C r y s t a l S t r u c t u r e o f a B a c t e r i a l R N A P o l y m e r a s e H o l o e n z y m e a t 2 . 6 Å R e s o l u t i o n C r y s t a l S t r u c t u r e o f a B a c t e r i a l R N A P o l y m e r a s e H o l o e n z y m e a t 2 . 6 Å R e s o l u t i o n Fig. 1. Holoenzyme crystal structure. The subunits colors are: β , sage; β ’, white ( β ’163-452, cyan; β ’ Z n - f i n g e r , g r e e n ) ; α I , b l u e ; α I I , l i g h t o r a n g e ; σ , m a g e n t a ; a n d ω , r e d . T w o c a t a l y t i c M g 2+ ( r e d ) and two Zn 2+ ions (blue) are shown as spheres. Fig. 1 The DNA-dependent RNA polymerase ( RNAP ) is the key enzyme of the transcription process, and is a fi na l ta rg et in ma ny re gu la to ry pa th wa ys th at c o n t r o l g e n e e x p r e s s i o n i n a l l l i v i n g o r g a n i s m s . B a c t e r i a l R N A P e x i s t s i n t w o f o r m s : c o r e a n d h o l o e n z y m e . T h e c o r e e n z y m e ( ~ 4 0 0 k D a ) consists of five subunits : α -dimer ( α 2 ), β, β ’ , and ω . T h e t r a n s c r i p t i o n c y c l e i n b a c t e r i a l c e l l s c a n b e d i v i d e d in to th re e ma jo r st ag es : in it ia ti on , el on ga ti on , an d te rm in at io n. Al th ou gh it is ca ta ly ti ca ll y ac ti ve , th e core enzyme is incapable of initiating transcription efficiently and with specificity. For this, it must bind a n i n i t i a t i o n f a c t o r , σ , t o f o r m a h o l o e n z y m e t h a t can recognize specific DNA sequences (promoters) [1,2]. During initiation, the holoenzyme specifically bind s to two cons erve d hexa mers in the prom oter at nucleotide ( nt ) positions –35 and –10 relative to t h e t r a n s c r i p t i o n s t a r t s i t e ( + 1 ) , t o f o r m a c l o s e d p r o m o t e r c o m p l e x . T h e n , i t u n w i n d s t h e d o u b l e - str and ed DNA aro und the –10 reg ion (be twe en nt – 1 2 a n d + 2 ) , r e s u l t i n g i n t h e o p e n p r o m o t e r complex, and initiates transcription in the presence of nucleoside triphosphate substrates [3]. After the synthesis of a 9 - 12 nt-long RNA, of which 8 - 9 are b a s e - p a i r e d w i t h t h e D N A t e m p l a t e s t r a n d ( R N A - DNA hybrid), the transcription complex passes from the initiation to elongation stage [4]. This transition is ch ar ac te ri ze d by th e es ca pe of th e RN AP fr om th e pr om ot er , th e di ss oc ia ti on of σ fr om th e co re , a n d t h e f o r m a t i o n o f a h i g h l y p r o c e s s i v e t e r n a r y elongation complex. The σ -factor plays a key role i n i n i t i a t i o n , b e i n g d i r e c t l y i n v o l v e d i n p r o m o t e r r e c o g n i t i o n , D N A m e l t i n g , a n d p r o m o t e r e s c a p e and clearance [2]. The family of related σ proteins s h a r e s f o u r r e g i o n s o f s e q u e n c e h o m o l o g y , d e s i g n a t e d 1 t o 4 , w h i c h a r e f u r t h e r d i v i d e d i n t o several subregions [5]. The regions 4.2, 2.3 - 2.4, and 2.5 were shown to recognize the –35, –10, and t h e s o - c a l l e d “ e x t e n d e d – 1 0 ” e l e m e n t s o f t h e promoter, respectively. We have determined the crystal structure of the T. thermophilus RNAP holoenzyme, containing the major σ -factor ( σ 70 ), at 2.6-Å resolution ( ) at b e a m l i n e B L 4 5 X U . T h e σ 7 0 s u b u n i t i s l o c a t e d a l m o s t e n t i r e l y o n t h e c o r e s u r f a c e , e x c e p t f o r a short segment ( σ σ 313 - 342), which is buried within the core molecule. The modeled structure of 70 14 Fig. 2 Fig. 2. σ domain organization and structure. (a) Scheme of structural dom ain s and con ser ved reg ion s. (b) Rib bon dia gra m of σ . Th e col or coding is the same as in (a) except for the non-conserved regions (grey). c o n s i s t s e n t i r e l y o f α - h e l i c e s c o n n e c t e d b y e i t h e r t u r n s o r l o o p s , a n d i t c a n b e d i v i d e d i n t o f o u r str uct ura l dom ain s: N-t erm ina l dom ain 1 ( ND1 ), N- ter min al dom ain 2 ( ND2 ), lin ker dom ain ( LD ), and C-terminal domain ( CD ) ( ). N D 1 c o n s i s t s o f e i g h t α - h e l i c e s ( σ 7 4 - 2 5 4 ) com pri sin g fou r hel ix- tur n-h eli x mot ifs ( HtH ). Thi s d o m a i n e n c o m p a s s e s r e g i o n 1 . 