ANALYTICALBIOCHEMISTRY206, 183-188 (1992)

Localization of in Vivo Ribosome Pause Sites Jeong-Kook Kim 1 and Margaret J. Hollingsworth 2 Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York 14260

Received May 7, 1992

A p r o t o c o l f o r t h e l o c a l i z a t i o n o f t h e 5' b o u n d a r i e s o f in v i v o r i b o s o m a l p a u s i n g s i t e s h a s b e e n d e v e l o p e d . These mapping experiments combine two basic techn i q u e s . T h e first is t h e i s o l a t i o n o f p o l y s o m a l t r a n scripts via centrifugation of tissue extracts through a sucrose cushion in the presence of translational elongation inhibitors. The second technique involves a microc o c c a l n u c l e a s e p r o t e c t i o n a s s a y first d e v e l o p e d b y Wol i n a n d W a l t e r for in v i t r o - b o u n d r i b o s o m e s ( E M B O J. 7, 3 5 5 9 - 3 5 6 9 , 1 9 8 8 ) . U s i n g t h i s m e t h o d , t h e 5' b o u n d a r i e s o f in v i v o r i b o s o m a l p a u s e s i t e s w e r e l o c a l i z e d o n spinach chloroplast mRNAs derived from the atpA g e n e . T h i s m e t h o d is e a s i l y a d a p t a b l e to t h e i d e n t i f i c a t i o n o f in v i v o r i b o s o m a l p a u s e s i t e s f r o m a n y o r g a n i s m . It c o u l d a l s o be a d a p t e d to t h e l o c a l i z a t i o n o f in v i v o b i n d i n g s i t e s for o t h e r n u c l e i c a c i d b i n d i n g p r o t e i n s . © 1 9 9 2 Academic Press, Inc.

t h r o u g h t o e p r i n t i n g (10) or n u c l e a s e p r o t e c t i o n a s s a y s (5). M a n y f u n d a m e n t a l c o n c e p t s c o n c e r n i n g t h e f a c t o r s t h a t influence t r a n s l a t i o n a l p a u s i n g h a v e b e e n r e v e a l e d t h r o u g h studies such as these. H o w e v e r , t h e s e experim e n t s h a v e b e e n limited to s y s t e m s in which t h e in vitro t r a n s l a t i o n s y s t e m s are t h o u g h t to faithfully m i m i c t h e in vivo situation. H e r e , we p r e s e n t a m e t h o d t h a t d e m o n s t r a t e s t h e applicability of the nuclease p r o t e c t i o n a s s a y of W o l i n a n d W a l t e r to in vivo s y s t e m s (5). A l t h o u g h t h e specific e x a m p l e given h e r e is identification of ribos o m a l p a u s e s on s p i n a c h c h l o r o p l a s t t r a n s c r i p t s , this m e t h o d is easily a d a p t a b l e to a n y s y s t e m f r o m which p o l y s o m e s c a n be isolated a n d for which a clone of t h e gene of i n t e r e s t is available. F u r t h e r a d a p t a t i o n s of this m e t h o d would allow t h e identification of in vivo t r a n script binding sites of a n y p r o t e i n for which a n a n t i b o d y is available. MATERIALS AND METHODS

T r a n s l a t i o n of a n m R N A is a f u n d a m e n t a l step in the control o f the e x p r e s s i o n of m a n y genes. A v a r i e t y of m e c h a n i s m s are utilized in living s y s t e m s to c o n t r o l the e x p r e s s i o n of a given gene at the t r a n s l a t i o n a l level. F o r e x a m p l e , v a r i a t i o n s in t h e efficiency of t r a n s l a t i o n initiation c a n be influenced by R N A p r i m a r y a n d / o r secondary s t r u c t u r a l factors (1-3). In addition, a cell c a n control t h e e x p r e s s i o n of a p a r t i c u l a r t r a n s c r i p t b y v a r y i n g the s p e e d a n d efficiency of t r a n s l a t i o n elongation. T r a n s l a t i o n a l e l o n g a t i o n can be influenced t h r o u g h a v a r i e t y of factors, including availability of c h a r g e d t R N A s , R N A s e c o n d a r y structure, a n d t r a n s l a t i o n a l p a u s e s due to i n t e r a c t i o n with trans-acting f a c t o r s such as t h e signal recognition particle ( S R P ) (4-8). R e s e a r c h into t r a n s l a t i o n a l p a u s i n g h a s p r i m a r i l y focused on s y s t e m s a m e n a b l e to in vitro t r a n s l a t i o n (5,9). R i b o s o m e binding regions in vitro are identified either t Present address: University of Pennsylvania School of Medicine, Howard Hughes Medical Institute, 328 Clinical Research Building, Philadelphia, PA 19104. 2 To whom correspondence should be addressed. 0003-2697/92 $5.00 Copyright © 1992by AcademicPress, Inc. All rights of reproduction in any form reserved.

