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10.1101/gad.5.11.1935Access the most recent version at doi:1991 5: 1935-1945Genes Dev.

B F Pugh and R TjianTFIID complex.Transcription from a TATA-less promoter requires a multisubunit

References

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T r a n s c r ip t io n f ro m a T A T A - I e ssp r o m o t e r r eq u ir es a m u l t i s u b u n i tT F I I D c o m p l e xB . F r a n k l i n P u g h 1 a n d R o b e r t T j i a nHoward H ughes M edical Insti tute, D epartm ent of Molecular and Cell Biology, Univ ersity of California, Berkeley,California 9 4720 USA

I n e u k a r y o te s , t h e T A T A b o x - b i n d i n g p r o t e i n ( T B P ) is r e s p o n s ib l e f o r n u c l e a t i n g a s s e m b l y o f t h e t r a n s c r i p t io ni n i t i a ti o n m a c h i n e r y . H e r e , w e r e p o r t th a t a T F I I D c o m p l e x c o n t a i n i n g T B P i s e s s e n ti a l f o r t r a n s c r ip t i o n e v e na t a p r o m o t e r t h a t la c k s a T A T A b o x . I m m u n o p u r i f i c a t i o n o f T F I I D r e v e a l s t h at t h e a c t i v e s p e c i e s i nr e c o n s t i t u t in g T A T A - l e s s t r a n s c r i p t io n i s a m u l t i s u b u n i t c o m p l e x c o n s i s t in g o f T B P a n d m a n y T B P - a s s o c i a t e df a c t o r s ( T A F s ) .[Key Words: T A T A b o x - b i n d i n g p r o t e i n ; t r a n s c r i p t i o n ; T F I I D c o m p l e x ; T A T A - i n d e p e n d e n t ; T B P ]Received August 20, 1991 ; revised version accepted September 13, 1991.

T h e p r o m o t e r s o f e u k a r y o t i c p r o t e i n - c o d i n g g e n e s d i f f e rw i d e l y b u t c a n b e g e n e r a l l y c l a s s i f i e d a s c o n t a i n i n g o rl a c k i n g a T A T A b o x ( c o n s e n s u s T A T A A A A ) , w h i c h i st y p i c a ll y l o c a te d 2 5 - 3 0 n u c l e o t i d e s u p s t r e a m f r o m t h et r a n s c r i p t i o n a l s t a r t s i t e ( o r i n i t i a t o r e l e m e n t ) . T h eT A T A b o x b i n d s T F I I D a n d , a l o n g w i t h t h e i n i t i a t o r ,h e l p s d i r e c t R N A p o l y m e r a s e I I a n d o t h e r r e q u i r e d b a s a lini t ia t ion factors (TFIIA, TFIIB, TFIIE, TFIIF , and TFIIG)t o a s s e m b l e e f f i c i e n t l y i n t o a s t a b l e p r e i n i t i a t i o n c o m -p l e x { S a l t z m a n a n d W e i n m a n n 1 9 8 9 ) .R e c e n t l y , c D N A c l o n e s o f t h e T A T A b o x - b i n d i n g p r o -t e i n ( T B P ) f r o m y e a s t , m a m m a l s , f li e s, a n d p l a n t s h a v eb e e n i s o la t e d , a n d p r o t e i n s e q u e n c e c o m p a r i s o n s r e v e a l as t r i k i ng b i pa r t i t e s t r uc t u r e (Cava l l i n i e t a l . 1989 ; E i sen-m a n n e t a l . 1 9 8 9 ; H a h n e t a l . 1 9 8 9 a ; H o r i k o s h i e t a l .1989 ; S chmi d t e t a l . 1989 ; F i kes e t a l . 1990 ; Gasch e t a l .1990 ; Hoey e t a l . 1990 ; Ho f fm ann e t a l . 1990a , b ; Kao e ta l . 1990 ; M uh i ch e t a l . 1990 ; P e t e r son e t a l . 1990}. T h ec a r b o x y - t e r m i n a l 1 8 0 r e s i d u e s a r e r i c h i n b a s i c a m i n oa c i d s , c o n t a i n a t a n d e m r e p e a t , a n d a r e > 8 0 % i d e n t i c a li n a m i n o a c i d s e q u e n c e a m o n g t h e d i f f e r e n t o r g a n i s m s .T h i s h i g h l y c o n s e r v e d c a r b o x y - t e r m i n a l d o m a i n i s n e c -e s s a r y a n d s u f f i c i e n t f o r t h e T B P t o b i n d t h e T A T A b o xa n d r e c r u i t T F I I A a n d T F II B t o t h e p r o m o t e r ( B u r a t o w s k iet a l. 19 89; Ho ey et a l . 1990; Hor iko shi e t a l . 1990 ; Petersone t a l . 1990) . M os t s i gn i f i can t l y , t h i s r eg i on a l so su f f i cest o r e p l a c e t h e e n d o g e n o u s h u m a n T F I I D f r a c t i o n i n s u p -p o r t i n g a b a s a l u n r e g u l a t e d l e v e l o f t r a n s c r i p t i o n i n i t i a -t i on (Ho ey e t a l . 1990 ; Ho r i ko sh i e t a l . 1990 ; P e t e r son e ta l. 1 9 9 0) . I n c o n t r a s t , t h e d i v e r g e n t a m i n o - t e r m i n a l p o r -t i o n o f t h e v a r i o u s T B P s s h a r e s v e r y l i t t l e s e q u e n c e s i m -

1Present address: Department of Molecular and C ell B iology, The P enn-sylvania State Univers ity , Univers ity Park, Pennsylvania 16802 USA.

i l a r i t y an d , i n s o m e c a s e s , is r i c h i n g l u t a m i n e r e s i d u e s .A l t h o u g h t h e f u n c t i o n o f t h e a m i n o - t e r m i n a l d o m a i n i sp o o r l y c h a r a c t e r i z e d , i n h i g h e r e u k a r y o t e s t h i s s p e c i e s -s p e c i f i c d o m a i n a p p e a r s t o i n d i r e c t l y m e d i a t e p r o m o t e rr e g u l a t i o n b y s e q u e n c e - s p e c i f i c t r a n s c r i p t i o n a l a c t i v a -t o r s such a s S p l (P e t e r son e t a l . 1990 ; P ugh and T j i an1990).B i o c h e m i c a l c h a r a c t e r i z a t i o n o f t h e r e c o m b i n a n t h u -m a n T B P o v e r e x p r e s s e d i n H e L a c e l l s r e v e a l s t h a t i t e x -i s t s a s a 3 8 - k D m o n o m e r i n s o l u t i o n ( P e t e r s o n e t a l .1 99 0 ). I n c o n t r a s t, i t s n a t u r a l o r e n d o g e n o u s c o u n t e r p a r tb e h a v e s a s a m u c h l a rg e r m a c r o m o l e c u l a r c o m p l e x d u r -i n g g e l f i l t r a t i o n c h r o m a t o g r a p h y a n d g l y c e r o l g r a d i e n ts e d i m e n t a t i o n ( P u g h a n d T j i a n 1 9 9 0 ; D y n l a c h t e t a l .1 9 91 ; N . T a n e s e a n d R . T j i a n , u n p u b l . ) . T h r o u g h o u t t h i sp a p e r w e w i l l re f er to t h e 3 8 - k D m o n o m e r a s t h e h u m a nT A T A b o x - b i n d i n g p r o t e i n ( h T B P ) a n d t o t h e h i g h - m o -l e c u l a r - w e i g h t e n d o g e n o u s p r o t e i n a s t h e T F I I D c o m -p l e x . P r e v i o u s f in d i n g s s u g g e s t t h a t T B P a l o n e i s n o t s u f -f i c i e n t t o c a r r y o u t a l l o f t h e a c t i v i t i e s g e n e r a l l y a t t r i b -u t e d t o t h e p a r t i a l l y p u r i f i e d f r a c t i o n c o n t a i n i n g t h eT F I I D c o m p l e x , e s p e c i a l l y t r a n s c r i p t i o n a l r e g u l a t i o n b yu p s t r e a m a c t i v a t o r s ( H o f f m a n n e t a l . 1 9 9 0 b ; K a m b a d u re t a l . 1990 ; P e t e r son e t a l . 1990 ; P ugh and T j i an 1990 ;D y n l a c h t e t a l . 1 9 9 1 ; T a n e s e a n d T j i a n 1 9 9 1 ; W o r k m a ne t a l . 1991) . T o account f o r t h i s d i f f e r ence , t r ansc r i p -t i o n a l a c t i v a t o r s s u c h a s S p l a n d C C A A T b o x t r a n s c r i p -t i o n f a c t o r ( C TF ) w e r e p r o p o s e d t o r e g u l a t e t r a n s c r i p t i o nt h r o u g h c o a c t i v a t o r s o r a d a p t o r s t h a t c o p u r i f i e d w i t h t h eT F I ID c o m p l e x ( P u g h a n d T j i a n 1 99 0 ).I f T BP i s c r i t i ca l f o r t r ansc r i p t i on and i s r ec ru i t ed t ot h e p r o m o t e r b y t h e T A T A b o x , h o w c a n s o m e p r o m o t -e r s f u n c t i o n i n t h e a b s e n c e o f a T A T A b o x ? Is T B P o r t h eT F I I D c o m p l e x r e q u i r e d a t s u c h p r o m o t e r s ? I f s o , h o wd o e s i t r e c o g n i z e t h e p r o m o t e r ? T h e s e s o - c a l l e d T A T A -

GE N E S & D E V E L OPME N T5:1935-1945 9 1991 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/91 $3.00 1935

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Pugh and Tjian

l e s s p r o m o t e r s r e g u l a t e a l a r g e v a r i e t y o f c e l l u l a r a n dv i r a l g e n e s , i n c l u d i n g t h o s e c o d i n g f o r d i h y d r o f o l a t e r e -d u c t a s e , c-H-ras, a d e n i n e d e a m i n a s e , T G F - a , t h y m i d y -l a t e s y n t h e t a s e , a n d S V 4 0 l a t e g e n e p r o d u c t s ( D y n a n1 9 8 6; S w i c k e t a l . 1 9 8 9 a n d r e f s. t h e r e in ) . O n e c o m m o na n d i n t r i g u i n g p r o p e r ty o f m a n y T A T A - l e s s p r o m o t e r s i st h e p r e s e n c e o f m u l t i p l e G C b o x e s ( c o n s e n s u sG G G G C G G G G C ) t h a t b in d t h e t r a n s c r ip t i o n a l a c t i v a to rS p 1 . T r a n s c r i p t i o n i n i t i a t i o n d i r e c t e d b y t h e s e t e m p l a t e si s c r i t i c a l l y d e p e n d e n t o n S p l ; t h e r e f o r e , u n d e r s t a n d i n gh o w S p l a c t i v a te s t r a n s c r i p t io n a t s u c h T A T A - l e s s pr o -m o t e r s m i g h t p r o v i de n e w i n s i g h t s i n to t h e m e c h a n i s mof i n i t i a t i on .

