J. Mol. Biol. (1976) 1{}8, 771-779
LETTERS TO THE EDITOR
Transcription of Bacteriophage r in Vitro: Analysis with Restriction Enzymes The three phage r promotors (P,., PA and Po) defined and mapped relative to one another by Axelrod (1976) have been mapped onto the bacteriophage genome. RNA chains were specifically initiated in vitro at each promotor with oligonucleotide primers, pulse-labeled with (a-aaP)-labeled ribonueleoside triphosphates, chased into frill-length molecules with an excess of unlabeled ribonucleoside triphosphates, and purified on polyacrylamide gels. The isolated RNA species were hybridized to a series of purified, tmlabeled DNA fragments generated by cleavage of r RFI~ DNA with restriction endonucleases from Hemophilus influenzae (endonuclease R) or H. aegyptius (endonuclease Z). The a2P-labeled RNA sequences (approx. 100 to 400 bases) originating from promotors A', A or G hybridized preferentially to DNA fragments R-4, R-8 and R-6b/Z-3, respectively. The DNA fragments have previously been aligned with the bacteriophage genetic map by independent methods in other laboratories (Chert et al., 1973 ; Hutchison et al., 1973 ; Lee & Sinsheimer, 1974a,b ; Borrias et al., 1976). Comparison of the above hybridization results to that fragment map shows that promotors A', A and G lie near the starts of cistrons A, B and D. I n the preceding paper (Axelrod, 1976), the R N A species generated by transcription in vitro of bacteriophage r R F I t D N A were m a p p e d relative to one another by initiation at specific promotors with oligonucleotide primers and analysis on polyacrylamide gels; three promotors (PA', PA and Po), two rho-independent termination sites (T2 and T4), and two rho-dependent termination sites (T1 and Ta) were defined. I n this letter, those promotor and termination sites are aligned with respect to the genetic map. Use is made of the defined set of D N A fragments generated b y cleavage of CX R F I D N A with restriction enzymes from Hemophilus influenzae (endonuclease R) or H. aegyptius (endonuclease Z); these fragments have been m a p p e d relative to one another and to the genetic m a p in other laboratories (Chen et al., 1973; Hutchison et al., 1973; Lee & Sinsheimer, 1974a,b; Borrias et al., 1976). To determine the m a p location of each promotor, R N A chains were initiated specifically at t h a t promotor with an appropriate oligonucleotide primer, pulselabeled over a short distance with a2P-labeled ribonucleoside triphosphates, chased into full-length molecules with an excess of unlabeled R N A precursors, and purified b y electrophoresis on a polyacrylamide gel. The double precaution of initiating synthesis with a specific oligonucleotide primer and purifying the R N A on a gel ensured t h a t each preparation of a2P-labeled R N A represented synthesis from a single, well-defined promotor. The R N A was then hybridized to each of the D N A fragments generated b y cleavage of CX R F I D N A with endonuclease R or, in some cases, endonuelease Z. I t was expected t h a t the a2p-labeled portion of each pulselabeled R N A would hybridize only to t h a t region of the D N A from which it was t r a n s c r i b e d - - t h a t is, to one, or at most two, of the D N A fragments---enabling one tAbbreviat~on used: RFI, the double-stranded closed circular supereoiled replicative form DNA of r 771
772
N. AXELI~OD
to locate the origin of transcriptioh. As an internal control, RNA uniformly labeled with 3H-labeled ribonucleoside triphosphates and containing all the r RNA sequences in approximately equal molar amounts was included in each hybridization reaction. This RNA hybridized to all of the DNA fragments, to each according to its molecular weight, and served as a standard against which the 32p hybridization could be compared. Although all CX RNA sequences may not have been present in equimolar amounts in the [3H]RNA preparation, the use of the same RNA preparation in all experiments ensured that the ratios of 32p to 3H cts/min hybridized were meaningful. As expected, the 32p pulse-labeled portion of each RNA species did hybridize preferentially to a limited number of DNA fragments, results which will be discussed below. However, the 32P-labeled RNA also hybridized to some extent to nearly all of the DNA fragments, in each case in proportion to the size of the fragment. Experiments conducted to determine the nature of this "general hybridization" background have revealed that (1) the "general hybridization" background is in fact the result of annealing and is not the result of non-specific trapping of labeled RNA onto the nitrocellulose filters, for when an equivalent amount of DNA or in vitro synthesized RNA from bacteriophage h was substituted for the corresponding r material in a hybridization reaction, non-specific trapping of heterologous nucleic acids was not observed. (2) The "general hybridization" does not result from radioactive precursors incorporated at a low level into the portion of the RNA synthesized during the chase, for when ternary complexes of DNA, RNA polymerase, and radioactively labeled nascent RNA were purified away from unincorporated (~-32P)-labeled ribonucleoside triphosphates before the chase period begun to ensure that no further radioactive label was incorporated into RNA, the labeled RNA again hybridized to some extent to nearly all of the DNA fragments. (3) The "general hybridization" is not a result of incomplete ribonuclease digestion, for it is not reduced by more extensive ribonuclease treatment. Nor does it represent an intrinsic property of the RNA itself, for when RNA is hybridized in a mock reaction with no DNA, or when RNA is added after the DNA has been subjected to such a mock hybridization, no "general hybridization" can be observed. Preliminary experiments performed to determine the nature of this "general hybridization" suggest that it reflects a short RNA sequence found within the first 400 bases or so of each promotor and repeated apparently at random m a n y times in the r DNA. In these experiments, pulse-labeled RNA from promotor G which had hybridized to several different DNA fragments as "general hybridization" was eluted from those DNA fragments, sized by electrophoresis on polyacrylamide gels, digested by pancreatic or T~ ribonuclease, and subjected to "fingerprint" analysis. By all of these criteria, the I~NAs eluted from the different DNA fragments appeared to be quite similar to one another. These studies will be discussed in more detail in a forthcoming publication. For the moment, I emphasize that the important variable to consider in evaluating the following experiments is not the total number of 32p cts/min hybridized to a given DNA fragment, but rather, the ratio of 32p to aH ets/min hybridized, which in effect compensates for the "general hybridization". Promotor A ' and promotor A
32p pulse-labeled RNAs originating at either promotor A' or at promotor A were prepared as described in the legend to Figure 1 and were hybridized, along with uniformly labeled [3H]RNA, to each of the DNA fragments generated b y cleavage
L E T T E R S TO T H E E D I T O R
773
of CX R F I DNA with endonuelease R. Figure 1 (a) shows that pulse-labeled RNA originating at promotor A' hybridized preferentially to DNA fragment R-4. T h a t is, the ratio of 32p to 3H cts/min hybridized to R-4 was significantly greater than for any other DNA fragment. This suggests t h a t promotor A' lies within the region of DNA defined by fragment R-4. Because cistron A is the only eistron whose N-terminus resides in fragment R-4 (the fragment map is presented in Fig 3), it seems likely that promotor A' is located at the start of t h a t cistron. Figure l(b) shows that when the pulse-labeled RNA was initiated at promotor A, the ratio of 32P/3H cts/min hybridized to fragment R-8 was 10 to 100-fold greater than the ratio of counts hybridized to any other fragment. Thus, RNA synthesis from promotor A begins in the region defined by fragment R-8. Consideration of this result in light of the fragment map shown in Figure 3 indicates that RNA synthesis initiated at promotor A probably begins at the distal end of eistron A or at the start of cistron B.