Spinach Growth Spinacia oleracea seeds were g e r m i n a t e d in v e r m i c u lite a n d t h e n t r a n s f e r r e d a n d grown h y d r o p o n i c a l l y . E n t i r e p l a n t s with a m a x i m u m leaf length of 5 c m were utilized for t h e s e e x p e r i m e n t s . Isolation and Verification of Polysomes I m m e d i a t e l y u p o n h a r v e s t , s p i n a c h leaves were frozen in liquid n i t r o g e n a n d g r o u n d to a fine p o w d e r with a m o r t a r a n d pestle. L e a f p o w d e r was m i x e d w i t h e x t r a c tion buffer at a ratio of 1 g to 0.5 ml of 200 mM T r i s - H C 1 , p H 8.5, 200 mM KC1, 30 mM MgC12, 10 mM E G T A , 200 mM sucrose, 2.5 mM dithiotreitol ( D T T ) , 3 0.5 m g / m l heparin, 5 tzg/ml p r o t e i n a s e K, a n d 100 # g / m l c h l o r a m phenicol. C h l o r a m p h e n i c o l inhibits e l o n g a t i o n of chlorop l a s t t r a n s l a t i o n , t h e r e b y " f r e e z i n g " t h e c h l o r o p l a s t rib o s o m e s in place. I f c y t o p l a s m i c m R N A s are to be k e p t in a p o l y s o m a l s t a t e as well, cycloheximide should be 3 Abbreviations used: DTT, dithiotrietol; DEPC, diethylpyrocarbonate. 183

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KIM AND HOLLINGSWORTH

a d d e d to a final c o n c e n t r a t i o n of 50 teg/ml. S a m p l e s were held a t r o o m t e m p e r a t u r e until the p o w d e r h a d t h a w e d a n d t h e n h o m o g e n i z e d in a W a r i n g b l e n d e r at t h e h i g h e s t s p e e d for 15 s. All s u b s e q u e n t steps were p e r f o r m e d at 0 - 4 ° C . H o m o g e n a t e s were filtered t h r o u g h two layers of M i r a c l o t h (Calbiochem) a n d cent r i f u g e d at 16,000g for 10 min. S u p e r n a t a n t f r o m the c e n t r i f u g a t i o n was divided into 1.5-ml aliquots. T h e aliq u o t s were either u s e d i m m e d i a t e l y or frozen in liquid n i t r o g e n a n d s t o r e d at - 7 5 ° C . E a c h 1.5-ml aliquot was l a y e r e d o n t o a 3-ml cushion c o n t a i n i n g 1.75 M sucrose, 40 mM T r i s - H C 1 , p H 9.0, 30 mM MgC12, 200 mM KC1, 5 mM E G T A a n d subjected to c e n t r i f u g a t i o n in a B e c k m a n S W 5 5 Ti r o t o r for 18 h at 53,000 r p m . T h e s u p e r n a t a n t was carefully d r a i n e d a f t e r c e n t r i f u g a t i o n a n d t h e pellets were w a s h e d twice with 200 mM T r i s - H C 1 , p H 8.5, 60 mM KC1, 30 mM MgC12, 100 teg/ml c h l o r a m p h e n i c o l . T h e w a s h e d pellet was r e s u s p e n d e d in 50 tel of t h e w a s h solution. At this point, t h e solubilized pellet could be frozen in liquid nit r o g e n a n d s t o r e d at - 7 5 ° C . T h e p o l y s o m e c o n t e n t of a sucrose cushion pellet was c h e c k e d b y layering a p o r t i o n of a solubilized pellet o n t o a 15-60% sucrose gradient. T h e g r a d i e n t was centrifuged in a B e c k m a n SW41 T i r o t o r for 2.5 h at 39,000 r p m . Sucrose g r a d i e n t s were f r a c t i o n a t e d u s i n g an I S C O M o d e l 185 g r a d i e n t f r a c t i o n a t o r c o n n e c t e d to an I S C O f r a c t i o n collector. A b s o r b a n c e of the g r a d i e n t was m o n i t o r e d at 254 nm. O n l y s a m p l e s with p o l y s o m a l profiles were u s e d in s u b s e q u e n t e x p e r i m e n t s (Fig. 1A).