I n a n e f f o r t t o u n d e r s t a n d h o w S p l c a n a c t i v a t e aT A T A - l e s s p r o m o t e r , w e p r e v i o u s l y a d d r e s s e d t h e f a c t o rr e q u i r e m e n t a t a p r o m o t e r c o n t a i n i n g o n l y S p l - b i n d i n gs i t e s a n d a n i n i t i a t o r e l e m e n t . S p l w a s f o u n d t o u t i l i z et h e s a m e f r a c t i o n a t e d b a s a l i n i t i a t i o n f a c t o r s , i n c l u d i n gt h e p a r t i a l l y p u r i fi e d T F I I D f r a c t io n , t h a t a r e r e q u i r e d f o rt r a n s c r ip t i o n a t T A T A - c o n t a i n i n g p r o m o t e r s ( P u g h an dT j i a n 1 9 90 ). I n a d d i t i o n , a n o v e l a c t i v i t y w a s d e s c r i b e dt h a t w a s p r e s e n t i n t h e H e L a T F I I D f r a c t i o n b u t d i s t i n c tf r o m T B P a n d t h e S p 1 c o a c t i v a t o r a n d w a s r e q u i r e d o n l yf o r a c t i v a t i o n o f a T A T A - l e s s p r o m o t e r b y S p l ( P u g h a n dT j i a n 1 9 90 ). I n t h e p r e s e n c e o f p r o m o t e r - b o u n d S p l , t h i ss o - c a l l e d t e t h e r i n g f a c t o r a p p a r e n t l y c o u l d f u n c t i o n a s as u b s t i t u t e f o r t h e T A T A b o x i n t r a n s c r i p t i o n a s s a y s i nv i t r o . T h e s e o b s e r v a t i o n s l e d t o t h e p r o p o s a l t h a t S p lp l a y s a n e s s e n t i a l r o l e in a s s e m b l i n g t h e b a s a l i n i t i a t i o nf a c to r s , p o s s i b l y b y a n c h o r i n g T B P t o t h e T A T A - l e s st e m p l a t e v i a t h e t e t h e r i n g f a c t o r . O u r h y p o t h e s i s i s t h a tT F I I D i s a c o m p l e x o f d i s t i n c t t r a n s c r i p t i o n f a c to r s , a n dT B P is t h e c o r e s u b u n i t t h a t i s r e q u i r e d f o r t r a n s c r i p t i o ne v e n a t a T A T A - l e s s t e m p l a t e . H o w e v e r , n e i t h e r o f t h e s ea s s u m p t i o n s h a s b e e n e x p e r i m e n t a l l y e st a b li s h e d .

I n t h i s p a p e r w e a d d r e s s w h e t h e r t h e p r o p o s e d T F I I Dc o m p l e x p l a y s a n i m p o r t a n t r o l e i n t r a n s c r i p t i o n a t aT A T A - l e s s p r o m o t e r . A f f i n i t y - p u r i f i e d a n t i b o d i e s d i -r e c te d a g a i n s t t h e r e c o m b i n a n t h T B P w e r e u s e d t o s p e -c i f ic a l l y i n h i b i t a n d / o r r e m o v e f r o m t h e H e L a n u c l e a re x t r a c t t h e e n d o g e n o u s T B P , a l o n g w i t h a n y f a c t o r s t h a tm i g h t b e t i g h t l y a s s o c i a te d w i t h i t, s u c h a s t h e t e t h e r i n gf a ct o r. T h e c o m p o s i t i o n o f t h e i m m u n o p r e c i p i t a t e dT F U D c o m p l e x w a s d e t e r m i n e d b y g e l e l e c t r o p h o r e s i s ,a n d t h e d e p l e t e d e x t r a c t s w e r e t h e n t e s t e d f o r t h e i r a b i l -i t y t o t r a n s c r i b e a n S p 1 - a c t i v a t e d T A T A - l e s s p r o m o t e r i nt h e p r e s e n c e o r a b s e n c e o f a d d e d p u r i f i e d r e c o m b i n a n tT B P o r p u r i f i e d i m m u n o p r e c i p i t a t e d T F I I D c o m p l e x .T h e r e s u l t s s u g g e s t s i m i l a r r e q u i r e m e n t s f o r t h e T F I I Dc o m p l e x a t p r o m o t e r s c o n t a i n i n g o r l a c k i n g t h e T A T Ab o x b u t d i s t i n c t m e c h a n i s m s o f t r a n s c r i p t i o n a l r e g u l a -t i o n b y S p l .Resul t sP r oper t i e s o f a T A T A - le s s pr omoterB e f o r e a d d r e s s i n g w h e t h e r t h e T B P o r t h e p r o p o s e dT P I I D c o m p l e x i s r e q u i r e d a t a T A T A - l e s s p r o m o t e r , i t i sf i r s t n e c e s s a r y t o e s t a b l i s h t h a t t h e t e m p l a t e b e i n g u s e di s b e h a v i n g a s a T A T A - l e s s p r o m o t e r . T h e l a c k o f a c a -

n o n i c a l T A T A - l i k e s e q u e n c e 3 0 n u c l e o ti d e s u p s t r e a m o ft h e t r a n s c r i p t i o n a l s t a r t s i t e m a y n o t b e s u f f i c i e n t c r i te -r i a t o d e f i n e a T A T A - l e s s p r o m o t e r . F o r e x a m p l e , t h ea d e n o v i r u s E I V a 2 p r o m o t e r h a s b e e n d e s c r i b e d a s aT A T A - l e s s p r o m o t e r ( C a r c a m o e t al . 1 98 9) . H o w e v e r , ar e c e n t s t u d y i d e n t i f i e d a T A T A b o x l o c a t e d a t p o s i t i o n+ 15 , w h i ch i s r equ i r ed f o r spec i f i c and e f f i c i en t i n i t i a -t i o n f r o m t h e + 1 s i te o f t h e p r o m o t e r ( C a r c a m o e t a l.1990) . I n add i t i on , T B P and f ac t o r s i n t he T FI I D f r ac t i onh a v e t h e p o t e n t i a l t o r e co g n i z e t h e p r o m o t e r t h r o u g h an u m b e r o f s e q u e n c e s u n r e l a t e d t o th e T A T A A A A m o t i f( H ahn e t a l . 1989b ; S i nge r e t a l . 1990) . A f unc t i ona l l yT A T A - l e s s p r o m o t e r s h o u l d h a v e n o i n t r i n s ic a b i l it y t os p e c i f i c a ll y b in d T B P o r t h e T F I I D c o m p l e x d i r e c t l y a n dt h u s d i r e c t v i r t u a l l y n o b a s a l l e v e l t r a n s c r i p t i o n i n t h ea b s e n c e o f a p r o m o t e r - b o u n d a c t i v a t o r s u c h a s S p l .T h e t e m p l a t e c h o s e n f o r t h i s s t u d y , G6I c o n s i s t s o fm u l t i p l e S p l - b i n d i n g s i t e s u p s t r e a m o f a n i n i t i a t o r e l e -m e n t ( S m a l e a n d B a l t i m o r e 1 9 8 9; P u g h a n d T j i a n 1 9 90 ).N o s e q u e n c e u p s t r e a m o r w i t h i n 3 0 n u c l e o t id e s d o w n -s t r e a m o f t h e t r a n s c r i p t i o n a l s t a r t s i t e r e m o t e l y r e s e m -b l e d t h e T A T A A A A s e q u e n c e ( se e F ig . 1 A ) . A s f u r t h e re v id e n c e, n e i t h e r t h e r e c o m b i n a n t h T B P n o r t h e e n d og -e n o u s H e L a T F I ID s p e c i f i c a ll y b o u n d t o a n y s e q u e n c e o nth e G6I p r o m o t e r , o v e r a w i d e p r o t e i n c o n c e n t r a t i o nr a n g e t h a t p r o t e c t e d a n e q u i v a l e n t p r o b e c o n t a i n i n g t h ea d e n o v i r u s m a j o r l a t e ( A d M L ) T A T A b o x ( d a t a n o ts h o w n ) . B y t h i s c r i t e r io n , n e i t h e r T B P n o r a n y c o m p o -n e n t i n t h e T F I I D c o m p l e x r e c o g n i z e d t h e G6I p r o m o t e r .I n t h e p r e s e n c e o f ex c e s s T B P o r T F I ID , n o n s p e c i f i c b i n d -i n g t h r o u g h o u t t h e p r o b e w a s o b s e r v e d , w i t h t h e c o n s e -q u e n c e o f a h i g h l e v e l o f r a n d o m t r a n s c r i p t i o n i n i t i a t i n gt h r o u g h o u t t h e t e m p l a t e ( d a ta n o t s h o w n ) .B a s al t r a n s c r i p t i o n o n G 6 I w a s t h e n a s s e s se d . I n a p a r -t i a l l y p u r i f ie d t r a n s c r i p t i o n s y s t e m d e v o i d o f S p l , s p e -c i fi c i n i t i a t i o n w a s < 0 . 1 % o f t h e l e v e l o b s e r v e d w i t h a ne q u i v a l e n t T A T A - c o n t a i n i n g p r o m o t e r ( F i g . 1 B, l a n e s1 ,2 ). I n t h e p r e s e n c e o f S p l , e f f i c i e n t t r a n s c r i p t i o n w a sr e s t o r e d on G 6 I a t t h e p r e v i o u s l y d e f i n e d i n v i v o s t a r ts i t e ( F i g . 1B, l ane 3 ; Sm a l e and B a l t i m or e 1989). T h e> 1 0 0 - f o l d s t i m u l a t i o n o f t r a n s c r i p t i o n o b s e r v e d o n G 6 Iw i t h t h e a d d i t i o n o f S p 1 e m p h a s i z e s t h e c e n t r a l r o l e t h a tS p l p l a y s in a c t i v a t i n g t r a n s c r i p t i o n f r o m t h i s T A T A -l e s s p r o m o t e r . T h e f a i l u r e o f G6 1 t o s u p p o r t a n y b a s a lt r a n s c r i p t i o n a n d s p e c i f i c a l l y b i n d h T B P o r t h e h u m a nT F I I D (h T F II D ) c o m p l e x r e l a t i v e t o i t s T A T A - c o n t a i n i n ga n a l o g l e a d s u s t o c o n c l u d e t h a t t h e G6I p r o m o t e r r e p re -s e n t s a b o n a f id e T A T A - l e s s t e m p l a t e t h a t h a s n o i n t r i n -s i c a b i l i t y t o r e c r u i t T B P o r t h e T F I I D c o m p l e x d i r e c t ly .