Promotor G 32p pulse-labeled RNA originating at promotor G was prepared as described in the legend to Figure 2 and was hybridized as before to each of the DNA fragments generated by cleavage of CX R F I DNA with endonuclease R. Figure 2(a) shows t h a t the 3~P-labeled RNA hybridized preferentially to fragment R-6. :Fragment R-6 actually consists of three fragments--R-6a, R-6b and R-6r cannot be separated physically but which are located at three different locations on the genome (see Fig. 3); and it is not possible to conclude from these data which of the three fragments has hybridized to the labeled RNA. In a further effort to localize promotor G, 32p pulse-labeled RNA originating at this promotor was prepared as described above and was hybridized to each of the DNA fragments generated by cleavage of CX R F I DNA with another restriction enzyme, endonuclease Z. Because each of the R-6 fragments overlaps a different Z fragment (see fragment map in :Fig. 3), it should be possible to determine in this manner which of the three R-6 fragments contains the sequences of promotor G. Recall that the method of preparing the RNA for these experiments ensures t h a t all of the G-specific RNA is initiated at one site on the genome, so one should see enhanced hybridization to only one of the Z fragments (Z-3/R-6b, Z-2/R-6e or Z-8 or Z-4/R-6a). The results are shown in Figure 2(b). The labeled RNA hybridized preferentially to fragment Z-3 (which overlaps R-6b), but also to a somewhat lesser extent to fragment Z-2 (which overlaps R-6r This experiment is difficult to interpret. Apparently, the RNA was initiated within the region corresponding to either Z-3 or Z-2, but the two DNA fragments were either cross-contaminated or contain regions of homology so that the 32p pulse-labeled RNA hybridized to both Z fragments. To resolve this question further, a new approach was taken in which the DNA fragment adjacent to the fragment containing the promotor was identified. RNA was initiated from promotor G as before but was pulse-labeled for two and one-haft times as long as in previous experiments, so that 82p was incorporated into a sequence of RNA of approximately 400 bases rather than the 150 or so bases labeled previously. The RNA was chased into full-length molecules, purified by electrophoresis on gels, and hybridized to the collection of Z fragments. I f the G promotor were located in the sequence defined by the 100 base-pair overlap of R-6b and Z-3, then some of the 32P-labeled RNA would be expected to hybridize to Z-3 (approx. 100 bases or less
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FIG. 1. H y b r i d i z a t i o n of pulse-labeled R N A o r i g i n a t i n g a t p r o m o t e r s A ' or A to D N A f r a g m e n t s g e n e r a t e d b y cleavage of CX R F I D N A w i t h e n d o n u c l e a s e R . (a) R N A c h a i n s were i n i t i a t e d in vitro a t p r o m o t e r A ' w i t h t h e "artificial o l i g o n u e l e o t i d e " A p A p A ( A p A + A T P ) ; p u l s e - l a b e l e d for 5 m i n w i t h (a-32P)-labeled C T P a n d U T P , d u r i n g w h i c h t i m e a p p r o x . 200 to 400 b a s e s were i n c o r p o r a t e d into R N A ; c h a s e d w i t h a n e x c e s s of u n l a b e l e d ribonucleoside t r i p h o s p h a t e s ; a n d s u b j e c t e d to p o l y a c r y l a m i d e gel electrophoresis. T h e m a j o r R N A species o r i g i n a t i n g f r o m prom o t o r A ' (the 1.87• l0 s mo]. w t R N A c h a r a c t e r i z e d b y A x e l r o d , 1976) w a s e l u t e d f r o m t h e gel a n d h y b r i d i z e d , in t h e p r e s e n c e of u n i f o r m l y labeled [ 3 H ] R N A c o n t a i n i n g all r sequences in a p p r o x i m a t e l y e q u a l a m o u n t s , to e a c h of t h e D N A f r a g m e n t s g e n e r a t e d b y c l e a v a g e of C X R F I D N A w i t h e n d o n u c l e a s e R. (b) R N A ws.s p r e p a r e d a n d h y b r i z e d as a b o v e , e x c e p t t h e R N A c h a i n s were i n i t i a t e d a t p r o m o t e r A w i t h t h e "artificial o l i g o n u c l e o t i d e " C p A p U p C (CpA + U T P + C T P ) , a n d t h e m a j o r species p r o d u c e d a n d e l u t e d f r o m t h e gel w a s t h e 1.60 • l0 s reel. w t R N A c h a r a c t e r i z e d b y A x e h ' o d (1976). E a c h s a m p l e w a s c o u n t e d in a B e c k m a n s c i n t i l l a t i o n c o u n t e r for 20 rain, to a n a c c u r a c y o f a t least 7~o. 3up pulse-labeled R N A w a s s y n t h e s i z e d e s s e n t i a l l y as described b y A x e l r o d (1976), e x c e p t t h a t reaction m i x t u r e s c o n t a i n e d 10 t i m e s as m u c h D N A a n d R N A p o l y m e r a s e as before, a n d t h e specific a c t i v i t y of t h e (~ 32p).labeled r i b o n u c l e o s i d e s w a s 10 to 20 t i m e s as h i g h as before. T h e R N A w a s purified b y e l e c t r o p h o r e s i s on p r e p a r a t i v e p o l y a c r y l a m i d e gels as in A x e l r o d (1976) followed b y e l u t i o n into 2 • SSC (SSC is 0-15 M-NaCI, 0.015 M-sodium c i t r a t e p H 7.2) -F 0 . 2 % s o d i u m d o d e c y l sulfate. 3H-labeled R N A w a s p r e p a r e d as a b o v e , e x c e p t t h a t t h e R N A w a s labeled c o n t i n u o u s l y w i t h [ a H ] C T P a n d t h e R N A w a s n o t gel-purified. r RFI DNA was cleaved with endonuclease R a n d s u b j e c t e d to e l e c t r o p h o r e s i s on 3.5~ to 7.5~ p o l y a c r y l a m i d e g r a d i e n t gels. T h e D N A f r a g m e n t s were v i s u a l i z e d b y s t a i n i n g w i t h m e t h y l e n e blue, c u t o u t w i t h a r a z o r blade, a n d r e m o v e d f r o m t h e gel slice b y e l e c t r o p h o r e s i s into 40 m M - T r i s - a c e t a t e , 20 m ~ - s o d i u m a c e t a t e , 2 m M - E D T A ( p H 7.9}. r R F I D N A cleaved w i t h e n d o n u c l e a s e Z w a s k i n d l y p r o v i d e d b y C. H u t c h i s o n a n d M. Edgell a n d w a s purified as a b o v e . H y b r i d i z a t i o n s were p e r f o r m e d as follows : for e a c h h y b r i d i z a t i o n , a p p r o x . 0.2 p m o l o f a D N A f r a g m e n t in 60 td 2 x SSC was d e n a t u r e d b y boiling for 2 rain a n d chilled r a p i d l y in solid CO2/acetone. To t h e frozen D N A were a d d e d 40 ~l of 2 • SSC c o n t a i n i n g 1"4 • 10 -3 p m o l of u n i f o r m l y labeled [ 3 H ] R N A a n d 2 • 10 -4 to 6 • 10 -4 p m o l o f pulse-labeled [ a 2 p ] R N A ; a n d t h e m i x t u r e w a s a g i t a t e d w i t h a V o r t e x m i x e r u n t i l it r e a c h e d r o o m t e m p e r a t u r e , i n c u b a t e d for 2 h a t 67~ a n d chilled. T h e r e a c t i o n w a s t h e n inc u b a t e d for 1 h a t 37~ w i t h e i t h e r 1 t~g p a n c r e a t i c R N A a s e ( W o r t h i n g t o n ) p l u s 5/zg T1 R N A a s e (Calbiochem) in a final v o h o f 125 td 2 • SSC or w i t h 15 t~g p a n c r e a t i c R N A a s e p l u s 5 tLg T1 R N A a s e in a final vol. of 1 m l 2 x SSC (the 2 p r o c e d u r e s g a v e identical results) a n d chilled. 25 tzg of b o v i n e s e r u m a l b u m i n were a d d e d as carrier, a n d t h e R N A w h i c h s u r v i v e d t h e R N A a s e d i g e s t i o n w a s p r e c i p i t a t e d w i t h 4 m l of 5 % triehloroacetic acid c o n t a i n i n g 0-01 M-sodium p y r o p h o s p h a t e , filtered o n t o a nitrocellulose filter (Millipore DA), w a s h e d w i t h 20 to 50 m l of acid, dried a n d c o u n t e d in toluene/liquifluor. T h e efficiency of h y b r i d i z a t i o n of t h e [ 3 H ] R N A b y t h i s m e t h o d , defined as t h e s u m of R N A h y b r i d i z i n g a b o v e b a c k g r o u n d to e a c h of t h e 13 D N A f r a g m e n t s , p l u s t h e b a c k g r o u n d , d i v i d e d b y t h e t o t a l a c i d - p r e e i p i t a b l e p o l y n u e l e o t i d e , is a p p r o x . 1.0. T h e b a c k g r o u n d ( R N A a s e - r e s i s t a n t m a t e r i a l in t h e R N A ) is u s u a l l y 2 to 5 % o f t h e a c i d - p r e e i p i t a b l e
LETTERS
TO T H E E D I T O R
775
of t h e [32P]RNA) a n d s o m e t o t h e a d j a c e n t f r a g m e n t Z-7 (the r e m a i n i n g 300 bases of [32P]RNA). A t l e a s t t h r e e t i m e s as m u c h 32p s h o u l d h y b r i d i z e t o Z-7 as to Z-3. B u t i f t h e G p r o m o t e r were in t h e r e g i o n defined b y t h e R-6c/Z-2 o v e r l a p , t h e n t h e [32P]RNA s h o u l d h y b r i d i z e t o Z-2 a n d p o s s i b l y t o p a r t o f t h e a d j a c e n t f r a g m e n t Z-6a. T h e e x p e r i m e n t a l results a r e s h o w n in F i g u r e 2(c). L i k e t h e s h o r t p u l s e - l a b e l e d R N A s h o w n in F i g u r e 2(b), t h e 32p a g a i n h y b r i d i z e d t o b o t h Z-2 a n d Z-3; b u t in a d d i t i o n , a b o u t t h r e e to four t i m e s as m u c h [82P]RNA h y b r i d i z e d t o Z-7 as well. This is c o n s i s t e n t w i t h t h e p o s s i b i l i t y t h a t p r o m o t e r G lies in t h e r e g i o n defined b y t h e R-6b/Z-3 o v e r l a p . B e c a u s e t h i s r e g i o n covers t h e N - t e r m i n u s o f gene D, i t is l i k e l y t h a t p r o m o t e r G is l o c a t e d a t t h e s t a r t o f t h a t cistron.