Production of Ribosome-Protected Fragments Determination of appropriate micrococcal nuclease digestion conditions. Several aliquots of the frozen polys o m e s were t h a w e d at r o o m t e m p e r a t u r e . One, to be u s e d as a negative control, was p h e n o l e x t r a c t e d a n d e t h a n o l p r e c i p i t a t e d . C a l c i u m chloride a n d D T T were a d d e d to each of the r e m a i n i n g aliquots to r e a c h final c o n c e n t r a t i o n s o f 3 a n d 2 mM, respectively. Zero to 1000 u n i t s of m i c r o c o c c a l n u c l e a s e ( B o e h r i n g e r - M a n n h e i m ) were a d d e d to t h e aliquots. E a c h reaction was i n c u b a t e d at 26°C for 30 min. At t h e e n d of t h e i n c u b a t i o n , E G T A was a d d e d to r e a c h a final c o n c e n t r a t i o n of 5 mM. E a c h reaction was divided into two p o r t i o n s a n d frozen on dry ice. To determine whether the nuclease had completely digested t h e p o l y s o m a l t r a n s c r i p t s , one of the aliquots f r o m each of t h e r e a c t i o n s was subjected to sucrose gradient analysis as outlined above. I n our hands, concent r a t i o n s of micrococcal n u c l e a s e f r o m 50 to 1000 u n i t s all p r o d u c e d m o n o s o m e s (Fig. 1B). T h e v o l u m e of t h e r e m a i n i n g h a l f of t h e micrococcal r e a c t i o n was raised to 200 #1 for a final c o n c e n t r a t i o n of 20 mM H e p e s , 150 mM p o t a s s i u m acetate, 10 mM m a g n e s i u m a c e t a t e , 5 mM E G T A , 2 mM D T T .