Inac t i va t ion o f T B PH a v i n g e s t a b l i s h e d t h a t t h e r e c o m b i n a n t h T B P a n d e n -d o g e n o u s h u m a n h T F I I D c o m p l e x h a v e n o i n t r i n s i cb i n d i n g s p e c i f i c i t y f o r th e G 61 p r o m o t e r , t h e n e x t s t e pw a s t o d e t e r m i n e w h e t h e r T B P p l a y e d a n y r o l e i n S p l -a c t i v a t e d t r a n s c r i p t i o n f r o m t h i s p r o m o t e r . O u r p r e v i o u ss t u d y r e v e a l e d t h a t t h e p a r t i a l l y p u r i f i e d H e L a T F I I Df r a c t i o n , w h i c h c o n t a i n e d a t l e a s t T B P , t h e S p l c o a c t i -v a t o r , a n d t h e t e t h e r i n g f a c t o r , w a s r e q u i r e d f o r r e c o n -

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Figure 1. In vitro transc riptio n of a TATA -less promoter. (A) Aschematic and nucleotide sequence of the TATA-less promoterG6I. The 21-bp repeats (GC boxes) of the S V40 early promoter(solid underline) were placed upstream of the AdML initiatorelement (broken underline), as described previously {Pugh andTjian 1990). The trans criptio nal start site is boxed. (B) Basal andactivated transcription on G 6I . HeLa basal initiation fractions(TFIIA, TFIIB, THID, and T HIE/F /polym erase II) were recons ti-tuted on G6T I (which is a TAT A-containing version of G6I) inthe absen ce of Spl (lane 1), and o n G 6 I in t he absence (lane 2) orpresence (lane 3) of Spl as described previou sly (Pugh and Tjia n1990). Initiation from the initiator yields a 377-nucleotide tran-script. Nucleotide size markers are show n at left .

t ran scr i p t io n (Fig . 2 , lane 1). Spec if ic in i t i a t i on was re -s to re d b y th e a d d i t i o n o f t h e p a r t i a l l y p u r i f i e d H e LaTFI ID ( l a n e 2 ); su rp r i s in g ly , h o w e v e r , n o a mo u n t o f r e-c o m b i n a n t T B P w a s a b le to r e c o n s t i t u t e t r a n s c r i p t i o n o nG 6 I ( l a n e 3 ; d a t a n o t sh o w n ) . Th i s su g g e s t e d th a t a n a d -d i t i o n a l h e a t - la b i l e fa c t or t h a t c o p u r i f ie d w i t h t h e H e L aT H I D f r a c t i o n i s r e q u i r e d f o r T A T A - l e s s t r a n s c r i p t i o n .T h i s a d d i t i o n a l h e a t - l a b i le a c t i v i t y i s l i k e l y t o b e a t l e a s tp a r t o f t h e p re v io u s ly d e f in e d t e th e r in g f a c to r , a s i t s a c -t i v i t y i s d i sp e n sa b le i n t h e p re se n c e o f a TA TA b o x ; t h a tis , as observed previously (Pe te rson e t a l . 1990) , the re -c o m b i n a n t T B P, a l o n g w i t h t h e h e a t - s t a b l e S p l c o a ct i -v a to r, r e s to re s b o th b a sa l a n d h i g h l e v e l s o f Sp 1 -a c t iv a t e dt r a n sc r ip t io n to t h e h e a t - t r e a t e d e x t r a c t i n t h e p re se n c eof a TATA box (Fig . 2 , c f . lanes 8 ,9 wi th 4 ,5) . Al thought h i s e x p e r i m e n t p r o v i d e s a d d i t i o n a l i n s i g h t i n t o t h e n a -tu re o f t h e t e th e r i n g f a c to r a n d fu r th e r d i s t i n g u i sh e s i tf ro m th e c o a c t iv a to r , i t d o e s n o t y i e ld i n fo rm a t io n r e-g a r di n g t h e r e q u i r e m e n t o f T B P .

I n a n a t t e m p t t o s p e c i f i c a l ly i n a c t i v a t e o r r e m o v e T B Pw i t h o u t i n d i s c r i m i n a t e l y a f f e c ti n g o t h e r i m p o r t a n t i n i -t i a t i o n f a c to rs , w e tu r n e d to t h e u se o f a n t i -TBP a n t ib o d -i es . T o m a x i m i z e s p e c i f i c i ty a nd m i n i m i z e a n y p o t e n t i a ln o n sp e c i f i c i n h i b i to ry ef f e c t s o n t r a n sc r ip t io n , t h e p o ly -c l o n a l a n t i b o d i e s w e r e p u r i f i e d b y h T B P a f f i n i t y c h ro m a -t o g r a p h y . B o t h t h e r e c o m b i n a n t h T B P a n d t h e r e s u l t i n gp u r i f i e d a n t i -h TBP a n t ib o d ie s w e re >9 5 % p u re , a s d e t e r-m i n e d b y s i l v e r - s t a i n e d S D S - p o l y a c r y l a m i d e g e l s ( d a t an o t s h o w n) . T h e h T B P a n t i b o d i e s i n h i b i t e d t h e p r o d uc -t i o n of - 9 5 % o f t h e R N A t r a n s c r i p t i n i t i a t i n g f r o m t h e+ 1 s t a r t s i t e o f t h e TA TA - le ss G 6 I t e mp l a t e (Fig. 3 , l a n e s3 -8 ) . In c o n t ra s t , e q u iv a l e n t a mo u n t s o f a f f i n i t y -p u r i f i e d

stituti ng TAT A-less transcription (Pugh and Tjian 1990).Be c a u se c o mp le t e p u r i f i c a t i o n o f t h e f a c to r s p re se n t i nt h i s f r a c t io n a n d r e c o n s t i t u t i o n o f t h e i r a c t i v i t y o n G 6 Ih a d n o t b e e n a c h i e v e d , w e w e r e u n a b l e t o d e t e r m i n eu n a m b i g u o u s l y w h e t h e r T B P w a s i n v o l v e d i n T A T A -l e s s t r a n s c r i p t io n . W e n o t e t h a t n e i t h e r t h e r e c o m b i n a n ty e a s t n o r h T B P c o u l d r e p l a c e t h e e n d o g e n o u s h T F I I Dfraction for transcription on G6I in a system reconsti-tuted w i th p a r t i a l l y p u r i f i e d I- te La b a sa l i n i t i a t i o n f a c to r s(Pu g h a n d T j i a n 1 9 9 0 ; d a t a n o t sh o w n ) .A p r e v i o u s l y d e s c r i be d h e a t t r e a t m e n t p r o to c o l ( N a k a -jiraa et al. 1988), whic h ina ctivates TBP in nuclear ex-tracts w h i l e l e a v i n g t h e o t h e r b a s a l i n i t i a t i o n f a c to r s a n dc o a c t iv a to r s r e l a t i v e ly u n a f fe c t e d , w a s fo u n d to su p p o r tSp l -a c t iv a t e d t r a n sc r ip t io n in t h e p re se n c e o f a d d e d r e -c o m b i n a n t h T B P o n a T A T A - c o n t a i n i n g p r o m o t e r(Pe te rson e t a l . 1990). In contrast , on G6I we fou nd th a tt h e h e a t - t r e a t e d n u c l e a r e x t r a c t w a s u n a b l e t o s u p p o r t

Figure 2. The tetherin g factor is heat labile. Nuc lear extractswere incubated at 47~ for 15 min (Nakajima et al. 1988)andassayed for transcriptio nal activity on G6I in the presence of Spl{lanes 1-3) or on G6T I in the absence or presence of Spl [lanes4--9). Lanes 1, 4j and 5 contain no added hTBP~ lanes 2, 6, and 7contai n added partia lly purified HeLa THID; lanes 3, 8, and 9contain 30 ng of hTBP. A broad range of hTBP titrations pro-duced no detectable + 1 initiation on G6I.

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Figure 3. hTBP antibo dies specifically inh ibi t TATA -less transcription. HeLa nuclear extracts (90% depleted of Sp 1) were assayed fortranscription on G6I in the absence (lane 1) or presence (lane 2) of added Spl by the runoff assay. Alth ough a nu mb er of transcr iptionfactors present i n the H eLa nuclea r extract bind to the SV40 21-bp repeats present on G6I (Mitchell et al. 1987), lanes 1 and 2 sh owthat Spl is th e do min ant activator. In creasing amou nts of affinity-purified hTBP antibodies (lanes 3-8), dTB P antibod ies (lanes 9-11 ),or anti-CTF antibodies (lanes 13-15) (as indicated above each lane) were preincubated wi th the n uclea r extract for 2 hr at 4~ beforeassaying the extracts for transcription. Tr anscripts a rising from the + 1 start site are 154 nucleo tides (nt). The ban d labeled internalstandard arises from an in depen dent reaction involv ing an inte rnal RN A and [a-g2P]UTP. It serves as a useful inter nal standard for RNArecovery and comparisons between experiments. The nucleotide molecular mass markers are shown at left. Unit definitions aredescribed in Materials and methods.

c o n t ro l a n t ib o d ie s r a i se d a g a in s t e i t h e r t h e d iv e rg e n ta m i n o - t e r m i n a l d o m a i n o f r e c o m b i n a n t Drosophila T BP( d T B P a n t i b o d i e s ) o r t h e u n r e l a t e d h u m a n t r a n s c r i p t i o nf a c to r C T F d i d n o t s i g n i f i c a n t l y i n h i b i t t r a n s c r i p t i o n(Fig . 3 , lanes 9-11 and 13-15, respec t ive ly) . In addi t ion ,t r a n s c r i p t i o n f r o m G 6 I w a s c o m p l e t e l y i n h i b i t e d b y 1l x g/ m l o f ~ - a m a n i t i n , c o n f i r m i n g t h a t t r a n s c r i p t i o n w a sc a r r i e d o u t b y RN A p o ly m e ra se I I ( la n e 1 2 ) . Th e se f i n d -in g s su g g e s t ed th a t TBP w a s in v o lv e d in Sp l -a c t iv a t e dT A T A - l e s s t r a n s c r i p t i o n o f th e G 6I t e m p l a t e .

Immunodepletion of TBPO n e a l t e r n a t i v e i n t e r p r e t a t i o n o f t h e d i r e c t i n h i b i t i o ne x p e r i m e n t s p r e s e n t e d a b o v e i s t h a t t h e i n h i b i t e d f a c to ri s n o t TBP b u t a d i f f e re n t f a c to r t h a t c o n ta in s o n e o rmo re c ro ss - re a c t in g e p i to p e s . To id e n t i fy t h e p ro t e in st h a t d i r e c t l y b i n d h T B P a n t i b o d i e s , n u c l e a r e x t r a c t s a m -p l e s w e r e s u b j e c t e d t o W e s t e m b l o t a n a l y s i s w i t h p u r i-f i e d h TBP a n t ib o d ie s (F ig . 4 A ). Th e ma jo r imm u n o re a c -t i v e s p e c i e s m i g r a t e d w i t h t h e s a m e m o l e c u l a r m a s s a st h e 3 8 - k D r e c o m b i n a n t T B P a n d w a s t h e r ef o r e l i k e l y t ob e th e e n d o g e n o u s TBP (c f. l a n e s 1 a n d 3) . Tw o w e a k lyc r o s s- r e a ct i n g p o l y p e p t i d e s of - 6 5 a n d - 9 0 k D w e r e a l sop r e s e n t i n t h e n u c l e a r e x t r a c t . T o d e t e r m i n e w h i c h o ft h e p r o t e i n s t a r g e t ed b y t h e a n t i b o d y r e s u l t e d i n i n h i b i -t i o n o f T A T A - l e s s t r a n s c r i p t i o n w e p e r f o r m e d t h e f o l-l o w i n g i m m u n o d e p l e t i o n a s s a y s. F i rs t , p u r i fi e d h T B P a n -t i b o d ie s w e r e m i x e d w i t h t h e H e L a n u c l e a r e x t r a c t. P r o-

t e i n s b o u n d t o t h e a n t i b o d i e s w e re t h e n r e m o v e d b ya d s o r p t io n o f t h e a n t i b o d y o n t o p r o t e i n A - S e p h a r o s er e s in . W h e n t h e d e p l e t e d e x t r ac t w a s e x a m i n e d b y W e s t -e m b lo t , t h e m a jo r d e p le t e d sp e c i e s w a s TBP [Fig. 4 A, c f .l a n e s 2 ,3 ) ; t h e l e v e l o f t h e 6 5 - a n d 9 0 -k D p ro t e in s r e -m a i n e d r e l a t i v e l y u n a l t e r e d . T h u s , t h e a n t i b o d y a p -p e a r ed t o i m m u n o d e p l e t e o n l y T B P . T h e o t h e r c r os s -r e-a c t i n g s p e c i e s m i g h t h a v e b e e n a b u n d a n t l o w - a f f i n i t ye p i to p e s t h a t w e re n o t d e p le t e d e f f i c i e n t ly a n d , t h e re -fo re , w e re n o t l i k e ly r e l e v a n t t o t r a n s c r ip t io n in i t i a t i o n .