A transcription map for r T h i s l e t t e r shows t h a t w h e n R N A chains a r e specifically i n i t i a t e d in vitro a t r p r o m o t o r s A ' or A a n d p u l s e - l a b e l e d o v e r a v e r y s h o r t sequence ( a p p r o x . 100 t o 400 bases), t h e l a b e l e d m a t e r i a l h y b r i d i z e s p r e f e r e n t i a l l y t o r e s t r i c t i o n e n d o n u c l e a s e g e n e r a t e d D N A f r a g m e n t s R - 4 or R-8, r e s p e c t i v e l y . W h e n R N A is specifically i n i t i a t e d a t p r o m o t o r G, t h e r e s u l t s a r e n o t as u n e q u i v o c a l as t h o s e g i v e n a b o v e b u t a r e c o n s i s t e n t w i t h t h e i n t e r p r e t a t i o n t h a t t h e p u l s e - l a b e l e d R N A is i n i t i a t e d pref e r e n t i a l l y w i t h i n t h e o v e r l a p o f D N A f r a g m e n t s R-6b/Z-3. B e c a u s e f r a g m e n t s R-4, R-8 a n d R-6b/Z-3 a r e each l o c a t e d n e a r t h e N - t e r m i n u s o f o n l y one c i s t r o n - - c i s t r o n s A , B a n d JO, r e s p e c t i v e l y (see Fig. 3 ) - - i t is l i k e l y t h a t t h e p r o m o t o r s a r e s i t u a t e d a t t h e s t a r t s o f t h o s e cistrons. H o w e v e r , i t is w o r t h n o t i n g s e v e r a l u n c e r t a i n t i e s in t h i s e x p e r i m e n t a l a p p r o a c h . (1) I t is n o t possible to d e t e r m i n e f r o m t h e s e d a t a e x a c t l y w h e r e a p r o m o t e r lies w i t h i n a D N A f r a g m e n t . (2) T h e D N A f r a g m e n t s a r e o n l y a p p r o x i m a t e l y a l i g n e d w i t h t h e b a c t e r i o p h a g e genetic m a p . E a c h f r a g m e n t h a s been m a p p e d on t h e basis o f i t s a b i l i t y to c o m p l e m e n t specific genetic m a r k e r s in a t r a n s f e c t i o n a s s a y , b u t t h e genetic m a r k e r s t h e m s e l v e s h a v e n o t been p r e c i s e l y l o c a t e d on the genome by physical means.
radioactivity. The efficiency of hybridization of the [32P]RNA, defined as the sum of RNA hybridizing above background to the 1 or 2 preferred DNA fragments, plus the background, divided by the total acid-preclpit~able polynucleotide, is approx. 0-25 to 0.50. This lower efllcieney in the second case might reflect the fact that, in general, less than one fourth as much [32p]RNA as [SH]RNA was present in each hybridization reaction. Note that the background referred to above, which is normally 2 to 5% of the acid-precipitable radioactivity, is RNAase-resistant material in the [32P]RNA, and was subtracted from the total ors/rain hybridized in each reaction before plotting the data (see below) ; the so called "general hybridization" discussed in the text is material which has hybridized to DNA above this RNAase-resistant background. To express the [SH]RNA hybridization results, the RNA hybridizing above background to each DNA fragment was plotted against the size of that DNA fragment; the points fall on a straight line, the smaller the fragment, the lower the amount of hybridization. Because fragment R-6 is a composite of three unresolvable fragments, the RNA hybridizing above background to R-6 was divided by 3 before it was plotted. To express the [32P]RNA hybridization results, the RNA hybridizing above background to each DNA fragment was plotted against the size of that DNA fragment, using an arbitrary scale on the ordinate so that the 3H cts/min and the 32p cts/min fall on the straight line. For any hybridization reaction in which the amount of [3~P]RNA hybridized falls considerably above the basic calibration straight line, the difference between the 32p and 3H cts/min is noted with an arrow extending from the 32p data point down to the 3H data point. For fragment R-6 (a composite of 3 unresolvable fragments) twice the amount of "general hybridization" calculated by extrapolation from the basic calibration straight line was subtracted from the RNA hybridizing to this fragment before it was plotted. ( 0 ) 32p (ors/rain) hybridized; (O) 3H (ets/min) hybridized. 51
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Fro. 3. Locations on the genetic m a p of ~bX174 of restriction fragments a n d of promoters a n d terminators for R N A synthesis in vitro. The h e a v y line represents t h e ~X174 genome (drawn here for convenience as a linear molecule). The 9 cistrons are arranged in the order determined b y genetic recombination (Benbow et al., 1971), their sizes calculated from published estimates of the molecular weights of the corresponding gene products (Burgess & D e n h a r d t , 1969; Gelfand & Hayashi, 1969; Borr~s et al., 1971; Godson, 1971; Benbow et al., 1972). The D N A fragments produced b y digestion of CX R F I D N A with endonuclease R or endonuelease Z h a v e been d r a w n w i t h respect to the genetic m a p as described elsewhere (Chen et ed., 1973; Lee & Sinsheimer, 1974a,b; Borrias et al., 1976). The three ~X174 promotors (PA', P ^ a n d Pa), 2 r h o - d e p e n d e n t t e r m i n a t i o n sites (T z a n d Ta), a n d 2 rho-independent t e r m i n a t i o n sites (T2 a u d T4) defined a n d m a p p e d relative to one a n o t h e r in the preceding paper (Axelrod, 1976) are n o t e d here b y arrows; the distances between t h e m are those established in t h a t paper. The promotors have been aligned with the genetic m a p on the basis of d a t a in the present paper. The direction of transcript~ion is clockwise (from left to right) (summarized b y D e n h a r d t , 1975).