Isolation of ribosome-protected fragments. T h e 200 tel of micrococcal nuclease- or m o c k - t r e a t e d p o l y s o m e s were l a y e r e d on top of a 120-tel sucrose cushion of 0.25 M sucrose in 20 mM H e p e s , 150 mM p o t a s s i u m acetate, 10 mM m a g n e s i u m acetate, 5 mM E G T A , a n d 2 mM D T T . T h e r i b o s o m e s a n d t h e i r b o u n d R N A s were pelleted b y c e n t r i f u g a t i o n for 30 m i n at 70k r p m at 4°C in a T L A 100.3 r o t o r ( B e c k m a n TL100). T h e t o p 240 tel were carefully r e m o v e d . T o the b o t t o m 80 tel, 200 tel of p r o t e i n a s e K solution (50 mM NaC1, 50 mM T r i s - H C 1 , p H 7.5, 5 mM E D T A , 0.5% sodium dodecyl sulfate, 200 teg/ml p r o t e i n a s e K (Sigma)) were added. Digestion of t h e r i b o s o m e s w a s allowed to proceed at 37°C for 30 min. Following p r o t e a s e digestion, t h e s a m p l e s were p h e n o l / c h l o r o f o r m e x t r a c t e d twice a n d e t h a n o l precipitated. E a c h pellet was dissolved in 10 tel of diethylpyroc a r b o n a t e ( D E P C ) - t r e a t e d w a t e r a n d s t o r e d at - 2 0 ° C . An a b s o r b a n c e r e a d i n g at 260 n m was u s e d to estim a t e t h e q u a n t i t y of t h e R N A of i n t e r e s t in the sample. F o r e x a m p l e , we e s t i m a t e d t h a t only 5% of the A26o of this s a m p l e was due to m R N A (the r e m a i n i n g 95% b e i n g r R N A a n d t R N A ) a n d of t h a t , only 1% was related to atpA, o u r gene of interest. T h i s r o u g h e s t i m a t e was u s e d to d e t e r m i n e the q u a n t i t y of s i n g l e - s t r a n d e d D N A c o m p l e m e n t a r y to the gene of i n t e r e s t to be u s e d in the n e x t step of the analysis. W e typically u s e d 10-fold m o r e sing l e - s t r a n d e d D N A t h a n our e s t i m a t e d a m o u n t of RNA. Isolation of Single-Stranded Antisense DNA S i n g l e - s t r a n d e d D N A utilized in this s t u d y was derived f r o m the p l a s m i d p J B 6 . T h i s p l a s m i d c o n t a i n s a f r a g m e n t of s p i n a c h c h l o r o p l a s t D N A t h a t encodes t h e genes, in order, rps2, a t p I , a t p H , a t p F , a n d a t p A (11); for n o m e n c l a t u r e see (12), in v e c t o r p B l u e s c r i p t S K + ( S t r a t a g e n e ) . W h e n Escherichia coli cells t r a n s f o r m e d with p J B 6 are i n f e c t e d with t h e V C S - M 1 3 helper p h a g e ( S t r a t a g e n e ) , p h a g e particles c o n t a i n i n g singles t r a n d e d J B 6 c a n be isolated f r o m t h e media. Singles t r a n d e d J B 6 is c o m p l e m e n t a r y to t h e R N A s encoded b y t h e five genes. S i n g l e - s t r a n d e d D N A s were isolated f r o m p h a g e - c o n t a i n i n g s u p e r n a t a n t b y two p h e n o l e x t r a c t i o n s followed b y c h l o r o f o r m : i s o a m y l alcohol (24:1) e x t r a c t i o n s until the i n t e r f a c e was no longer visible. D N A was precipit a t e d f r o m the a q u e o u s p h a s e in t h e p r e s e n c e of 0.75 M a m m o n i u m a c e t a t e a n d 70% ethanol.

Oligonucleotide Labeling T h e single oligonucleotide u s e d in t h e e x p e r i m e n t s r e p o r t e d here is identical to b a s e s - 1 2 7 t h r o u g h - 1 0 8 (where t h e a d e n o s i n e of the a t p A initiation codon is +1) of t h e large A T P s y n t h a s e gene cluster f r o m t h e s p i n a c h c h l o r o p l a s t g e n o m e (11). Its sequence is C A T T T A C G G A C C A T C A A T G C . T h i s oligonucleotide was s y n t h e -

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LOCALIZATION OF RIBOSOMAL PAUSING I N V I V O

sized on an Applied Biosystems Oligonucleotide Synthesizer Model 391. T h e oligonucleotide was 5'-end-labeled using T4 polynucleotide kinase (NE Biolabs) and [3,-32p]ATP per supplier's protocols.

A254

Mapping the Positions of the Protected Fragments using T4 DNA Polymerase Hybridization. Single-stranded DNA encoding the complement of the RNA of interest was hybridized to the protected fragments and to a 5'-end-labeled oligonucleotide t h a t would hybridize upstream of the area of interest. T h e concentrations of the DNA and the oligonucleotide were 10- and 20-fold, respectively, higher than the estimated concentration of the ribosome-protected RNA fragments. T h e mixture also included 1 #1 of 10× hybridization buffer (330 mM Tris-acetate, pH 7.7, 670 mM potassium acetate), with D E P C - w a t e r added for a total of 10 #l. T he reaction was incubated at 65°C for 5 min and t hen slowly cooled to 30°C over 30 min. Extension of hybridized primers. Eleven microliters of 33 mM Tris-acetate, pH 7.7, 67 mM potassium acetate, 20 mM magnesium acetate, 1 mM D T T , 1 mM ATP, 334 pM each of dATP, d T T P , dGTP, and dCTP, and 3 units of T4 DNA polymerase (New England Biolabs) were carefully mixed into the hybridization. T h e polymerization was allowed to proceed at 37°C for 30 min. T h e extension reaction was phenol/chloroform extracted twice and ethanol precipitated. T h e dried pellet was dissolved in 6 #l of 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA. F o u r microliters of gel-loading buffer were added to that. Gel-loading buffer consisted of 0.1% xylene cyanol, 0.1% bromophenol blue, 10 mM EDTA, 95% formamide. Analysis of extension products. Samples were incubated at 80°C for 2 min and loaded on an 8% polyacrylamide-7 M urea gel in a lane adjacent to a sequencing ladder derived from the same primer and singlestranded DNA. Sequencing ladders were generated using the Sequenase 2.0 kit (U.S. Biochemicals). Frequently, sequencing was performed with [asS]dATP, to adjust the intensity of the sequencing bands down to that of the extended radiolabeled primers. After electrophoresis, gels were dried and subjected to autoradiography. RESULTS