T h e n e x t c r i t i c a l t e s t i n d e m o n s t r a t i n g t h e i m p o r t a n c eo f T B P w a s to s h o w t h a t i m m u n o d e p l e t i o n o f TB P f r o mth e e x t r a c t c o r re sp o n d e d to a l o ss o f TA T A - le ss t r a n -sc r ip t io n . A s sh o w n in F ig u re 4 B [ l a n e s 1-7), i n c r e a s i n ga m o u n t s o f h T B P a n t i b o d y f o l l o w e d b y r e m o v a l w i t hp r o t e i n A - S e p h a r o s e e f f i c i e n t ly d e p le t e d t h e a b i l i t y o fth e e x t r a c t t o t r a n sc r ib e G 6 I . Th e c o n t ro l (Drosophila)d TBP a n t ib o d y h a d l i t t l e e f f e c t ( l a n e 8 ) .

T h e s p e c i f ic i t y of t h e h T B P a n t i b o d y w a s e v a l u a t e df u r t h e r b y s e l e c t i v e l y b l o c k i n g t h e a n t i b o d y w i t h p u r i-f i ed a n t ig e n . I n c r e a s i n g a m o u n t s o f p u r i f i e d r e c o m b i n a n th TBP o r c o n t ro l d TBP w e re p re in c u b a te d w i th t h e a n t i -b o d y p ri o r to i m m u n o d e p l e t i o n o f t h e e x t r a ct . T h e a b i l -i t y of e x t r a c t s t r e a t e d w i th t h e b lo c k e d a n t ib o d y to su p -p o r t t r a n sc r ip t io n in i t i a t i n g f ro m th e + 1 s t a r t s i t e o f G 6 Iw a s q u a n t i t a t e d a n d s h o w n i n F i g u r e 4 C . U n d e r t h e s ec o n d i t i o n s , b e t w e e n 4 a n d 1 3 n g o f r e c o m b i n a n t h T B Pw a s s u f f i c i e n t to n e u t ra l i z e a l l o f t h e s p e c i f i c a n t ib o d y inth e r e a c t io n su c h th a t n o lo ss o f t r a n sc r ip t io n w a s o b -

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Figure 4. Imm uno dep let io n of the endo genous hT BP. (A} Weste rn blot of TBP. Nuclea r extrac t {NE~ 21 tzg}, wh ich w as ei the rmock -depleted ( lane 3) or TBP-depleted {as described below) wit h 20 ng of hTBP a ntibod y ( lane 2), was e lec t rophoresed on an SD S-10%polyacry lamide ge l , t rans fe r red on to n i t roce l lu lose , and p robed wi th a f f in ity -pur i fi ed hTBP an t ibod ies and a lk a l ine phospha tase -conjugated goat an ti-rabbit 2 ~ antibody. Lane 1 is the same as lane 2 but also contain s 1 ng of purif ied bacte rial ly expressed hTBP. T heendogenous TBP was es t imated to be -0 .007 % pure in the nuc lea r ex t ract . P ro te in molecu la r mass {kD) markers a re show n a t right.{B) Effect of im mu nod epl et io n on TA TA-less tran script ion. Increasing amo unts of hTBP antib ody {lanes 2-7) or dTBP antibody ( lane8) [as indicate d above each lane) were incubate d w ith 84 ~xg of nuclea r extract for 1 hr at 4~ Protein A -Seph arose {2 ~I hydrated byvolume} was then added and mixed in te rmi t ten t ly fo r 1 h r a t 4~ The m ix ture was cen t r i fuged a t 14 ,000 rpm (microcen tr i fuge) fo r 2min . The superna tan t was assayed fo r t r ansc r ip t ion as desc r ibed in Mate r ia l s and methods . Iden t ica l resu l t s were ob ta ined wi thantibodies derived from two different rabbits . (C) Antibody neutral izat ion, hTBP antibody (80 ng) was preincubated with 0, 4, 13, or40 ng of recombinant hTBP (bars 2-5) or with 0, 4, 13, or 40 ng of recom binan t dTBP (bars 6-7) for 1 hr at 4~ The treated hTB Pant ibody was then used fo r imm unode ple t ion o f the nuc lea r ex t rac t a s desc ribed in B . The m ock reac t ion con ta ined no an t ibod ies andno recom binan t TBP. Trea ted ex t rac t s were assayed for t r ansc r ip t ion on G6I as described in Fig. 3. Transcrip ts ar is ing from the + 1 starts i t e were quan t i t a ted by au torad iography fo l lowed by scann ing dens i tom et ry and a re p resen ted in ba r g raph fo rm. In the exper imen tswhere the neu t ra l i z ing TBP an t igen was e i the r in excess o r no t recognized by the an t ibody (bars 5 -7) , a h igh background of nonspec i f ict ransc r ip t ion was observed th ro ughout the t em pla te inc lud ing near the s ta r t s i t e as a resu l t o f the h igh degree of nonspec i f ic b ind ing .This had the e f fec t o f a r t i f ic ia l ly inc reas ing the apparen t s igna l of these reac t ions . (D) Ef fec t o f immunod eple t ing the TFI ID f rac t ionon TATA-less t ransc r ip t ion and basa l TATA-conta in ing t ransc r ip t ion . The endogenous TFI ID complex was par t i a l ly pur i f i ed asdescribed previou sly (Pugh and Tjian 1990). An al iq uot of TFIID (containing 1 ng of TBP as mea sured by Wester n blot) was incub atedwi th the ind ica ted amount o f hTBP an t ibody and immunodeple ted wi th p ro te in A-Sepharose as desc r ibed in B . The superna tan t wasassayed for t rans cript i on wi th the o ther basal ini t ia t ion fract ions (TFIIA-TFIIF) and Spl on G6I {Q) or for basal TATA-containingtransc ript ion in the abs ence of Spl on G6TI (C)) as described previou sly (Pugh and Tjian 1990). Data qua nti t at io n is described in C.

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Pugh and Tjian

served (c f . bars 3 and 4 wi th 1 and 2) . As a contro l , dTBPw a s l a rg e ly i n e f fe c t iv e a t b lo c k in g th e h TBP a n t ib o d y(b a r s 6 a n d 7 ) . Th e se e x p e r ime n t s p ro v id e d a d d i t i o n a ls u p p o r t t h a t t h e h T B P a n t i b o d y w a s s p e c i f i c a ll y a n dq u a n t i t a t i v e l y r e m o v i n g t h e e n d o g e n o u s T B P, t h e r e b yr e n d e r i n g t h e e x t r a c t s i n c a p a b l e o f t r a n s c r ib i n g G6 I .

In a n e f fo r t t o c o r re l a t e t h i s a p p a re n t TBP d e p e n d e n c eo f T A T A - l e s s t r a n s c r i p t i o n w i t h t h e r e q u i r e m e n t f o rTBP a t a TA TA -c o n ta in i n g p ro mo te r , Sp 1 -a c t iv a t e d tr a n -s c r i p t i o n o n G 6 I w a s c o mp a re d s id e b y s id e t o b a sa l (n oS p l p r e s e n t ) t r a n s c r i p t i o n o n G 6 T I r e c o n s t i t u t e d w i t hf r a c t i o n a te d b a s a l i n i t i a t i o n f a c to r s . I n c r e a s i n g a m o u n t so f h T B P a n t i b o d y w e r e p r e i n c u b a t e d w i t h a p a r t i a l ly p u-r i f ie d TF I I D f r a c t io n a n d w e r e s u b s e q u e n t l y r e m o v e dw i t h p r o t e i n A - S e p h a r o s e . T h e a n t i b o d y - d e p l e t e d T F II Df ra c t io n s w e re a ssa y e d fo r t r a n sc r ip t io n , a n d th e d a t aw e re q u a n t i t a t e d (Fig. 4D ) . In c re a s in g a m o u n t s o f a n t i -b o d y d e c r ea s e d t r a n s c r i p t i o n a l a c t i v i t y f r o m b o t h t y p e so f p ro mo te r s w i th n e a r ly i d e n t i c a l p ro f i l e s , su g g e s t in gt h a t T B P w a s e q u a l l y i m p o r t a n t a t b o t h t y p e s o f p r o m o t -e r s. To g e th e r , t h e d a t a p re se n te d th u s f a r p ro v id e c o m-p e l l i n g e v id e n c e th a t TBP a n d fa c to rs t i g h t ly a sso c i a t e dw i th i t a r e e sse n t i a l fo r t r a n sc r ip t io n a t b o th t h e TA TA -le ss G6I a n d T A T A - c o n t a i n i n g G 6 T I p ro mo te r s .

T BP-assoc ia ted fac tor sT h e a p p a r e n t s i z e d i f f e r e n c e b e t w e e n r e c o m b i n a n t T B Pa n d t h e n a t i v e T F II D c o m p l e x s u g g e s te d t h a t T B P m i g h tb e o n l y o n e c o m p o n e n t o f a m u l t i s u b u n i t T F I I D c o m -p le x . To a d d re ss t h i s p o ss ib i l i t y , t h e c o mp o s i t i o n o f t h ei m m u n o p r e c i p i t a t e d T F I ID c o m p l e x w a s e x a m i n e d b yp o ly a c ry l a m id e g e l e l e c t ro p h o re s i s . Be c a u se o u r s t a r t i n gma te r i a l w a s a p a r t i a l l y p u r i f i e d TFI ID f ra c t io n i t w a sf i r s t n e c e ssa ry t o e s t a b l i sh t h a t TBP i s t h e o n ly a n t ig e nin t h i s f r a c t io n th a t i s c a p a b le of b e in g r e c o g n iz e d b y th ea n t i - h T B P a n t i b od i e s . F i g ur e 5 A s h o w s a W e s t e m b l o t o fth e TFI ID f ra c t io n a n d p u r i f i e d TBP. O n ly TBP (a n d mi -n o r b re a k d o w n p ro d u c t s ) i n t h e TFI ID f ra c t io n h a d a v id -i t y f or t h e a n t i b o d y , t h e r e b y e s t a b l i s h i n g i t a s t h e o n l yp ro t e in i n t h e TFI ID f ra c t io n l i k e l y t o b e t a rg e t e d d i -r e c t ly b y th e a n t ib o d y .H a v in g e s t a b l i sh e d TBP a s t h e so l e a n t ib o d y t a rg e t i nt h e T F I I D f r a c t i o n , w e t h e n e x a m i n e d t h e i m m u n o p r e -c ip i t a t e b y SD S - -p o ly a c ry l a mid e ge l e l e c t ro p h o re s i s fo l-l o w e d b y s i l v e r s t a in in g . F ig u re 5 B, l a n e 1 , sh o w s th ep o l y p e p t i d e d i s t r i b u t i o n o f < 0 . 3 % o f t h e i n p u t T F I I Df r a c t i o n . W h e n t h e i m m u n o p r e c i p i t a t e w a s w a s h e d e x -t e n s i v e l y an d e l u t e d f ro m t h e p r o t e i n A - S e p h a r o s e w i t h

Figure 5. Puri fica tion of TBP -associated factors. (A)Western blot of recomb inant bacterial hTBP and thephosphocellulose TFIID fraction. (B) Five micro-grams of the partially purified TFIID fraction start-ing material is shown i n lane 1. It represents