The promotor locations determined above are in good agreement with the model of transcription proposed in the preceding paper (Axelrod, 1976). A transcription map combining the two sets of data is presented in Figure 3. Note that the only fixed points on this map are the distances between the various promotors and terminators, determined in the previous paper on the basis of the molecular weights of RNAs extending between these sites, and the localization of the promotors with regard to the restriction fragments. The exact molecular weight of some of the gene products is uncertain, particularly of genes C, E and J (IV[. Hayashi, personal communication), and the precise alignment of the restriction enzyme fragments has not yet been completed. Thus, the accurate positioning of the promotors with regard to the genetic map cannot be determined until some of these other variables have been defined. The proposed transcription map accords well with several aspects of r behavior in vivo. (1) Mutations in cistrons D, E, F and G show polar effects upon the genes downstream from them as far as but not including gene A (Hayashi & Hayashi, 1970; Benbow et al., 1972; Vanderbilt et al., 1972). This suggests that cistrons D, E, F, G and H, but not A, are coded for by a single polycistronic messenger, transcribed clockwise. (2) After infection by r the bacteriophage gene 19 protein product is. made in the highest molar amounts, followed in descending order by the products of genes E, F, etc., around the circle (Godson, 1971; Benbow et al., 1972). This implies that there is a translation origin (and perhaps a transcription origin) at the start of gene D, and that synthesis proceeds in a clockwise direction.
778
N. A X E L R O D
F u r t h e r m o r e , M. H a y a s h i e t a l . '(1976) h a v e r e c e n t l y c h a r a c t e r i z e d a n u m b e r o f ,]~X174 R N A s in vivo with respect, to t h e ir ,nolecular weights a n d h y b r i d i z a t i o n to D N A cl eav ed hy r e s t r i c t i o n enzymes. A c o m p a r i s o n of t h ese t r a n s c r i p t s ill, vivo a n d t h e t r a n s c r i p t s i n vitro described in this l e t t e r is s h o w n in T a b l e 1. T h e following conclusions can be d r a w n . (1) R N A s i n i t i a t e d a t p r o m o t o r A ' are r a p i d l y d e g r a d e d i n vivo a n d t h u s c a n n o t be c o m p a r e d t o t h e i r in vitro c o u n t e r p a r t s . (2) B o t h in vivo a n d i n vitro, one can d e t e c t R N A species w h i c h i n i t i a t e a t p r o m o t e r A or a t p r o m o t o r G, a n d t e r m i n a t e a t t h e r h o - i n d e p e n d e n t sites T2 or T4. (3) T h e r h o - d e p e n d e n t in vitro t e r m i n a t o r T a is a p p a r e n t l y n o t u t i l i z e d in vivo, n o r do R N A p o l y m e r a s e
TABLE 1
Comparison of R2VAs in v i t r o with the R N A species in v i v o characterized by M . H a y a s h i e t a l . (1976)
Promotor
Terminator
Molecular weight ( x 10 -6)
Name
A RNA species seen both in vitro and in vivo RNAs in vitro A T4 1.87 I A T4 1-60 II G T4 1.34 III A T2 0"52 IX G Ta 0"21 XIII
Promoter
Terminator
Molecular weight ( • 10 -6)
RNAs in vivo ? ? A T4 G T4 A T2 G T2
1-8 1"5 1-3 0.60 0.23
IV
A
1-10
V
A
VI
(,'
B RNA species seen only in vivo~ start of gone H start of gone G start of gone (l
0.98 0.82
C RNA species seen only in vitro$ A' Ta (rho) 1"00 A" Ta 0-84 A' PG 0.62 A' PA 0.30 A' T1 (rho) 0-07 A Ta (rho) 0.70 G Ta (rho) 0.42 G Ta (rho) 0-35 A PG 0.27 Each of the in vitro RNA species characterized in this and the accompanying paper is listed with respect to its molecular weight and its sites of origin and termination. Each of the in vivo RNA species characterized by Hayashi etal. is listed by the name given to it by those authors, its molecular weight, and its sites of origin and termination. ~RNAs VII, VIII, X, XI, and X I I appearing in vivo may be mixtures and have not yet been mapped. SRNAs starting at promoter A' are apparently degraded rapidly in vivo and thus cannot be detected under most labeling conditions. A possible exception is RNA species I, a unit length R N A present in minor amounts which might originate at promotor A' and terminate at T 4. The remaining in vitro RNA species which are not seen in vivo all terminate at rho-dependent termination sites or at the site of downstream promoters.
L E T T E R S TO T H E E D I T O R
779
molecules stop at the sites of downstream promotors in vivo. By contrast, R N A molecules seem to terminate in vivo at sites near the beginnings of genes F, G and H which are not utilized in vitro under the conditions used above. Chen et al. (1973) have reported t h a t the E. coti R N A polymerase binds in vitro to CX R F I D N A at sites which m a p within D N A fragments R-2, R-4, and in the overlap of fragments R-6b and Z-3. The latter two binding sites probably correspond to promotors A' and G. The other R N A polymerase binding site a p p a r e n t l y does not act as a promotor for R N A synthesis in vitro under the conditions employed here. I t is possible t h a t an additional initiation factor is required in order for t h a t site to act as a promotor. Alternatively, it m a y serve some other function entirely. Chen et al. observed no R N A polymerase binding site which corresponds to promotor A. This is not surprising, for I also find (unpublished observations) t h a t when CX R F I D N A and E. coli R N A polymerase are incubated in the high concentrations used in their experiments, p r o m o t o r A is a great deal less active t h a n promotors A' and G; the binding could well h a v e been below the level of detection. I thank J. D. Watson, Joe Sambrook and Jeff Roberts for their help throughout this research and the preparation of this manuscript, and Marshall Edgell, Clyde Hutchison and their colleagues for generously providing me with bacterial strains, experimental preparations, and unpublished data. This investigation was supported b y National Institutes of Health Training grant TO1 GM00036 from the National Institute of General Medical Sciences, and by grant GM09541 held first by J. D. Watson and later by Waiter Gilbert. The Biological Laboratories Harvard University, Cambridge, Mass. 02138, U.S.A.
NANCY
~XELR0 D
Received 29 July 1974, and in revised form 9 August 1976
REFERENCES
Axelrod, N. (1976). J. Mol. Biol. 108, 797-814. Benbow, R. M., Hutehison, C. A. I I I , Fabricant, J. D. & Sinsheimer, R. L. (1971). J. Virol. 7, 549-558. Benbow, R. M., Mayol, R. F., Picehi, J. C. & Sinsheimer, R. L. (1972). J. Virol. I0, 99-114. Borrgm, M. T., Vanderbilt, A. S. & Tessman, E. {1971), Virology, 45, 802-803. Borrias, W. E., Weisbeek, P. J., van Arkel, G. A. (1976). Nature (London), 261, 245-249. Burgess, A. B. & Denhardt, D. T. (1969). J. Mol. Biol. 44, 377-386. Chen, C. Y., Hutehison, C. A. I I I & Edgell, M. (1973). Nature New Biol. 243, 233-236. Denhardt, D. T. (1975). GRC Critical Reviewa in Microbiology (Laskin, A. I. & Lechevalier, H., eds.), pp. 161-223, CRC Press, Cleveland. Edgell, M. H., Hutchison, C. A. I I I & Sclair, M. {1972). J. Virol. 9, 574-582. Geffand, D. H. & Hayashi, M. (1969). J. Mol. Biol. 44, 501-516. Godson, N. G. (1971). J. Mol. Biol. 57, 541-552. Hayashi, M., Fujimura, F. & Hayashi, M. N. (1976). Proc. Nat. Acad. Sci., U.S.A., in the press. Hayashi, Y. & Hayashi, M. (1970). Cold Spring Harbor Syrup Quant. Biol. 35, 171-174. Hutehison, C. A. I I I , Middleton, J. H. & Edgell, M. H. (1972). Biophys. Soc. Abstr., Biophys. J. 12(3), 31a. Lee, A. S. & Sinsheimer, R. L. (1974a). J. Virol. 14, 872-877. Lee, A. S. & Sinsheimer, R. L. (1974b). Proc. Nat. Acad. Sci., U.S.A. 71, 2882-2886. Vanderbilt, A. S., BorroWs, M. T., Germeraad, S., Tessman, I. & Tessman, E. (1972). Virology, 50, 171-179.
