by auto- catalysis is much more specific than the classical methods of labeling by reactive Substrate analogues because other Substrate binding sites such as regulatory sites are excluded. The gener- al nature of affinity labeling by autocatalysis suggests an application of this method to com- pletely different classes of enzymes provided they catalyse a condensation reaction according to the scheme

enzyme + A e n z y m e - A e n z y m e - A + B * e n z y m e - C * + D

Table 3. Comparison of the amino-acid sequence between L e u1 2 3 ? and L y s1 2 4 2 in the ß sub- unit of E. coli R N A Polymerase with a similar region in labeled Polypeptides from other R N A polymerases.

The subunit, its approximate molecular mass, the position of the first amino acid of the given sequence and the source of the sequence is indicated.

Lit.

E. coli ß (150 k) 1233 L K L N H L V D D K (50)

Yeast B140 Q R L R H M V D D K (52)

Mb. thermo*B' (78 k) 481 Q K L H H M T T D R c S. acido.^b B (126 k) 958 L K L G H L P D S T d

a Methanobacterium thermoautotrophicum Winter.

b Sulfolobus acidocaldarius.

c Berghöfer, B., Schallenberg, J. & Klein, A . , Fachbereich Biologie - Molekulargenetik, Universi- tät, D-3550 Marburg, personal communication.

d Pühler, G . & Zillig, W., Max-Planck-Institut für Biochemie, D-8033 Martinsried, personal com- munication.