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Subuni ts o f human T FIID

0 . 1 % S D S , a l a rg e n u m b e r o f p r o t e i n s w e r e o b s e r v e d t oc o i m m u n o p r e c i p i t a t e w i t h t h e e n d o g e n o u s T B P ( F ig . 5 B,l a n e 1 2 ) . O b v i o u s l y , m a n y o f t h e s e p r o t e i n s c o u l d h a v eb e e n n o n s p e c i f i c a l l y a d s o r b e d t o t h e r e si n , t h e a n t i b o d y ,o r T B P . T h e r e f o r e , w e s o u g h t t o d e m o n s t r a t e t h e s p e c i -f i c i t y o f t h e s e a n t i b o d i e s t h r o u g h a p a ra l l e l i m m u n o p r e -c i p i t a t i o n i n w h i c h t h e a n t i g e n - b i n d i n g s i t e s o f t h e a n -t i b o d y w e r e b l o c k e d w i t h r e c o m b i n a n t h T B P . P r e l i m i -n a r y s t u d i e s i n d i c a t e d t h a t t h e t e t h e r i n g f a c t o r a n dc o a c t i v a t o r a c t i v i t i e s w e r e t i g h t l y a s s o c i a t e d w i t h t h ee n d o g e n o u s T B P a n d n o t e x c h a n g e a b l e o n t o e x o g e n o u sT B P. T h e re f o re , w e r e a s o n e d t h a t i m m u n o p r e c i p i t a t i o nc a r r i e d o u t w i t h T B P - b l o c k e d a n t i b o d i e s w o u l d r e c o v e rf e w , i f a n y , s p e c i f i c f a c t o r s a n d t h u s w o u l d s e r v e a s av e r y s t r i n g e n t c o n t r o l f o r s p e c i f i c it y . I n a d d i t i o n , t o v i -s u a l i z e t h e v a r i o u s a s s o c i a t e d f a c t o r s a p a r t f r o m t h e l a rg ea m o u n t o f a n t i b o d y p r e se n t , w e a t t e m p t e d t o e l u t e t h ev a r i o u s s u b u n i t s f r o m t h e i m m u n o p r e c i p i t a t e d T F I I Dc o m p l e x w i t h i n c r ea s i n g c o n c e n t r a t i o n s o f t h e d e n a t u r -a n t g u a n i d i n e h y d r o c h l o r i d e ( G u H C 1 ) . P a r a l l e l s t u d i e sw i t h Drosophila T F I I D s u g g e s t e d t h a t t h i s r e a g e n t w a se f f ec t i ve i n s t r i pp i ng T B P- as soc i a t ed f ac t o r s ( T A Fs ) f r omd T B P ( B .D . D y n l a c h t a n d R . T j ia n , u n p u b l . ) . A s s h o w n i nF i gur e 5B , 1 M G uH C 1 ( l ane 7 ), bu t n o t 0 .2 M G uH C 1 ( l ane5), e f f ec t i ve l y e l u t ed a t l e a s t 6 ma j o r T A Fs and a t l e a s t 10a d d i t i o n a l m i n o r T A F s . W e e s t i m a t e a 2 0 0 0 - f o l d p u r i f i -c a t i o n o f t h e s e T A F s b y i m m u n o p r e c i p i t a t i o n , a n d- 2 0 , 0 0 0 - f o l d e n r i c h m e n t f r o m t h e c r u d e n u c le a r e x t ra c t .I n l a n e 9 , t h e r e m a i n i n g p r o t e i n s w e r e e l u t e d w i t h 0 . 1 %S D S . I n t h e c o n t r o l l a n e s w h e r e t h e a n t i b o d y w a sb l o c k e d w i t h r e c o m b i n a n t h T B P , n o m a j o r p r o t e in s w e r eobse r v ed i n t he 0 .2 M G uH C 1 ( l ane 4 ), 1 M G uH C 1 { lane 6 ),o r 0 .1% SD S { l ane 8 ) e l ua t e , w i t h t he excep t i on o f t hea n t i b o d y a n d r e c o m b i n a n t T B P . T h u s , t h e 1 6 o r m o r eT A F s e l u t e d i n l a n e 7 w e r e n o t c o n t a m i n a n t s n o n s p e ci f-i c a l l y a d s o r b e d t o t h e r e s i n b u t , i n s t e a d , a p p e a r e d t o b ep h y s i c a l l y a s s o c i a t e d w i t h t h e e n d o g e n o u s T B P . F u r t h e r -m o r e , b e c a u s e t h e a n t ib o d i e s b l o c k e d w i t h r e c o m b i n a n th T B P d i d n o t b e c o m e a s s o c i a t e d w i t h T A F s , t h e T A F sm u s t b e t i g h t l y a s s o ci a t ed w i t h t h e e n d o g e n o u s T B P a n du n a b l e t o e x c h a n g e f r e e l y .

M o l e c u l a r w e i g h t d i s t r ib u t i o n o f t h e h u m a n T A F s( F ig . 5B , l anes 7 ,12 ) can be c om par e d t o t h a t o f t he Droso-phila T A F s ( l a n e 1 0 ) p u r i f i e d i n a s i m i l a r m a n n e r . A l -t h o u g h t h e r e i s l i t t l e c o r r e s p o n d e n c e i n s i z e of i n d i v i d u a lT A F s b e t w e e n t h e t w o s p e c i e s , a g e n e r a l t r e n d i n t h ep a t t e r n o f T A F p o l y p e p t i d e s i s a p p a r e n t . T h u s , T F I I Dc o m p l e x e s i s o l a t e d f r o m h u m a n s a n d f l i e s h a v e a q u a l i -t a t i v e l y s i m i l a r c o m p o s i t i o n a n d m i g h t e x p l a i n w h ym a n y h u m a n t r a n s c r i p ti o n f a c t o rs f u n c t i o n e f f ic i e n tl y i nDrosophila c e l l s . I n F i g u r e 5 C , a d e n s i t o m e t r i c s c a n o ft h e h u m a n T A F s i s s h o w n a l o ng w i t h t h e i r a p p a r e n t m o -l e c u l a r m a s s e s . A l t h o u g h t h e r e l a t i v e i n t e n s i t i e s o f t h ev a r i o u s T A F s r e f le c t t h e a m o u n t o f T A F s p r e s e n t , t h ed i f f e r e n c e s i n m o l e c u l a r m a s s a n d t h e p o t e n t i a l v a r i a b l es i l v e r s t a i n i n g e f f i c i e n c y o f t h e b a n d s m a y c o m p l i c a t ea n y q u a n t i t a t i v e a s s e s s m e n t o f s t o i c h i o m e t r i e s i n t h eT F I I D c o m p l e x . W i t h t h a t c a v e a t , s o m e T A F s a p p e a r t ob e p r e s e n t i n a n e a r s t o i c h i o m e t r i c r a t i o w h i l e o t h e r s a r ec l e a rl y s u b s t o i c h i o m e t r ic . T h e s e s t u d i e s e s t a b l is h t h a t

e n d o g e n o u s H e L a T F I I D e x i s ts a s a c o m p l e x c o n t a i n i n g al ar g e n u m b e r o f s u b u n i ts , s o m e o f w h i c h w e s u s p e c t t ob e n e c e s s a r y f o r t e t h e r i n g f a c t o r a n d c o a c t i v a t o r f u n c -t i on .

Imm unop uri f ied TFIID complexes reconst i tu teTA TA-less transcriptionP a r a l le l s t u d i e s w i t h t h e Drosophila T F I ID h a v e d e m o n -s t r a t e d t h a t e l u t e d T A F s c a n s u p p o r t a c t i v a t o r - r e s p o n -s i v e t r a n s c r i p t i o n ( c o a c t i v a t o r a c t i v i t y ; D y n l a c h t e t a l .1 9 9 1 ) . H o w e v e r , t h e s a m e T A F s w e r e u n a b l e t o r e s t o r et h e t e t h e r i n g f a c t o r a c t i v i t y o n t h e T A T A - l e s s G 6 I pr o -m o t e r ( d a t a n o t s h o w n ) . T h i s d i d n o t c o m e a s a s u r p r is eb e c a u s e w e h a v e f o u n d t h e t e t h e r i n g f a c t o r a c t i v i t y t o b eq u i t e l a b i l e , w h e r e a s t h e c o a c t i v a t o r a c t i v i t y a p p e a r s t obe mor e r e s i l i en t ( s ee F i g . 2D ; Pugh and T j i an 1990) .B e c a us e o f t h e s e t e c h n i c a l l i m i t a t io n s w e a t t e m p t e d t or e c o n s t i t u t e T A T A - l e s s t r a n s c ri p t i o n w i t h t h e i n t a c t i m -m u n o p u r i f i e d T F I I D c o m p l e x a t t a c h e d t o t h e p r o t e i nA - S e p h a r o s e r e s i n . O u r r a t i o n a l e w a s b a s e d o n t w o p r e-v i o u s o b s e r v a t i o n s . F i rs t, w o r k b y A r i a s a n d D y n a n( 1 9 8 9 ) d e m o n s t r a t e d t h a t t r a n s c r i p t i o n c o u l d b e r e c o n -s t i t u t e d o n a s o l i d s u p p o r t s u c h a s S e p h a r o s e . S e c o n d ,c l o s e i n s p e c t i o n o f F i g u r e s 3 a n d 4 B r e v e a l t h a t t h ea m o u n t o f a n t i b o d y re q u i r e d to i m m u n o p r e c i p i t a t eT F II D w a s s i g n i f ic a n t l y l o w e r t h a n t h e a m o u n t r e q u i r e dt o d i r e c t l y i n h i b i t a c t i v i t y . T h u s , p r o v i d e d e x c e s s a n t i -b o d i e s a r e n o t u s e d t o i m m u n o p r e c i p i t a t e T F I I D , i ts h o u l d r e t a i n a s i g n i f i c a n t a m o u n t o f t r a n s c r i p t i o n a c -t i v i t y . T h e i m m u n o p r e c i p i t a t e d T F I I D c o m p l e x w a sw a s h e d e x t e n s i v e ly t o r e m o v e a n y p r o t e i n s n o n s p e c if i-c a l l y a d s o r b e d o n t o t h e r e s i n a n d a d d e d b a c k t o a t r a n -s c r ip t i o n r e a c t io n i m m u n o d e p l e t e d o f T F I ID a c t i v it y . A sshow n i n F i gur e 6 , l ane 3 vs . l ane 1 , h i gh l eve l s o f t r an -s c r i p t i o n w e r e r e s t o r e d o n G 6 I w h e n t h e p u r i f i e d i m m u -n o p r e c i p i t a t e d T F I ID c o m p l e x w a s a d d e d t o t h e r e a c t i o n .N o S p l a c t i v a t i o n w a s o b ta i n e d w h e n p r o t e i n A - S e p h a -r o s e a l o n e w a s a d d e d t o t h e r e a c t i o n , i n d i c a t i n g t h a tt r a n s c r i p t i o n a l s t i m u l a t i o n w a s s p e c i f i c t o t h e T F I I Dc o m p l e x . N o t s u r p r i s i n g l y , p u r i f i e d r e c o m b i n a n t T B Pw a s u n a b l e t o r e s t o r e S p l - d i r e c t e d t r a n s c r i p t i o n t o t h eT A T A - l e s s G 6 I t e m p l a t e ( l an e 2 v s . la n e 1 ) b u t r e c o n s t i -t u t e d t r a n s c r i p t io n t o a T A T A - c o n t a i n i n g p r o m o t e r ( la n e5 vs . la n e 4 ). T h e s e r e s u l t s i n d i c a t e t h a t a n i n t a c t T F I I Dc o m p l e x i s e s s e n t i a l f o r t r a n s c r i p t i o n a t t h e T A T A - l e s sG 61 p r o m o t e r . M o r e o v e r , T B P a p p e a r s t o b e a s u b u n i t o ft h i s c o m p l e x a n d , b y i ts e l f, i s c a p a b l e o f p e r f o r m i n gsome o f t he ac t i v i t i e s ( i . e . , ba sa l t r ansc r i p t i on ) a t t r i bu t edt o t h e T F I I D c o m p l e x b u t n o t a l l of t h e m ( i. e. , T A T A - l e s st r ansc r i p t i on ) .