LETTERS TO THE EDITOR
Transcription of Bacteriophage r in Vitro: Analysis with Restriction Enzymes The three phage r promotors (P,., PA and Po) defined and mapped relative to one another by Axelrod (1976) have been mapped onto the bacteriophage genome. RNA chains were specifically initiated in vitro at each promotor with oligonucleotide primers, pulse-labeled with (a-aaP)-labeled ribonueleoside triphosphates, chased into frill-length molecules with an excess of unlabeled ribonucleoside triphosphates, and purified on polyacrylamide gels. The isolated RNA species were hybridized to a series of purified, tmlabeled DNA fragments generated by cleavage of r RFI~ DNA with restriction endonucleases from Hemophilus influenzae (endonuclease R) or H. aegyptius (endonuclease Z). The a2P-labeled RNA sequences (approx. 100 to 400 bases) originating from promotors A', A or G hybridized preferentially to DNA fragments R-4, R-8 and R-6b/Z-3, respectively. The DNA fragments have previously been aligned with the bacteriophage genetic map by independent methods in other laboratories (Chert et al., 1973 ; Hutchison et al., 1973 ; Lee & Sinsheimer, 1974a,b ; Borrias et al., 1976). Comparison of the above hybridization results to that fragment map shows that promotors A', A and G lie near the starts of cistrons A, B and D. I n the preceding paper (Axelrod, 1976), the R N A species generated by transcription in vitro of bacteriophage r R F I t D N A were m a p p e d relative to one another by initiation at specific promotors with oligonucleotide primers and analysis on polyacrylamide gels; three promotors (PA', PA and Po), two rho-independent termination sites (T2 and T4), and two rho-dependent termination sites (T1 and Ta) were defined. I n this letter, those promotor and termination sites are aligned with respect to the genetic map. Use is made of the defined set of D N A fragments generated b y cleavage of CX R F I D N A with restriction enzymes from Hemophilus influenzae (endonuclease R) or H. aegyptius (endonuclease Z); these fragments have been m a p p e d relative to one another and to the genetic m a p in other laboratories (Chen et al., 1973; Hutchison et al., 1973; Lee & Sinsheimer, 1974a,b; Borrias et al., 1976). To determine the m a p location of each promotor, R N A chains were initiated specifically at t h a t promotor with an appropriate oligonucleotide primer, pulselabeled over a short distance with a2P-labeled ribonucleoside triphosphates, chased into full-length molecules with an excess of unlabeled R N A precursors, and purified b y electrophoresis on a polyacrylamide gel. The double precaution of initiating synthesis with a specific oligonucleotide primer and purifying the R N A on a gel ensured t h a t each preparation of a2P-labeled R N A represented synthesis from a single, well-defined promotor. The R N A was then hybridized to each of the D N A fragments generated b y cleavage of CX R F I D N A with endonuclease R or, in some cases, endonuelease Z. I t was expected t h a t the a2p-labeled portion of each pulselabeled R N A would hybridize only to t h a t region of the D N A from which it was t r a n s c r i b e d - - t h a t is, to one, or at most two, of the D N A fragments---enabling one tAbbreviat~on used: RFI, the double-stranded closed circular supereoiled replicative form DNA of r 771
772
N. AXELI~OD
to locate the origin of transcriptioh. As an internal control, RNA uniformly labeled with 3H-labeled ribonucleoside triphosphates and containing all the r RNA sequences in approximately equal molar amounts was included in each hybridization reaction. This RNA hybridized to all of the DNA fragments, to each according to its molecular weight, and served as a standard against which the 32p hybridization could be compared. Although all CX RNA sequences may not have been present in equimolar amounts in the [3H]RNA preparation, the use of the same RNA preparation in all experiments ensured that the ratios of 32p to 3H cts/min hybridized were meaningful. As expected, the 32p pulse-labeled portion of each RNA species did hybridize preferentially to a limited number of DNA fragments, results which will be discussed below. However, the 32P-labeled RNA also hybridized to some extent to nearly all of the DNA fragments, in each case in proportion to the size of the fragment. Experiments conducted to determine the nature of this "general hybridization" background have revealed that (1) the "general hybridization" background is in fact the result of annealing and is not the result of non-specific trapping of labeled RNA onto the nitrocellulose filters, for when an equivalent amount of DNA or in vitro synthesized RNA from bacteriophage h was substituted for the corresponding r material in a hybridization reaction, non-specific trapping of heterologous nucleic acids was not observed. (2) The "general hybridization" does not result from radioactive precursors incorporated at a low level into the portion of the RNA synthesized during the chase, for when ternary complexes of DNA, RNA polymerase, and radioactively labeled nascent RNA were purified away from unincorporated (~-32P)-labeled ribonucleoside triphosphates before the chase period begun to ensure that no further radioactive label was incorporated into RNA, the labeled RNA again hybridized to some extent to nearly all of the DNA fragments. (3) The "general hybridization" is not a result of incomplete ribonuclease digestion, for it is not reduced by more extensive ribonuclease treatment. Nor does it represent an intrinsic property of the RNA itself, for when RNA is hybridized in a mock reaction with no DNA, or when RNA is added after the DNA has been subjected to such a mock hybridization, no "general hybridization" can be observed. Preliminary experiments performed to determine the nature of this "general hybridization" suggest that it reflects a short RNA sequence found within the first 400 bases or so of each promotor and repeated apparently at random m a n y times in the r DNA. In these experiments, pulse-labeled RNA from promotor G which had hybridized to several different DNA fragments as "general hybridization" was eluted from those DNA fragments, sized by electrophoresis on polyacrylamide gels, digested by pancreatic or T~ ribonuclease, and subjected to "fingerprint" analysis. By all of these criteria, the I~NAs eluted from the different DNA fragments appeared to be quite similar to one another. These studies will be discussed in more detail in a forthcoming publication. For the moment, I emphasize that the important variable to consider in evaluating the following experiments is not the total number of 32p cts/min hybridized to a given DNA fragment, but rather, the ratio of 32p to aH ets/min hybridized, which in effect compensates for the "general hybridization". Promotor A ' and promotor A
32p pulse-labeled RNAs originating at either promotor A' or at promotor A were prepared as described in the legend to Figure 1 and were hybridized, along with uniformly labeled [3H]RNA, to each of the DNA fragments generated b y cleavage
L E T T E R S TO T H E E D I T O R
773
of CX R F I DNA with endonuelease R. Figure 1 (a) shows that pulse-labeled RNA originating at promotor A' hybridized preferentially to DNA fragment R-4. T h a t is, the ratio of 32p to 3H cts/min hybridized to R-4 was significantly greater than for any other DNA fragment. This suggests t h a t promotor A' lies within the region of DNA defined by fragment R-4. Because cistron A is the only eistron whose N-terminus resides in fragment R-4 (the fragment map is presented in Fig 3), it seems likely that promotor A' is located at the start of t h a t cistron. Figure l(b) shows that when the pulse-labeled RNA was initiated at promotor A, the ratio of 32P/3H cts/min hybridized to fragment R-8 was 10 to 100-fold greater than the ratio of counts hybridized to any other fragment. Thus, RNA synthesis from promotor A begins in the region defined by fragment R-8. Consideration of this result in light of the fragment map shown in Figure 3 indicates that RNA synthesis initiated at promotor A probably begins at the distal end of eistron A or at the start of cistron B.