2 u p t o t h e N - t e r m i n a l h a l f o f r e g i o n 2 . 4 , i n c l u d i n g t h e n o n - conserved segment between regions 1 and 2. ND1 has a U-shaped structure, and is connected to ND2 ( σ 2 6 1 - 3 1 2 ) b y a s h o r t l i n k e r l o o p ( σ 2 5 5 - 2 6 0 ) , T h a t l i e s a t t h e C - t e r m i n u s o f r e g i o n 2 . 4 . N D 2 , corr espo ndin g to cons erve d regi ons 2.4 - 2.5 and 3.1, consists of three α -helices that fold into an α - h e l i c a l b u n d l e . T h e C - t e r m i n a l h e l i x o f N D 1 a n d the N-terminal helix of ND2 ( σ 234 - 281) form a V- shaped structure near the opening of the upstream DNA binding channel, which is likely to be a binding s i t e f o r t h e – 1 0 e l e m e n t o f p r o m o t e r . T h e 3 0 r e s i d u e - l o n g “ l i n k e r ” d o m a i n , L D ( σ 3 1 3 - 3 3 9 ) , intervenes between the globular N- and C-terminal portions of σ and has a mostly extended, unfolded conformation. Roughly at its midpoint, LD forms a h a i r p i n l o o p ( σ 3 1 8 - 3 2 9 ) t h a t p r o t r u d e s i n t o t h e active site cleft. The C-terminal domain, CD ( σ 340 15 Fig. 3 Fi g. 3. Mo de ll in g of th e ho lo en zy me /c lo se d pr om ot er co mp le x. Th e pr ot ei n colour coding is the same as in Fig. 1( c) , except all of β ’ is shown in white. The d s D N A ( g r e e n ) c o n t a i n s t h e – 3 5 ( c y a n ) , – 1 0 ( r e d ) , e x t e n d e d – 1 0 ( o r a n g e ) promoter regions. The Mg 2+ (red) and Zn 2+ (blue) ions are shown as spheres. References [1] R.R. Burgess et al. , Nature 221 (1969) 43. [2] C. Gross et al. , Transcriptional Regulation ( eds S . R . M c K n i g h t & K . R . Y a m a m o t o ) - C o l d S p r i n g H a r b o r L a b o r a t o r y P r e s s , C o l d S p r i n g H a r b o r (1992) 129. [ 3 ] M . T . J . R e c o r d e t a l . , E s c h e r i c h i a c o l i a n d S a l m o n e l l a ( e d F . C . N e i d h a r t ) - A S M P r e s s , Washington, D.C. (1996) 792. [4] P. von Hippel, Science 281 (1998) 660. [5] M. Lonetto et al. , J. Bacteriol. 174 (1992) 3843. - 42 3) , wh ic h in cl ud es co ns er ve d re gi on s 4. 1 an d 4.2, contains four α -helices, which are arranged as a pair of HtH motifs. This site is about 57 Å from the N-terminal half of the protein, containing regions 2 . 3 - 2 . 5 , t h a t a l l o w s u n a m b i g u o u s m o d e l i n g o f pr om ot er bound to the holoenzyme ( ). In the holoenzyme, certain σ structural elements gr ea tl y re du ce th e av ai la bl e sp ac e in th e fu nc ti on al ly Dmitry G. Vassylyev a , Sergei Borukhov b and Shigeyuki Yokoyama a,c,d (a) SPring-8 / RIKEN (b) SUNY Health Science Center, USA (c) RIKEN Genomic Sciences Center (d) The University of Tokyo E-mail: dmitry @ yumiyoshi.harima.riken.go.jp i m p o r t a n t p r o t e i n c a v i t i e s a n d c h a n n e l s t h a t a c c o m m o d a t e p r o m o t e r D N A , R N A - D N A h y b r i d , and RNA product. Thus, the holoenzyme structure a d d i t i o n a l l y r e s t r a i n s p o s s i b l e o r i e n t a t i o n s a n d c o n f o r m a t i o n s o f t h e n u c l e i c a c i d s i n t h e t r a n s c r i p t i o n i n t e r m e d i a t e s , a l l o w i n g b e t t e r u n d e r s t a n d i n g o f v a r i o u s s t e p s o f t r a n s c r i p t i o n initiation. 16 σ region 2.4 σ region 2.5 β ' Zn-finger 53-81 σ region 4.2
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