D i s c u s s i o nPromoter assembly v ia prote in-prote in in terac tionsI n a n e f fo r t t o u n d e r s t a n d h o w R N A p o l y m e r a s e I I c a nr e c o g n i z e a n d t r a n s c r i b e t h e w i d e v a r i e t y o f p r o m o t e r st h a t e x i s t i n a c e l l , w e h a v e f o c u s e d o n t w o a p p a r e n t l yd i s t i n c t c l as s e s o f p r o m o t e r s : T A T A - c o n t a i n i n g a n d

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Figure 6. Recon stitution of TATA-less transcription with pu-rified THID complexes. Tra nscription reactions were performedon the G6I template (lanes 1-3) and G6TI template (lanes 4,5), asdescribed in Materials and methods, by using immunodepletednuclear extracts supplemented with Spl. In lanes 2 and 5, reac-tions also contained 20 ng of recomb inant hTBP . In lane 3,immunopurified TFIID complexes containing 20 ng of endoge-nous TBP were added. The resin con taining the im muno purifiedTFIID complexes (as described in Fig . 5B) was resuspended inbuffer G.0 (see Materials and methods) and the indicatedamount was assayed.

TATA-les s . We sugges ted p rev ious ly tha t the m a jor d i f -fe rence be tween the two types o f t em pla te s i s the m ech-a n i s m b y w h i c h t h e y r e c r u i t T F II D t o t h e t e m p l a t e. W eproposed tha t TF IID i s a com plex o f m any regu la to ryfactors including the TBP {Pugh and Tjian 1990). AtTATA-conta in ing p rom ote rs , TF IID i s rec ru i t ed d i rec t lyto the DNA tem pla te v ia s equence -spec i f i c in te rac t ionsbe tween the TATA box and TBP . Var ia t ions on th i st h e m e m i g h t i n c l u d e a n y D N A e l e m e n t i n t h e p r o m o t e rtha t has an in t r ins ic b ind ing spec i f i c i ty fo r com ponen tsof the TF IID com plex . In con t ra s t , a t t rue TATA-les sprom ote rs tha t have no in t r ins ic spec i f i c i ty fo r TF IID,ce r ta in ac t iva to rs such a s S p l , when bound to a GC box ,a re p roposed to rec ru i t th e TF IID com plex v ia a t e the r ingfac to r phys ica l ly a s soc ia ted wi th TBP (shown schem at i -cal ly in F ig. 7) . Because b oth types of promo ters appearedto requ i re the s am e basa l in i t i a t ion func t ions , the boundTF IID com plex m os t l ike ly a s sem bles a s im i la r p re in i t i -a t ion com plex a t bo th types o f p rom ote rs . P ro m ote rstha t do no t b ind S p l (or a func t io na l ly eq u iva len t fac to r )bu t b ind o the r ac t iva to rs m igh t no t be ab le to rec ru i tT F I ID e f f i c ie n t l y w i t h o u t t h e h e l p o f t h e T A T A b o x.

T w o i m p o r t a n t a s s u m p t i o n s o f t h i s t e t h e ri n g m o d e la re tha t (1 ) TF IID i s requ i red a t a TATA-les s p rom ote r ,and {2) TF IID i s com posed o f m ul t i p le subun i t s tha t in -c lude a t l ea s t the t e the r ing fac to r and TBP . To addres swhe the r TF IID i s requ i red a t a TATA-les s p rom ote r wespec i f i ca l ly inh ib i t ed TATA-les s t ransc r ip t ion wi th a f -f in i ty pur i f i ed an t ibod ie s d i rec ted aga ins t TBP. Wes te rnb l o t a n a ly s i s a n d i m m u n o d e p l e t i o n s t u d ie s f u r t h e r c o n -f i rm tha t the los s o f TATA-les s t ransc r ip t ion i s a re su l to f spec i f i c inac t iva t ion o f TBP by the an t ibody . Mos t

i m p o r t a n t l y , i m m u n o p r e c i p i t a t i o n o f T B P r e v e a le d t h a ti t i s phys ica l ly a s soc ia ted wi th a t l ea s t 6 m a jor and 10m inor subun i t s in add i t ion to TBP . F ur the rm ore , wehave recons t i tu ted t ransc r ip t ion on the TATA-les s G6Item pla te wi th the pur i f i ed TF IID com plex . F rom theda ta p re sen ted in th i s pape r i t i s rea sonab le to p roposetha t p rom ote rs t ransc r ibed by RNA po lym erase I1 , in -c lud ing those tha t l ack a TATA box , are l ike ly to requ i reone fo rm or ano the r o f the TF IID com plex fo r e f f i c ien tt ransc r ip t ion in i t i a t ion . Recen t s tud ie s ind ica te tha tTBP is a l so an e s sen t i a l com p onen t o f RN A po lym eraseI II in i t i a t ion com plexes ; the re fo re , the TBP m ay se rve asa m ore gene ra l euka ryo t ic t ransc r ip t ion in i t i a t ion fac to rthan be l i eved p rev ious ly (Marg o t t in e t al . 1991 ; S im m enet al. 1991).The expe r im ents p re sen ted in th i s pape r dem ons t ra tea requ i rem ent fo r a TF IID com plex fo r TATA-les s t ran-sc r ip t ion bu t do no t iden t i fy the im por tan t subun i t s .Thus , i t i s fo rm a l ly pos s ib le tha t TBP m igh t no t be d i -rec t ly invo lved . The idea tha t TBP i s p re sen t in the com -p lex bu t no t ac tua l ly pa r t i c ipa t ing in the in i t i a t ion reac -t ion i s , however , incons i s ten t wi th the f ind ing tha tTATA-Ies s p rom ote rs requ i re bo th TF IIA and TF IIB(Pugh and Tjian 1990), which direct ly bind hTBP at aTATA-conta in ing p rom ote r (Bura towsk i e t a l . 1989 ;Peterson e t a l . 1990). In addi t ion, direct inhibi t ion ofTATA-less t ranscript ion by TBP ant ibodies (F ig. 3) ar-gues a func t iona l ro le fo r TBP , m u ch a s an t ibody inh ib i -t ion o f the l a rge subu ni t o f RNA po lym erase I1 im pl i -cates i ts role in t ranscript ion (Thompson e t a l . 1989). IfTBP were no t pa r t o f the in i t i a t ion p roces s , i t would beneces sa ry to p ropose an ad hoc nove l c i rcu i t ry o f in te r -act ions for TFIIA and TFIIB that bypasses TBP. Ourm ode l p rov ides a s im ple a l t e rna t ive in which the bas icpre in i t i a t ion com plex rem a ins e s sen t i a l ly una l t e red a t

Figure 7. Multiple distinct TFIID complexes. A schematic offactor assembly at a TATA-less promoter involving the TFIIDcomplex. (Below} Schematics of functionally distinct THIDcomplexes that m ight be generated by a specific arrang ement ofTAFs onTBP (shown in blackl. Some TAFs might be present inall TFIID complexes. O thers mig ht be present in only a subsetand are therefore earm arked for certain promoters.

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Subunits of human TFIID

b o t h T A T A - c o n t a i n i n g a n d T A T A - l e s s p r o m o t e r s b u tt h e p r o m o t e r - b i n d i n g s p e c i f i c i t y o f t h e T F I I D c o m p l e x i sa c h i e v e d t h r o u g h p r o t e i n - p r o t e i n i n t e r a c t i o n s w i t h i t st e t h e r i n g f a c t o r a n d p r o m o t e r - b o u n d S p l i n s t e a d o f d i -r e c t p r o t e i n - D N A i n t e r a c t i o n s b e t w e e n t h e T B P s u b u n i tand t h e T A T A b ox {F i g. 7 ). I t is l i ke l y t h a t T B P can i n -t e r a c t d i r e c t ly w i t h t h e D N A a t a T A T A - l e s s p r o m o t e ro n c e i t i s r e c r u i t e d b y p r o t e i n - p r o t e i n i n t e r a c t i o n s . C o n -s i s t e n t w i t h t h i s p r o p o s a l , S p l , t o a l i m i t e d e x t e n t , a p -p e a r s t o h e l p s t a b l y a s s e m b l e a p r e i n i t i a t i o n c o m p l e x a tt h e p r o m o t e r ( B .F . P u g h , u n p u b l . ) . I n p r e l i m i n a r y s t u d i e sw e a l s o l o o k e d f o r r e c r u i t m e n t o f t h e T F I I D c o m p l e x t oG 6 I i n t h e p r e s e n c e o f S p 1 b y D N a s e I f o o t p r i n t a n a l y s i sa n d f o u n d t h a t T F I ID c a u s e d D N a s e I h y p e r s e n s i t i v es i te s b e t w e e n - 3 0 a n d - 4 5 , b u t w e w e r e u n a b l e to de -t e c t s i g n i f i c a n t p r o t e c t i o n . W h e t h e r t h i s r e f l e c t s t h eneed f o r add i t i ona l f ac t o r s ( e .g ., T FI I A ) t o p r o m ot e s t ab l eb i n d in g r e m a i n s t o b e d e t e r m i n e d .T h e p o s s i b i l i t y t h a t t h e T F I I D c o m p l e x g a i n s b in d i n gs p e c i fi c it y t h r o u g h p r o t e i n - p r o t e i n i n t e r a c ti o n s w i t hp r o m o t e r - b o u n d S p 1 h a s a n e a r l y p r e c e d e n t i n p r o k a r y -o t i c s y s t e m s . A n a l o g o u s t o a T A T A - l e s s p r o m o t e r , t h ep h a g e k P RE p r o m o t e r l a c k s a c o n s e n s u s - 3 5 s e q u e n c e sn e c e s s a r y t o t e t h e r t h e Escherichia coli R N A p o l y m e r as ei n i t i a t i o n c o m p l e x t o t h e p r o m o t e r . A s a s u b s t i t u t e t h e kt r a n s c r i p t i o n a l r e g u l a t o r y p r o t e i n c I I b i n d s t o i t s r e co g -n i t i o n s e q u e n c e in t h e - 3 5 r e g io n a n d r e c r u it s R N Ap o l y m e r a s e p r e s u m a b l y t h r o u g h p r o t e i n - p r o t e i n i n t e r a c-t i o n ( S h i m a t a k e a n d R o s e n b e r g 1 9 8 1 ) . S u b s t i t u t i n g t h ec II p r o t e i n f o r t h e - 3 5 D N A s e q u e n c e a l l o w s c I1 t o s p e-c i f ic a l l y c o n t r o l t h e i n i t i a l e x p r e s s i o n o f cI a n d t h u s t h ed e c i s i o n t o w a r d l y s o g e n y o r l y s i s .