Promotor G 32p pulse-labeled RNA originating at promotor G was prepared as described in the legend to Figure 2 and was hybridized as before to each of the DNA fragments generated by cleavage of CX R F I DNA with endonuclease R. Figure 2(a) shows t h a t the 3~P-labeled RNA hybridized preferentially to fragment R-6. :Fragment R-6 actually consists of three fragments--R-6a, R-6b and R-6r cannot be separated physically but which are located at three different locations on the genome (see Fig. 3); and it is not possible to conclude from these data which of the three fragments has hybridized to the labeled RNA. In a further effort to localize promotor G, 32p pulse-labeled RNA originating at this promotor was prepared as described above and was hybridized to each of the DNA fragments generated by cleavage of CX R F I DNA with another restriction enzyme, endonuclease Z. Because each of the R-6 fragments overlaps a different Z fragment (see fragment map in :Fig. 3), it should be possible to determine in this manner which of the three R-6 fragments contains the sequences of promotor G. Recall that the method of preparing the RNA for these experiments ensures t h a t all of the G-specific RNA is initiated at one site on the genome, so one should see enhanced hybridization to only one of the Z fragments (Z-3/R-6b, Z-2/R-6e or Z-8 or Z-4/R-6a). The results are shown in Figure 2(b). The labeled RNA hybridized preferentially to fragment Z-3 (which overlaps R-6b), but also to a somewhat lesser extent to fragment Z-2 (which overlaps R-6r This experiment is difficult to interpret. Apparently, the RNA was initiated within the region corresponding to either Z-3 or Z-2, but the two DNA fragments were either cross-contaminated or contain regions of homology so that the 32p pulse-labeled RNA hybridized to both Z fragments. To resolve this question further, a new approach was taken in which the DNA fragment adjacent to the fragment containing the promotor was identified. RNA was initiated from promotor G as before but was pulse-labeled for two and one-haft times as long as in previous experiments, so that 82p was incorporated into a sequence of RNA of approximately 400 bases rather than the 150 or so bases labeled previously. The RNA was chased into full-length molecules, purified by electrophoresis on gels, and hybridized to the collection of Z fragments. I f the G promotor were located in the sequence defined by the 100 base-pair overlap of R-6b and Z-3, then some of the 32P-labeled RNA would be expected to hybridize to Z-3 (approx. 100 bases or less
774
N. AXELROD 900 800 700
600 "2
"O "C
B 2, ,~ ~ 7 0
o o o -~
5OO
c:
4oo
,500
~ ~
5o
F~
"/
13. "r
2oo
.
~ I I
R 9
8
I
I
7 5 4
I
t
5
2
1,~176176
I I
I
500
"
/oo
I/
I0
R 98/\65
6
4
5
2
7.(2) 7.(I) DNA frogment no. (o)
(b)
FIG. 1. H y b r i d i z a t i o n of pulse-labeled R N A o r i g i n a t i n g a t p r o m o t e r s A ' or A to D N A f r a g m e n t s g e n e r a t e d b y cleavage of CX R F I D N A w i t h e n d o n u c l e a s e R . (a) R N A c h a i n s were i n i t i a t e d in vitro a t p r o m o t e r A ' w i t h t h e "artificial o l i g o n u e l e o t i d e " A p A p A ( A p A + A T P ) ; p u l s e - l a b e l e d for 5 m i n w i t h (a-32P)-labeled C T P a n d U T P , d u r i n g w h i c h t i m e a p p r o x . 200 to 400 b a s e s were i n c o r p o r a t e d into R N A ; c h a s e d w i t h a n e x c e s s of u n l a b e l e d ribonucleoside t r i p h o s p h a t e s ; a n d s u b j e c t e d to p o l y a c r y l a m i d e gel electrophoresis. T h e m a j o r R N A species o r i g i n a t i n g f r o m prom o t o r A ' (the 1.87• l0 s mo]. w t R N A c h a r a c t e r i z e d b y A x e l r o d , 1976) w a s e l u t e d f r o m t h e gel a n d h y b r i d i z e d , in t h e p r e s e n c e of u n i f o r m l y labeled [ 3 H ] R N A c o n t a i n i n g all r sequences in a p p r o x i m a t e l y e q u a l a m o u n t s , to e a c h of t h e D N A f r a g m e n t s g e n e r a t e d b y c l e a v a g e of C X R F I D N A w i t h e n d o n u c l e a s e R. (b) R N A ws.s p r e p a r e d a n d h y b r i z e d as a b o v e , e x c e p t t h e R N A c h a i n s were i n i t i a t e d a t p r o m o t e r A w i t h t h e "artificial o l i g o n u c l e o t i d e " C p A p U p C (CpA + U T P + C T P ) , a n d t h e m a j o r species p r o d u c e d a n d e l u t e d f r o m t h e gel w a s t h e 1.60 • l0 s reel. w t R N A c h a r a c t e r i z e d b y A x e h ' o d (1976). E a c h s a m p l e w a s c o u n t e d in a B e c k m a n s c i n t i l l a t i o n c o u n t e r for 20 rain, to a n a c c u r a c y o f a t least 7~o. 3up pulse-labeled R N A w a s s y n t h e s i z e d e s s e n t i a l l y as described b y A x e l r o d (1976), e x c e p t t h a t reaction m i x t u r e s c o n t a i n e d 10 t i m e s as m u c h D N A a n d R N A p o l y m e r a s e as before, a n d t h e specific a c t i v i t y of t h e (~ 32p).labeled r i b o n u c l e o s i d e s w a s 10 to 20 t i m e s as h i g h as before. T h e R N A w a s purified b y e l e c t r o p h o r e s i s on p r e p a r a t i v e p o l y a c r y l a m i d e gels as in A x e l r o d (1976) followed b y e l u t i o n into 2 • SSC (SSC is 0-15 M-NaCI, 0.015 M-sodium c i t r a t e p H 7.2) -F 0 . 2 % s o d i u m d o d e c y l sulfate. 3H-labeled R N A w a s p r e p a r e d as a b o v e , e x c e p t t h a t t h e R N A w a s labeled c o n t i n u o u s l y w i t h [ a H ] C T P a n d t h e R N A w a s n o t gel-purified. r RFI DNA was cleaved with endonuclease R a n d s u b j e c t e d to e l e c t r o p h o r e s i s on 3.5~ to 7.5~ p o l y a c r y l a m i d e g r a d i e n t gels. T h e D N A f r a g m e n t s were v i s u a l i z e d b y s t a i n i n g w i t h m e t h y l e n e blue, c u t o u t w i t h a r a z o r blade, a n d r e m o v e d f r o m t h e gel slice b y e l e c t r o p h o r e s i s into 40 m M - T r i s - a c e t a t e , 20 m ~ - s o d i u m a c e t a t e , 2 m M - E D T A ( p H 7.9}. r R F I D N A cleaved w i t h e n d o n u c l e a s e Z w a s k i n d l y p r o v i d e d b y C. H u t c h i s o n a n d M. Edgell a n d w a s purified as a b o v e . H y b r i d i z a t i o n s were p e r f o r m e d as follows : for e a c h h y b r i d i z a t i o n , a p p r o x . 0.2 p m o l o f a D N A f r a g m e n t in 60 td 2 x SSC was d e n a t u r e d b y boiling for 2 rain a n d chilled r a p i d l y in solid CO2/acetone. To t h e frozen D N A were a d d e d 40 ~l of 2 • SSC c o n t a i n i n g 1"4 • 10 -3 p m o l of u n i f o r m l y labeled [ 3 H ] R N A a n d 2 • 10 -4 to 6 • 10 -4 p m o l o f pulse-labeled [ a 2 p ] R N A ; a n d t h e m i x t u r e w a s a g i t a t e d w i t h a V o r t e x m i x e r u n t i l it r e a c h e d r o o m t e m p e r a t u r e , i n c u b a t e d for 2 h a t 67~ a n d chilled. T h e r e a c t i o n w a s t h e n inc u b a t e d for 1 h a t 37~ w i t h e i t h e r 1 t~g p a n c r e a t i c R N A a s e ( W o r t h i n g t o n ) p l u s 5/zg T1 R N A a s e (Calbiochem) in a final v o h o f 125 td 2 • SSC or w i t h 15 t~g p a n c r e a t i c R N A a s e p l u s 5 tLg T1 R N A a s e in a final vol. of 1 m l 2 x SSC (the 2 p r o c e d u r e s g a v e identical results) a n d chilled. 25 tzg of b o v i n e s e r u m a l b u m i n were a d d e d as carrier, a n d t h e R N A w h i c h s u r v i v e d t h e R N A a s e d i g e s t i o n w a s p r e c i p i t a t e d w i t h 4 m l of 5 % triehloroacetic acid c o n t a i n i n g 0-01 M-sodium p y r o p h o s p h a t e , filtered o n t o a nitrocellulose filter (Millipore DA), w a s h e d w i t h 20 to 50 m l of acid, dried a n d c o u n t e d in toluene/liquifluor. T h e efficiency of h y b r i d i z a t i o n of t h e [ 3 H ] R N A b y t h i s m e t h o d , defined as t h e s u m of R N A h y b r i d i z i n g a b o v e b a c k g r o u n d to e a c h of t h e 13 D N A f r a g m e n t s , p l u s t h e b a c k g r o u n d , d i v i d e d b y t h e t o t a l a c i d - p r e e i p i t a b l e p o l y n u e l e o t i d e , is a p p r o x . 1.0. T h e b a c k g r o u n d ( R N A a s e - r e s i s t a n t m a t e r i a l in t h e R N A ) is u s u a l l y 2 to 5 % o f t h e a c i d - p r e e i p i t a b l e
LETTERS
TO T H E E D I T O R
775
of t h e [32P]RNA) a n d s o m e t o t h e a d j a c e n t f r a g m e n t Z-7 (the r e m a i n i n g 300 bases of [32P]RNA). A t l e a s t t h r e e t i m e s as m u c h 32p s h o u l d h y b r i d i z e t o Z-7 as to Z-3. B u t i f t h e G p r o m o t e r were in t h e r e g i o n defined b y t h e R-6c/Z-2 o v e r l a p , t h e n t h e [32P]RNA s h o u l d h y b r i d i z e t o Z-2 a n d p o s s i b l y t o p a r t o f t h e a d j a c e n t f r a g m e n t Z-6a. T h e e x p e r i m e n t a l results a r e s h o w n in F i g u r e 2(c). L i k e t h e s h o r t p u l s e - l a b e l e d R N A s h o w n in F i g u r e 2(b), t h e 32p a g a i n h y b r i d i z e d t o b o t h Z-2 a n d Z-3; b u t in a d d i t i o n , a b o u t t h r e e to four t i m e s as m u c h [82P]RNA h y b r i d i z e d t o Z-7 as well. This is c o n s i s t e n t w i t h t h e p o s s i b i l i t y t h a t p r o m o t e r G lies in t h e r e g i o n defined b y t h e R-6b/Z-3 o v e r l a p . B e c a u s e t h i s r e g i o n covers t h e N - t e r m i n u s o f gene D, i t is l i k e l y t h a t p r o m o t e r G is l o c a t e d a t t h e s t a r t o f t h a t cistron.