Isolation of Polysomes and Ribosome-Protected Fragments from Spinach A typical polysome profile of a solubilized aliquot of a spinach sucrose cushion pellet is outlined in Fig. 1A. Polysomes are clearly separated from ribosomal subunits and monosomes in this system. T h e profile in Fig. 1B results from micrococcal nuclease t r e a t m e n t of the

A Top B

~

~

Bottom po[ysomes

~j~

Bottom

A254

plolysomes FIG. 1. Sucrose gradient profile following isolation of spinach polysomes before (A) or after (B) micrococcal nuclease digestion. Y-axis is the absorbance of the sucrose gradient fractions at 254 nm. X-axis represents distance into the gradient. The gradient was collected from top (left) to bottom {right). Labels under the X-axis designate the boundary between polysomes and smaller species (monosomes and subunits) in the gradient.

sucrose cushion pellet prior to polysome analysis. As can be seen in Fig. 1B, micrococcal nuclease t r e a t m e n t resulted in the elimination of polysomal species. All 254-nm absorption peaks shifted to a section of the gradient corresponding to species with a velocity less th a n or equal to t hat of monosomes.

Localization of Ribosome Pause Sites in the Spinach Chloroplast atpA mRNA Monosomes resulting from micrococcal nuclease digestion of RNA/protein complexes isolated from spinach sucrose cushion pellets were examined to determine whether there were any ribosomal pause sites in the 5' region of the spinach chloroplast atpA mRNA. P r i m e r extension reactions were also performed using RNA purified from mock micrococcal nuclease-treated ribosomes. These mock-treated samples were the equivalent of total extracted spinach RNA. Therefore, this control could also be used to localize the 5' ends of chloroplast RNAs. Results of such an analysis are shown in Fig. 2. An additional control in which the RNAs isolated from sucrose cushion pellets were phenol extracted prior to micrococcal nuclease t r e a t m e n t was performed. This negative control (data not shown) looked identical to controls where extension was performed in the absence of RNA (Fig. 2). A total of 420 bases were inspected for ribosomal pausing. T h e areas investigated included the entire 68-

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KIM AND HOLLINGSWORTH Mock- NucleaseTreated Treated RNA RNA

Mock- NucleaseTreated Treated RNA RNA G A TC

G A TC

- .1 .3 1.0.1 .31.0

-.1

.31.0.1 .31.0

• 345 • 299 • 290 • 249 • 228

"-4

"-40

Irrrilrirlii 1 23

4 567

8910

11

1 2 3 4 567

891011

FIG. 2. Primer extension of an oligonucleotide identical to bases -127 through -108 upstream of the spinach chloroplast atpA gene. Lanes 1-4, dideoxy sequencing ladder using the same oligonucleotide and single-stranded DNA as in lanes 5-11. Dideoxy GTP, ddATP, ddTTP, or ddCTP were used in lanes 1, 2, 3, and 4, respectively. Lane 5, extension reaction in the absence of RNA, lanes 6-8, extension reaction with T4 DNA polymerase in the presence of 0.1, 0.3, or 1.0 ttg, respectively, of mock micrococcal nuclease-treated, phenol-extracted spinach RNA; lanes 9-11, extension reaction with T4 DNA polymerase in the presence of 0.1, 0.3, or 1.0 #g, respectively of RNA fragments derived from ribosome-bound, micrococcal nuclease-treated spinach RNA. The lanes on the left are generated from exactly the same samples as those on the right, but have been subjected to electrophoresis for a longer period of time. The 5' boundaries of pauses caused by protection of a fragment of atpA mRNA are noted to the right of each set of lanes. The numbering system to the right of the lanes designates the approximate base at which each pause occurs, where +1 is the AUG translation initiation site.