Dis t ingu ish ing TBP, te ther ing fac to r , and coac t iva to rA l t h o u g h T F I ID c o n t a i n s m u l t i p l e s u b u n i t s , a t t r i b u t in ga c t i v i t i e s t o e a c h h a s n o t b e e n s t r a i g h t f o r w a r d . T h e f o l -l o w i n g a c t i v i t i e s h a v e b e e n a t t r i b u t e d t o t h e T F I I D c o m -p l ex : ( 1 ) T A T A box- b i nd i ng f ac t o r ; ( 2 ) ba sa l i n i t i a t i onf ac t o r ; ( 3 ) coac t i va t o r ; and ( 4 ) t e t he r i ng f ac t o r . T he f i r s tt w o c a n b e c a r r ie d o u t b y T B P a l o n e . T h e c o a c t i v a t o ra c t i v i t y i s r e q u i r e d f o r s e q u e n c e - s p e c i f i c a c t i v a t o r s s u c ha s S p l t o s t i m u l a t e t h e b a s a l l e v e l t r a n s c r i p t i o n p r o v i d e dby T B P ( Pe t e r son e t a l. 1990 ; Pugh an d T j i an 1990 ;D y n l a c h t e t a l. 1 9 91 ). D e n a t u r a n t s s u c h a s u r e a a n d g u a -n i d i n e c a n b e u s e d t o s e p a r a t e T B P f r o m c o a c t i v a t o r s( D y n l a e h t e t a l. 1 9 9 1 ; T a n e s e a n d T i i a n 1 9 9 1; t h i s p a p e r l.T he te the r ing fac to r i s requ i re d fo r Sp 1 to ac t iva te a p ro -m o t e r t h a t l a c k s a f u n c t i o n a l T A T A b o x a n d i s d i s ti n c tf r o m t h e c o a c t i v a t o r b y t h e f o l l o w i n g c r i t e ri a : ( 1) M o n oS c h r o m a t o g r a p h y ( P u g h a n d T j i a n 1 99 0) o r 1 M G u H C 1( B. F. P u g h , u n p u b l . ) d e s t r o y s t e t h e r i n g a c t i v i t y b u t n o tc o a c t i v a t o r o r T B P a c t i v i t y ; a n d ( 2 ) h e a t i n g a n u c l e a re x t r a c t i n a c t i v a t e s T B P { N a k a j i m a e t a l . 1 9 8 8 ) a n d t h et e t h e r i n g f a c t o r ( t h is p a pe r ) b u t n o t t h e S p l c o a c t i v a t o r .T h e s e c r i t e r i a d i s t i n g u i s h t h e t e t h e r i n g f a c t o r , c o a c t i v a -t o r, a n d T B P a c t i v i t i e s f r o m o n e a n o t h e r , b u t t h e y d o n o td i s t in g u i s h w h i c h T A F s a n d h o w m a n y c o m p r i s e t h et e t h e r i n g f a c t o r a n d c o a c t i v a t o r a c t i v i t i e s . A l t h o u g ht h e s e t w o a c t i v it i e s m i g h t s h a r e c o m m o n s u b u n i t s, t h e

i m p o r t a n t p o i n t i s t h a t a l l o f t h e s e a c t i v i t i e s r e s i d e i n am u l t i s u b u n i t T F I ID " h o l o e n z y m e . "

I m p l i c a t i o n s o f a m u l t i s u b u n i t T F I ID c o m p l e xin gene regu la t ionT h e c o n c e p t o f a m u l t i s u b u n i t T F I I D c o m p l e x h a s i m -p o r t a n t r a m i f i c a t i o n s i n t h e r e g u l a t i o n a n d s p e c i f i c i t y o ft r a n s c ri p t io n . T F I ID c o m p l e x e s m i g h t b e h e t e r o g e n e o u si n compos i t i on ( i l l u s t r a t ed i n F i g . 7 ) , e ach bea r i ng a d i s -t i n c t c o m b i n a t i o n o f T A F s a n a l og o u s t o t h e c o m b i n a t o -r i a l a r r ay o f cis-regulatory e l e m e n t s t h a t m a k e e a c h p r o-m o t e r u n i q u e . T h e p r o p e r a r ra n g e m e n t s o f T A F s m i g h tp r o v i d e t h e c o m p l e m e n t a r y s u r f a c e f o r t h e u n i q u e a r -r a n g e m e n t o f s e q u e n c e - s p e c i f i c t r a n s c r i p t i o n a l r e g u l a -t o r s a t t h e p r o m o t e r . F i g u r e 7 s h o w s o n e p o s s i b l e a r -r a n g e m e n t f o r a T A T A - l e s s p r o m o t e r . E a c h p r o m o t e rm i g h t a s s e m b l e a m o r e o r l e s s d i s t i n c t r e g u l a t e d i n i t i a -t i o n c o m p l e x t h a t c a n d i c t a t e , a m o n g o t h e r t h i n g s , t h echo i ce o f R N A p o l y me r ase (I , I I, o r I I I ) and t he ove r a l le f f i c ie n c y of t r a n s c r i p t i o n i n i t i a t i o n . A l t h o u g h t h e 1 6 o rm o r e T A F s t h a t c o m p r i s e t h e H e L a T F I I D c o m p l e xm i g h t s e e m u n g a i n l y a t f i r s t , w e a r e r e m i n d e d t h a t an u m b e r o f m a c r o m o l e c u l a r r e a c t i o n s s u c h a s p r o t e i ns y n t h e s i s a n d R N A s p l i c i n g r e q u i r e a l a r g e n u m b e r o fp r o t e i n a n d R N A c o m p o n e n t s . B a c t e ri a a n d y e a s t h a v ep r o v i d e d u s e f u l p a r a d i g m s f o r s t u d y i n g h i g h e r e u k a r y -o t i c t r a n s c r i p t i o n , b u t t h e b a c t e r i a l g - f a c t o r s a n d y e a s tT F I I D a p p e a r t o b e m o n o m e r i c . T r a n s c r i p t i o n a l r e g u l a -t i o n i n y e a s t m i g h t i n v o l v e c o a c t i v a t o r s o r m e d i a t o r s n o tt i gh t l y a s s oc i a t ed w i t h T FI I D ( B e rge r e t al . 1990 ; K e l l e -h e r e t a l. 1 9 9 0) . A l a rg e m u l t i s u b u n i t T F I I D c o m p l e x i nh i g h e r e u k a r y o t i c c e l l s m i g h t h a v e e v o l v e d f o r t h e e x -t e n s i v e g e n e t i c r e g u l a t i o n n e e d e d b y h i g h l y s p e c i a l i z e dc e l l s i n a m u l t i - c e l l u l a r o r g a n i s m .M a t e r i a l s a n d m e t h o d sProteinsPublished procedures were used to prepare the fol lowing re-agents: HeL a nuclear ex tracts {Dignam et al . 198 3) 90% de-pleted of Spl (Jackson and Tjian 1989); He La basal ini t iat io nfractions (TFIIA, TFIIB, TFIID, TF IIE/F/polymerase II) (Pugh andTjian 1990); and recom binant vacc inia Spl (Jackson and Tjian1989); recombin ant bac terial dTBP (Hoey et al . 1990). hTBP andTFIID purificat ions are described below, hTB P, dTBP, and CTFpeptide rabbit polyclonal antibodies we re raised against gel-pu-rified pro te ins and were an t igen /a f f in i ty -pu r i f ied a t low pH(Dyn lac h t e t al . 1991). Pu r i f ied p ro te ins were quan t i ta te d onsi lver-stained gels and were judged >95% pure. Nuclear ex-tracts were qu anti tated b y Bradford, using gam ma globulin as astandard.TranscriptionTranscript ion reactions shown in Figure 2 contained 84 ~g ofSpl-depleted nuclear extract , 150 ng of Spl (when present) , 200ng of plasmid tem plates, 4 mM spermidine, 4 mM MgC12, 30 mMKC1, 10 mM HEPES--KOH (pH 7.9), 10% glycerol, 0.5 m M DTT ,0.5 mM ATP, 0 .5 mM CT P, 0 .5 mM GTP, and 0.5 mM UTP in avolum e of 20 ~1. Mix tures were preincuba ted for 30 m in a t 30~

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in the absence of NTPs. Reactions were termin ated 10 min afterthe addition of NTPs, and RNA products were measured byprime r exte nsion (Jones et al. 1985}.Transcription reactions shown in Figures 3, 4, and 6 wereperformed as described above except for the following changes:0.012 mM [e~-3ZP]UTP (5 ~Ci) replaced the 0.5 mM UTP; 150 ngof poly[d(G-C)] was added; and 20 ng (110 fmoles) of the G6Ipromoter fragment s hown in Figure 1A was used as a template.Runoff transcripts were analyz ed as described previously (Peter-son et al. 1990).U n i t c a l c u l a t i o n sThis section applies to Figures 3 and 4A. The dTBP antibody had130-fold more avidity than the hTBP antibody against their re-spective antigens. To correct for this avidity difference, theamount of dTBP antibody is reported in units, which was cal-culated by multi plyin g the am ount of antibody {e.g., 60 ng as inlane 11) by the r elative avidity (130). The hTBP antibody avidi tywas arbitrarily set at 1. Relative avidity was determined byWestern blots, comparing the amount of antibody needed togenerate equal signal intensit ies from equal amounts of antigen.The anti-CTF antibod y is presented in terms of nanograms. ThedTBP antibody cross-reacts with hTBP at 1-3% of the level itreacts with the dTBP and specifically inhibits transcription in aDro so p h i la nuclear extract (T. Hoey, and R. Tjian, unpubl.).R e c o m b i n a n t h T B P p u r i f ic a t io npARhTFIID-recombinant E. coli BL21 was grown in 12 liters ofYT medi um, 0.4% glucose, and 200 ~g/ml of ampici llin at 37~to an OD6o o of 0.5. The culture was induced with 0.1 mM IPTGand grown for an additional 60 rain at 30~ All subsequentsteps were perfor med at 4~ The harvested cells were resus-pended in 100 ml of lysis buffer [25 mM HEPES at pH 7.6, 200mM KC1, 0.1 mM EDTA, 12. 5 mM KCI, 10% glycerol, 0.1%NP-40, 1 mM DTT, 0.1 mM phen ylme thy lsu lfo nide fluoride(PMSF), 0.1 mM sodium metabisulfite] and incubated with 50mg of lysozyme for 15 min. The cells and DNA were disruptedby sonication in two 30-sec bursts at max imu m output. Nuclei cacids and bound proteins were precipitated by a slow additi on ofpolym in P to a final concentra tion of 0.25% (vol/vol). The ma-terial was sti rred for 20 min and cen trifuged for 15 min at 15,000rpm (SS-34 rotor). KC1 was added to t he supernatant unt il itsconductivity was equal to that of buffer H.35 (20 mM HEPES-KOH at pH 7.9, 10% glycerol , 2 mM MgC12, 1 mM DTT, 0.5 mMEDTA, 0.1 mM PMSF, 0.35 M KC1) and was t hen applied to a40-ml (2.5 x 8 cm) col umn of phosphocellulos e equilibratedwith buffer H.35. TBP was eluted with buffer H1 (which con-tained 1 M KC1). The TBP pool was brought to 0.86 M KC1 and0.5 M ammo ni um sulfate by the add ition of one-sixth volume ofbuffer H.0 containi ng 4 M amm on iu m sulfate. The material wasthen applied to a 3.7-ml phenyl-5PW high-performance liquidchromatography {HPLC) colu mn equilibrated in the samebuffer. The column was washed with buffer H.43 containing0.25 M ammo ni um sulfate. The colum n was developed with a20-ml linear salt gradient extending down to buffer H.0. TBPelut ed at 0.24 M KC1/0.12 M am mo ni um sulf ate and was >95%pure. Peak fractions were pooled and stored at - 80~ whereactivity was stable for at least 15 months.I m m u n o p u r i f i c a t i o n o f T F I I DNuclear extracts were chromatographed over phosphocellu losein buffer H.1 and washed with buffer H.5, and TFIID activityeluted with buffer H.7. Four micrograms of affinity-purified

hTBP antibodies was added to 400 ~g of the phos phocellul oseTFIID fraction and incubated on ice for 3 hr. In the controlincubation, the antibody was preincubated for 3 hr on ice wit h800 ng of recombinant hTBP purified from E. coli, then added tothe TFIID fraction. Protein A-Sepharose resin (20 }~1 hydra tedbed volume) was then added to each, and incubation at 4~ ona rotating wheel contin ued overnight. The resin was centrifugedat 2000 rpm {microcentrifuge) for 2 min at 4~ The resi n waswashed five times, each with 1 ml of buffer H.7. The resin wasthen sequentially eluted with 60 ~1 of buffer G.0, G.2, G1.0,and, finally, with protein gel loading buffer containing 0.1%SDS. Buffer G is the same as buffer H but also cont ains 100 mMpotas sium glut amate, 0.1% NP-40, no KC1, and the i ndic atedamount of GuHC1 {e.g., G1.0 denotes 1 M GuHC1). Eluted sam-ples were precipitated with trichloroacetic acid, washed withacetone, and loaded onto a 7.5% polyacryl amide prot ein gel.After electrophoresis, the gel was silver stained.