A transcription map for r T h i s l e t t e r shows t h a t w h e n R N A chains a r e specifically i n i t i a t e d in vitro a t r p r o m o t o r s A ' or A a n d p u l s e - l a b e l e d o v e r a v e r y s h o r t sequence ( a p p r o x . 100 t o 400 bases), t h e l a b e l e d m a t e r i a l h y b r i d i z e s p r e f e r e n t i a l l y t o r e s t r i c t i o n e n d o n u c l e a s e g e n e r a t e d D N A f r a g m e n t s R - 4 or R-8, r e s p e c t i v e l y . W h e n R N A is specifically i n i t i a t e d a t p r o m o t o r G, t h e r e s u l t s a r e n o t as u n e q u i v o c a l as t h o s e g i v e n a b o v e b u t a r e c o n s i s t e n t w i t h t h e i n t e r p r e t a t i o n t h a t t h e p u l s e - l a b e l e d R N A is i n i t i a t e d pref e r e n t i a l l y w i t h i n t h e o v e r l a p o f D N A f r a g m e n t s R-6b/Z-3. B e c a u s e f r a g m e n t s R-4, R-8 a n d R-6b/Z-3 a r e each l o c a t e d n e a r t h e N - t e r m i n u s o f o n l y one c i s t r o n - - c i s t r o n s A , B a n d JO, r e s p e c t i v e l y (see Fig. 3 ) - - i t is l i k e l y t h a t t h e p r o m o t o r s a r e s i t u a t e d a t t h e s t a r t s o f t h o s e cistrons. H o w e v e r , i t is w o r t h n o t i n g s e v e r a l u n c e r t a i n t i e s in t h i s e x p e r i m e n t a l a p p r o a c h . (1) I t is n o t possible to d e t e r m i n e f r o m t h e s e d a t a e x a c t l y w h e r e a p r o m o t e r lies w i t h i n a D N A f r a g m e n t . (2) T h e D N A f r a g m e n t s a r e o n l y a p p r o x i m a t e l y a l i g n e d w i t h t h e b a c t e r i o p h a g e genetic m a p . E a c h f r a g m e n t h a s been m a p p e d on t h e basis o f i t s a b i l i t y to c o m p l e m e n t specific genetic m a r k e r s in a t r a n s f e c t i o n a s s a y , b u t t h e genetic m a r k e r s t h e m s e l v e s h a v e n o t been p r e c i s e l y l o c a t e d on the genome by physical means.
radioactivity. The efficiency of hybridization of the [32P]RNA, defined as the sum of RNA hybridizing above background to the 1 or 2 preferred DNA fragments, plus the background, divided by the total acid-preclpit~able polynucleotide, is approx. 0-25 to 0.50. This lower efllcieney in the second case might reflect the fact that, in general, less than one fourth as much [32p]RNA as [SH]RNA was present in each hybridization reaction. Note that the background referred to above, which is normally 2 to 5% of the acid-precipitable radioactivity, is RNAase-resistant material in the [32P]RNA, and was subtracted from the total ors/rain hybridized in each reaction before plotting the data (see below) ; the so called "general hybridization" discussed in the text is material which has hybridized to DNA above this RNAase-resistant background. To express the [SH]RNA hybridization results, the RNA hybridizing above background to each DNA fragment was plotted against the size of that DNA fragment; the points fall on a straight line, the smaller the fragment, the lower the amount of hybridization. Because fragment R-6 is a composite of three unresolvable fragments, the RNA hybridizing above background to R-6 was divided by 3 before it was plotted. To express the [32P]RNA hybridization results, the RNA hybridizing above background to each DNA fragment was plotted against the size of that DNA fragment, using an arbitrary scale on the ordinate so that the 3H cts/min and the 32p cts/min fall on the straight line. For any hybridization reaction in which the amount of [3~P]RNA hybridized falls considerably above the basic calibration straight line, the difference between the 32p and 3H cts/min is noted with an arrow extending from the 32p data point down to the 3H data point. For fragment R-6 (a composite of 3 unresolvable fragments) twice the amount of "general hybridization" calculated by extrapolation from the basic calibration straight line was subtracted from the RNA hybridizing to this fragment before it was plotted. ( 0 ) 32p (ors/rain) hybridized; (O) 3H (ets/min) hybridized. 51
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Fro. 3. Locations on the genetic m a p of ~bX174 of restriction fragments a n d of promoters a n d terminators for R N A synthesis in vitro. The h e a v y line represents t h e ~X174 genome (drawn here for convenience as a linear molecule). The 9 cistrons are arranged in the order determined b y genetic recombination (Benbow et al., 1971), their sizes calculated from published estimates of the molecular weights of the corresponding gene products (Burgess & D e n h a r d t , 1969; Gelfand & Hayashi, 1969; Borr~s et al., 1971; Godson, 1971; Benbow et al., 1972). The D N A fragments produced b y digestion of CX R F I D N A with endonuclease R or endonuelease Z h a v e been d r a w n w i t h respect to the genetic m a p as described elsewhere (Chen et ed., 1973; Lee & Sinsheimer, 1974a,b; Borrias et al., 1976). The three ~X174 promotors (PA', P ^ a n d Pa), 2 r h o - d e p e n d e n t t e r m i n a t i o n sites (T z a n d Ta), a n d 2 rho-independent t e r m i n a t i o n sites (T2 a u d T4) defined a n d m a p p e d relative to one a n o t h e r in the preceding paper (Axelrod, 1976) are n o t e d here b y arrows; the distances between t h e m are those established in t h a t paper. The promotors have been aligned with the genetic m a p on the basis of d a t a in the present paper. The direction of transcript~ion is clockwise (from left to right) (summarized b y D e n h a r d t , 1975).