b a s e 5' n o n c o d i n g r e g i o n u p s t r e a m o f a t p A a n d t h e first 350 b a s e s a t t h e 5' e n d o f t h e a t p A o p e n r e a d i n g f r a m e . A t o t a ! o f t h r e e p a u s e s i t e s w e r e d e t e c t e d a t p o s i t i o n s 228, 249, a n d 345, w h e r e t h e first b a s e o f t h e o p e n r e a d i n g f r a m e is + 1 . T h e r e l a t i v e q u a n t i t i e s o f e a c h o f t h e s e b a n d s were d e t e r m i n e d by d e n s i t o m e t r y u s i n g a Shim a d z u C S 9 0 0 0 U l a s e r s c a n n e r . T h e p a u s e site a t b a s e 228 w a s f o u n d t o b e b y f a r t h e m a j o r one, a p p r o x i m a t e l y 5-fold m o r e a b u n d a n t t h a n the n e x t m o s t a b u n d a n t at b a s e 345. T h e p a u s e a t p o s i t i o n 249 w a s t h e l e a s t a b u n d a n t , 5 - f o l d less t h a n t h e p a u s e a t 345. T h e r e l a t i v e ra-

t i o s o f t h e r i b o s o m e p a u s e s a t 228, 249, a n d 345, r e s p e c t i v e l y , a r e a p p r o x i m a t e l y 25:1:5. T h e s e e x p e r i m e n t s also r e v e a l e d f o u r 5' e n d s i n t h e s e 420 b a s e s of m R N A , a t l o c a t i o n s - 4 0 , - 4 , 290, a n d 299. T h e a t p A g e n e is c o t r a n s c r i b e d w i t h t h r e e u p s t r e a m genes. T h e a p p r o x i m a t e l y 6.3-kb t r a n s c r i p t undergoes extensive R N A cleavage a n d splicing to yield approxim a t e l y 30 s m a l l e r m R N A s ( S t o l l a r a n d H o l l i n g s w o r t h , s u b m i t t e d for p u b l i c a t i o n ) . T h e r e f o r e , t h e 5' e n d s detected in these e x p e r i m e n t s are m o s t likely the result of R N A c l e a v a g e . All f o u r o f t h e 5' e n d s a r e also d e t e c t e d i n

LOCALIZATION OF RIBOSOMAL PAUSING IN VIVO the ribosome-protected lanes. This implies that these 5' ends are bound by protein in vivo. DISCUSSION Several interesting features of the expression of this particular gene were discovered in the process of examining its ribosome pause sites. First, no ribosome pauses were detected at the putative ribosome binding site for this open reading frame. Chloroplast ribosome binding sites have been hypothesized to be similar to the S h i n e Dalgarno site in prokaryotes (13,14). T h e locations of these sites have been predicted by inspection of sequences 5' to open reading frames for complementarity to the 3' end of the chloroplast 16S rRNA (14). When ribosomal pause sites are inspected after ribosome binding in vitro, pauses are commonly detected at translation initiation sites (5,9). It is unknown whether ribosomes pause at those sites in vivo. We did not detect chloroplast ribosomes pausing in vivo at the putative ribosome binding sequence for atpA. We do not know, however, if this is because the chloroplast ribosome does not specifically pause at ribosome binding sites or whether the ribosome binding site for this particular gene is not the one previously hypothesized. T h e best-fit ribosome binding site for atpA is an AGG located 47 bases from the AUG initiation codon (14). Since this does not fit the consensus of GGAGG spaced 7 to 12 bases upstream of the initiation codon especially well, it is possible th at the suggested ribosome binding site for atpA is incorrect. Another intriguing feature of the expression of this cluster revealed by these experiments is t hat all the 5' ends of the RNAs detected here are protected from micrococcal nuclease digestion when isolated as prot ei n/ RNA complexes. T h e question then becomes whether the 5' ends are protected by ribosomes or other RNA binding proteins. In Chlamydomonas, a group of proteins t h a t interact with the 5' ends of the psbA mRNA and regulate its expression in response to light has been discovered (15). Similar types of interaction s may be the cause of the protection of atpA 5' ends t hat we observe. T h e sucrose cushion isolation procedure used after micrococcal nuclease digestion is such t hat only ribonucleoprotein complexes, such as ribosomes, should pellet through the cushions. However, it is possible t h a t extremely dense protein complexes surrounding a single protected RNA fragment might also pellet through the cushion. In most systems, a typical control to determine whether ribosomes are the protecting species would be to perform an identical set of experiments on puromycin-treated polysomal RNAs. Puromycin is an aminoacyl-tRNA analog t h a t promotes the specific dissociation of ribosomes from mRNAs. However, none of the puromycin t r e a t m e n t protocols that we have tried have been successful in dissociating chloroplast ribosomes