A c k n o w l e d g m e n t sWe thank G. Peterson for the hTBP antisera and K. Kilomanskifor the template constructs. We also thank H. Echols, J. Kadon-aga, C. Gross, and the members of the Tjian laboratory for nu-merous helpful comme nts on th e manuscript. B.F.P is a fellowof the Leukemia Society of America. This work was supportedin part by a grant from the National Institutes of Health.The publication costs of this article were defrayed in part bypayment of page charges. This article mus t therefore be herebymarked "adve rtis ement" in accordance wit h 18 USC section1734 solely to indicate this fact.

R e f e r e n c e sArias, J.A. and W.S. Dynan. 1989. Prom oter -depende nt tran-scription by RNA polymerase II using imm obil ized enzyme

complexes. I . Bio l . Chem. 264: 3223-3229.Berger, S.L., W.D. Cress, A. Cress, S.J. Triezenber g, and L.Guarente. 1990. Selective inhibition of activated but notbasal transcripti on by t he acidic activati on dom ain of VP16:Evidence for transcriptional adaptors. C e l l 61: 1199-1208.Buratowski, S., S. Hahn, L. Guarente, and P.A. Sharp. 1989. Fiveintermediate complexes in transcription initiation by RNApolymer ase II. C e l l 56: 549-561.Carcamo, J., S. Lobos, A. Merino, L. Buckbinder, R. Weinmann,V. Natarajan, and D. Reinberg. 1989. Factors involved inspecific transcription by mammalian RNA polymerase II:Role of factors IID and MLTF in transcription from the ad-enovirus major late and IVa2 promoters. J. Bio l . Chem.264: 7704-7714.Carcamo, J., E. Maldonado, P. Cortes, M.H. Ahn , I. Ha, Y. Kasai,J. Flint, and D. Reinberg. 1990. A TATA-like sequence lo-cated downstream of the transcription initiation site is re-quired for expression of an RNA polymerase II transcribedgene. G e n e s & D e v . 4: 1611-1622.Cavallini, B., I. Faus, H. Matthes, J.M. Chipoulet, B. Winsor,J.M. Egly, and P. Chambon. 1989. Cloning of the gene en-coding the yeast pro tein BTF 1Y, whic h can substitu te for thehuman TATA box-binding factor. Pro c . Na t l . Aca d . S c i .86: 9803-9807.Dignam, J.D., R.M. Lebovitz, and R.G. Roeder. 1983. Accuratetranscription initiation by RNA polymerase II in a solubleextract from isolated mammal ian nuclei. N u c l e i c A c i d s R e s .11: 1475-1489.Dynan, W.S. 1986. Promoters for hou sekeepin g genes. T r e n d sGen e t . 2: 196--197.

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Dy n lach t , B .D . , T . Ho ey , an d R . T j ian 1 9 9 1 . Co ac t iv a to r s a s so -c i a t e d w i t h t h e T A T A b i n d i n g p r o t e i n m e d i a t e t r a n s c r i p -t i o n a l a c t i v a t i o n i n D r o s o p h i l a . C e l l 6 6 : 5 6 3 -5 7 6 .

Eisen man n , D .M. , C . Do l la rd , an d F . Win s to n . 1 9 8 9 . SPT1 5 , th eg e n e e n c o d i n g t h e y e a s t T A T A b i n d i n g f a c t o r T F I I D , i s r e -q u i r e d f o r n o r m a l t r a n s c r i p t i o n i n i t i a t i o n i n v i v o . C e l l5 8 : 1 1 8 3 -1 1 9 1 .

Fik e s , J .D . , D .M. Beck e r , F . Win s to n , an d L . Gu a ren te . 1 9 9 0 .S t r i k i n g c o n s e r v a t i o n o f T F I I D i n S c h i z o s a c c h a r o m y c e sp o m b e a n d S a c c h a r o m y c e s c e r e vi s i ae . N a t u r e 3 4 6 : 2 9 1 - 2 9 4 .

Gasch , A . , A . Ho f fman n , M. Ho r ik o sh i , R .G . Ro ed e r , an d N . -H .Gh u a . 1 9 9 0 . Arab id o p s i s th a l i an a co n ta in s two g en es fo rTFI ID. N a t u r e 3 4 6 : 3 9 0 -3 9 4 .

Hah n , S . , S . Bu ra to wsk i , P .A . Sh a rp , an d L . Gu a ren te . 1 9 8 9 a .I s o l a t i o n o f t h e g e n e e n c o d i n g t h e y e a s t T A T A b i n d i n g p r o -t e i n T F I I D : A g e n e i d e n t i c a l t o t h e S P T 1 5 s u p p r e s s o r o f T ye l e m e n t i n s e r t i o n s . C e l l 5 8 : 1 1 7 3 - 1 1 8 1 .

Hah n , S . , S . Bu ra to wsk i , P .A . Sh a rp , an d L . Gu a ren te . 1 9 8 9 b .Y e a s t T A T A - b i n d i n g p r o t e i n T F I ID b i n d s to T A T A e l e m e n t sw i t h b o t h c o n s e n s u s a n d n o n c o n s e n s u s D N A s e qu e n ce s .P roc . N at l . A c ad . Sc i . 8 6 : 5 7 1 8 -5 7 2 2 .

Ho ey , T . , B .D . Dy n lach t , M.G . Pe te r so n , B .F . Pu g h , an d R . T j ian .1 99 0. I s o l a t i o n a n d C h a r a c t e r i z a t i o n o f t h e D r o s o p h i l a g e n ee n c o d i n g t h e T A T A b i n d i n g p r o t e i n , T F I I D . C e l l 6 1 : 1 1 7 9 -1186.

Ho f fman n , A . , M. Ho r ik o sh i , C .K . Wan g , S . Sch ro ed e r , P .A .Wei l , an d R .G. Ro ed e r . 1 9 90 a . C lo n in g o f th e S c h i z o s a c c h a -r o m y ce s p o m b e TFIID g en e rev ea l s a s t ro n g co n se rv a t io n o ff u n c t i o n a l d o m a i n s p r e s e n t i n S a c c h a r o m y c e s c e r e v i s i a eTFIID. G e n e s & D e v . 4 : 1 1 4 1 - 1 1 4 8 .

Ho f fm an n , A . , E . S in n , T . Yam amo to , J . Wan g , A . Ro y , M. Ho r i -k o sh i , an d R .G. Ro ed e r . 1 9 90 b . H ig h ly co n se rv ed co re d o-m a i n a n d u n i q u e N t e r m i n u s w i t h p r e s u m p t i v e r e g u l a t o r ym o t i f s i n a h u m a n T A T A f a c t o r ( T F I I D ) . N a t u r e 3 4 6 : 3 8 7 -390.

Ho r ik o sh i , M . , C .K . Wan g , H . Fu j i i , J .A . Cro m l i sh , P .A . We i l ,an d R .G. Ro ed e r . 1 9 8 9 . C lo n in g an d s t ru c tu re o f a y ea s t g en ee n c o d i n g a g e n e r a l t r a n s c r i p t i o n i n i t i a t i o n f a c t o r T F I ID t h a tb i n d s t o t h e T A T A b o x . N a t u r e 3 4 1 : 2 9 9 -3 0 1 .H o r i k o s h i , M . , T . Y a m a m o t o , Y . O h k u m a , P . A . W e i l , a n d R . G .Ro ed e r . 1 9 90 . An a ly s i s o f s t ru c t u re - fu n c t io n re la t io n sh ip s o fy e a s t T A T A b o x b i n d i n g f a c t o r T F I I D . C e l l 6 1 : 1 1 7 1 - 1 1 7 8 .

Jack so n , S .P . an d R . T j ian . 1 9 89 . Pu r i f i c a t io n an d an a ly s i s o fR N A p o l y m e r a s e I I t r a n s c r i p t i o n f a c t o r s b y u s i n g w h e a tg e r m a g g l u t i n i n a f f i n i t y c h r o m a t o g r a p h y . P r o c . N a t l . A c a d .Sc i . 8 6 : 1 7 8 1 - 1 7 8 5 .

Jo n es , K .A. , K .R . Yamamo to , an d R . T j ian . 1 9 8 5 . Two d i s t in c tt r a n s c r i p t i o n f a c t o r s b i n d t o t h e H S V t h y m i d i n e k i n a s e p r o -m o t e r i n v i t r o . C e l l 4 2 : 5 5 9 -5 7 2 .Kamb ad u r , P , . , V . Cu lo t ta , an d D . Ham er . 1 9 90 . C lo n ed y ea s t

a n d m a m m a l i a n t r a n s c r i p t i o n f a c t o r T F I I D g e n e p r o d u c t ss u p p o r t b a s a l b u t n o t a c t i v a t e d m e t a l l o t h i o n e i n g e n e t r a n -sc r ip t io n . P r o c . N a t l . A c a d . S c i . 8 7 : 9 1 6 8 -9 1 7 2 .

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n o v e l m e d i a t o r b e t w e e n a c t i v a t o r p r o t e i n s a n d t h e R N Ap o l y m e r a s e I I t r a n s c r i p t i o n a p p a r a tu s . C e l l 6 1 : 1 2 0 9 -1 2 1 5 .

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Nak a j ima , N . , M. Ho r ik o sh i , an d R .G. Ro ed e r . 1 9 8 8 . Fac to r si n v o l v e d i n s p e c i fi c t r a n s c r i p t i o n b y m a m m a l i a n R N A p o l y -m e r a s e I I: P u r i f i c a t io n , g e n e t i c s p e c i f i c it y , a n d T A T A b o x -p r o m o t e r i n t e r a c t i o n s o f T F I I D . M ol . C e l l . B i o l . 8 : 4 0 2 8 -4040.

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a n a l y s i s o f G C e l e m e n t b i n d i n g a n d t r a n s c r i p t i o n i n t h eh a m s t e r d i h y d r o f o l a t e r e d u c t a s e g e n e p r o m o t e r . N u c l e i c A c -i ds R e s . 1 7 : 9 2 9 1 -9 3 0 4 .

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G E N E S & D E V E L O P M E N T 1 9 4 5


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