The promotor locations determined above are in good agreement with the model of transcription proposed in the preceding paper (Axelrod, 1976). A transcription map combining the two sets of data is presented in Figure 3. Note that the only fixed points on this map are the distances between the various promotors and terminators, determined in the previous paper on the basis of the molecular weights of RNAs extending between these sites, and the localization of the promotors with regard to the restriction fragments. The exact molecular weight of some of the gene products is uncertain, particularly of genes C, E and J (IV[. Hayashi, personal communication), and the precise alignment of the restriction enzyme fragments has not yet been completed. Thus, the accurate positioning of the promotors with regard to the genetic map cannot be determined until some of these other variables have been defined. The proposed transcription map accords well with several aspects of r behavior in vivo. (1) Mutations in cistrons D, E, F and G show polar effects upon the genes downstream from them as far as but not including gene A (Hayashi & Hayashi, 1970; Benbow et al., 1972; Vanderbilt et al., 1972). This suggests that cistrons D, E, F, G and H, but not A, are coded for by a single polycistronic messenger, transcribed clockwise. (2) After infection by r the bacteriophage gene 19 protein product is. made in the highest molar amounts, followed in descending order by the products of genes E, F, etc., around the circle (Godson, 1971; Benbow et al., 1972). This implies that there is a translation origin (and perhaps a transcription origin) at the start of gene D, and that synthesis proceeds in a clockwise direction.
778
N. A X E L R O D
F u r t h e r m o r e , M. H a y a s h i e t a l . '(1976) h a v e r e c e n t l y c h a r a c t e r i z e d a n u m b e r o f ,]~X174 R N A s in vivo with respect, to t h e ir ,nolecular weights a n d h y b r i d i z a t i o n to D N A cl eav ed hy r e s t r i c t i o n enzymes. A c o m p a r i s o n of t h ese t r a n s c r i p t s ill, vivo a n d t h e t r a n s c r i p t s i n vitro described in this l e t t e r is s h o w n in T a b l e 1. T h e following conclusions can be d r a w n . (1) R N A s i n i t i a t e d a t p r o m o t o r A ' are r a p i d l y d e g r a d e d i n vivo a n d t h u s c a n n o t be c o m p a r e d t o t h e i r in vitro c o u n t e r p a r t s . (2) B o t h in vivo a n d i n vitro, one can d e t e c t R N A species w h i c h i n i t i a t e a t p r o m o t e r A or a t p r o m o t o r G, a n d t e r m i n a t e a t t h e r h o - i n d e p e n d e n t sites T2 or T4. (3) T h e r h o - d e p e n d e n t in vitro t e r m i n a t o r T a is a p p a r e n t l y n o t u t i l i z e d in vivo, n o r do R N A p o l y m e r a s e
TABLE 1
Comparison of R2VAs in v i t r o with the R N A species in v i v o characterized by M . H a y a s h i e t a l . (1976)
Promotor
Terminator
Molecular weight ( x 10 -6)
Name
A RNA species seen both in vitro and in vivo RNAs in vitro A T4 1.87 I A T4 1-60 II G T4 1.34 III A T2 0"52 IX G Ta 0"21 XIII
Promoter
Terminator
Molecular weight ( • 10 -6)
RNAs in vivo ? ? A T4 G T4 A T2 G T2
1-8 1"5 1-3 0.60 0.23
IV
A
1-10
V
A
VI
(,'
B RNA species seen only in vivo~ start of gone H start of gone G start of gone (l
0.98 0.82
C RNA species seen only in vitro$ A' Ta (rho) 1"00 A" Ta 0-84 A' PG 0.62 A' PA 0.30 A' T1 (rho) 0-07 A Ta (rho) 0.70 G Ta (rho) 0.42 G Ta (rho) 0-35 A PG 0.27 Each of the in vitro RNA species characterized in this and the accompanying paper is listed with respect to its molecular weight and its sites of origin and termination. Each of the in vivo RNA species characterized by Hayashi etal. is listed by the name given to it by those authors, its molecular weight, and its sites of origin and termination. ~RNAs VII, VIII, X, XI, and X I I appearing in vivo may be mixtures and have not yet been mapped. SRNAs starting at promoter A' are apparently degraded rapidly in vivo and thus cannot be detected under most labeling conditions. A possible exception is RNA species I, a unit length R N A present in minor amounts which might originate at promotor A' and terminate at T 4. The remaining in vitro RNA species which are not seen in vivo all terminate at rho-dependent termination sites or at the site of downstream promoters.
L E T T E R S TO T H E E D I T O R
779
molecules stop at the sites of downstream promotors in vivo. By contrast, R N A molecules seem to terminate in vivo at sites near the beginnings of genes F, G and H which are not utilized in vitro under the conditions used above. Chen et al. (1973) have reported t h a t the E. coti R N A polymerase binds in vitro to CX R F I D N A at sites which m a p within D N A fragments R-2, R-4, and in the overlap of fragments R-6b and Z-3. The latter two binding sites probably correspond to promotors A' and G. The other R N A polymerase binding site a p p a r e n t l y does not act as a promotor for R N A synthesis in vitro under the conditions employed here. I t is possible t h a t an additional initiation factor is required in order for t h a t site to act as a promotor. Alternatively, it m a y serve some other function entirely. Chen et al. observed no R N A polymerase binding site which corresponds to promotor A. This is not surprising, for I also find (unpublished observations) t h a t when CX R F I D N A and E. coli R N A polymerase are incubated in the high concentrations used in their experiments, p r o m o t o r A is a great deal less active t h a n promotors A' and G; the binding could well h a v e been below the level of detection. I thank J. D. Watson, Joe Sambrook and Jeff Roberts for their help throughout this research and the preparation of this manuscript, and Marshall Edgell, Clyde Hutchison and their colleagues for generously providing me with bacterial strains, experimental preparations, and unpublished data. This investigation was supported b y National Institutes of Health Training grant TO1 GM00036 from the National Institute of General Medical Sciences, and by grant GM09541 held first by J. D. Watson and later by Waiter Gilbert. The Biological Laboratories Harvard University, Cambridge, Mass. 02138, U.S.A.
NANCY
~XELR0 D
Received 29 July 1974, and in revised form 9 August 1976
REFERENCES
Axelrod, N. (1976). J. Mol. Biol. 108, 797-814. Benbow, R. M., Hutehison, C. A. I I I , Fabricant, J. D. & Sinsheimer, R. L. (1971). J. Virol. 7, 549-558. Benbow, R. M., Mayol, R. F., Picehi, J. C. & Sinsheimer, R. L. (1972). J. Virol. I0, 99-114. Borrgm, M. T., Vanderbilt, A. S. & Tessman, E. {1971), Virology, 45, 802-803. Borrias, W. E., Weisbeek, P. J., van Arkel, G. A. (1976). Nature (London), 261, 245-249. Burgess, A. B. & Denhardt, D. T. (1969). J. Mol. Biol. 44, 377-386. Chen, C. Y., Hutehison, C. A. I I I & Edgell, M. (1973). Nature New Biol. 243, 233-236. Denhardt, D. T. (1975). GRC Critical Reviewa in Microbiology (Laskin, A. I. & Lechevalier, H., eds.), pp. 161-223, CRC Press, Cleveland. Edgell, M. H., Hutchison, C. A. I I I & Sclair, M. {1972). J. Virol. 9, 574-582. Geffand, D. H. & Hayashi, M. (1969). J. Mol. Biol. 44, 501-516. Godson, N. G. (1971). J. Mol. Biol. 57, 541-552. Hayashi, M., Fujimura, F. & Hayashi, M. N. (1976). Proc. Nat. Acad. Sci., U.S.A., in the press. Hayashi, Y. & Hayashi, M. (1970). Cold Spring Harbor Syrup Quant. Biol. 35, 171-174. Hutehison, C. A. I I I , Middleton, J. H. & Edgell, M. H. (1972). Biophys. Soc. Abstr., Biophys. J. 12(3), 31a. Lee, A. S. & Sinsheimer, R. L. (1974a). J. Virol. 14, 872-877. Lee, A. S. & Sinsheimer, R. L. (1974b). Proc. Nat. Acad. Sci., U.S.A. 71, 2882-2886. Vanderbilt, A. S., BorroWs, M. T., Germeraad, S., Tessman, I. & Tessman, E. (1972). Virology, 50, 171-179.