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(N. Stollar and J. K. Kim, unpublished). Other types of ribosome dissociation methods, such as incubation in EDTA, are not specific for the dissociation of ribosomes and would be expected to dissociate other RNA binding proteins as well. Experiments to determine whether the protection of the atpA mRNA 5' ends t h a t we observed is due to ribosomes or other proteinaceous species are currently in progress. T h e primary and secondary structure at and around the identified ribosome pause sites was inspected by computer-based comparative analysis. T h e r e was no significant primary structure similarity regardless of whether the sequences 5' to the ribosome, the 30-35 bases thought to be covered by the ribosome, or sequences 3' of the ribosome were compared. Secondary structures downstream of each of the ribosome binding sites were predicted by comparative analysis. T h e r e are several other regions of this mRNA t h a t might also be expected to produce stem-loop structures of stability similar to t h a t of those downstream of the ribosome pausing sites. However, no ribosome pauses were detected in conjunction with these putative structures. We cannot convincingly suggest any consensus primary or secondary structure associated with these pausing sites. If there were multiple ribosomes paused in a particular region, there could be a primary or secondary structure downstream of the final ribosome t h a t triggered the pausing of several ribosomes. Since this method detects only the 5' edge of a ribosome-protected fragment, downstream ribosomes and structures t hat might contribute to their pausing would not be recognized. Kim and colleagues have recently observed ribosomal pausing along the barley chloroplast psbA transcript, which codes for the photosystem II protein D1 (4). As with atpA, no structures were observed to be in common at all the pausing sites. T h e y propose t h a t these pauses may be the result of insertion of the nascent polypeptide into the thylakoid membrane. Although atpA is not a m e m b r a n e - b o u n d protein, it is incorporated into the A T P synthase, which is a multisubunit enzyme complex. Perhaps the process of folding and association with the rest of the A T P synthase subunits is related to the ribosomal pausing t hat we observe here. F u r t h e r experiments are in progress to test whether there is a correlation between complex assembly and ribosomal pausing. We have dem onst rat ed here a m et hod t h a t provides a straightforward set of techniques to detect the 5' boundaries of in vivo ribosomal pause sites. This method could also be adapted to the investigation of binding sites for any protein for which an antibody is available. Such a modification would entail precipitation of the p r o t e i n / RNA complex after digestion by micrococcal nuclease with antibodies specific for the protein in question. Extraction of the RNA, hybridization, and primer extension would be performed as for the ribosomal protection

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experiments above. This versatile procedure may prove to be v a l u a b l e in the study of R N A / p r o t e i n c o m p l e x e s in vivo, w h e r e f o o t p r i n t i n g e x p e r i m e n t s c a n o f t e n b e p r o b lematic. ACKNOWLEDGMENTS The authors thank Drs. Jim Berry, Paul Gollnick, and Jerry Koudelka for critical reading of the manuscript and many helpful comments. We are grateful to Neil Stollar for generation of the densitometry data. This work was supported by National Foundation Grant MCB 91-05726.

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