in vitro transcription of adenovirus 2 dna

Molec. gen. Genet. 143, 167- 175 (1976) OIGG © by Springer-Verlag 1976

In vitro Transcription of Adenovirus 2 DNA I. Characterization of Promoters for E. coli RNA Polymerase

S.J. Surzycki, J.A. Surzycki, and G.N. Gussin

Departments of Botany and Zoology, The University of Iowa, Iowa City, Iowa 52242, USA

Summary. E. coli RNA polymerase holoenzyme is able to recognize transcription initiation sites on Adenovi- rus 2 DNA that are functionally indistinguishable from promoters for the enzyme on phage DNAs. The complexes formed between the polymerase and the DNA at these sites can exist in two states- either as I (initiation) complexes, from which rapid RNA chain initiation is not possible, or as RS (rapid starting) "rifampicin resistant" complexes, from which rapid RNA chain initiation can occur. When transcription is limited to that initiated from stable, rifampicin-resistant pre-initiation complexes, initiation is strictly dependent on the presence of sigma factor; in addition, the frequency of initiation exhibits sigmoidal dependence on the temperature at which pre-initiation complexes are allowed to form, with a transition temperature of 26-28 ° C. The average half-time for initiation of RNA chains from sites on Ad 2 DNA is shown to be comparable to half-times for initiation of RNA chains from promoters on T7 and 2 DNAs. At saturating levels of enzyme, the half-times are 0.6, 0.9, and 1.6 sec for 2 b2, Ad 2 and T7 DNAs, respectively.

The existence of efficient, phage-like promoters for E. coli RNA polymerase on Ad 2 DNA suggests to us that such promoters may be closely related functionally and spatially to promoters for mammalian RNA polymerases.

Introduction

A major mechanism for the regulation of gene expression consists of the selection of specific sites (promoters) at which transcription may be initiated. The initiation specificity of E. coli RNA polymerase is dependent on the presence of sigma protein (Burgess et al., 1969), which preferentially enhances transcription initiated from stable complexes (Hinkle and Chamberlin, 1972a and b) between polymerase holoen-

zyme (core enzyme plus sigma factor) and template DNA. Detailed studies of transcription of phage T7 DNA have demonstrated that initiation is a three-step process: (1) RNA polymerase holoenzyme binds stably to specific sites on T7 DNA, forming " I " (initiation) complexes (Mangel and Chamberlin, 1974a, b, and c); (2) " I " complexes are converted spontaneously into "RS" (rapid starting) complexes, perhaps by denaturation of a short stretch of DNA at the initiation site (see Chamberlin, 1974); (3) in the presence of ribonucleoside triphosphates, each RS complex cata- lyzes the rapid formation of the first diester bond in a newly-synthesized RNA chain (Mangel and Cham- berlin, 1974a).

Correspondingly detailed information about the initiation of transcription in eucaryotic systems is lac- king. In preparation for studies of transcription of a well-defined eucaryotic template by mammalian RNA polymerases, we have investigated the interac- tion of E. coli RNA polymerase with Adenovirus 2 (Ad 2) DNA. The coli enzyme previously has been shown to recognize specific strong initiation sites in animal viral DNAs (Westphal, Delius and Mulder, 1973; Zain etal., 1973; Allet etal., 1974), although in no case has it been proved that the transcription of viral DNA by E. coli RNA polymerase accurately reflects viral transcription in vivo.

In the experiments reported here, we confined our attention to transcription of Ad2 DNA from stable, rapidly-starting, rifampicin-resistant 1 pre-initiation complexes (Bautz and Bautz, 1970; Sippel and Hart- mann, 1970; Chamberlin and Ring, 1972; Mangel and Chamberlin, 1974a). Studies using phage DNAs indicate that transcription from such complexes is generally restricted to that initiated at known pro-

Mangel and Chamberlin (1974a) point out that pre-initiation complexes able to initiate transcription in the presence of rifampicin are not "rifampicin-resistant ", although they are much less sensitive to the drug than are free polymerase molecules or polymerase molecules bound to Type B binding sites. However, because of previous usage of the term "rifampicin-resistant", we use this term and the more precise term ° ' rapid-starting" interchangeably.

168 S.J. Surzycki et al. : Promoters for E. coli RNA Polymerase on Adenovirus 2 DNA

m o t e r s ( W u et al., 1972 ; H ink l e , M a n g e l a n d C h a m b e r -

lin, 1972; Del ius , W e s t p h a l a n d A x e l r o d , 1973). W e

h a v e f o u n d tha t r i f amp ic in - r e s i s t an t , r ap id ly - s t a r t i ng

p r e - i n i t i a t i o n c o m p l e x e s f o r m e d b e t w e e n R N A poly-

m e r a s e h o l o e n z y m e a n d A d 2 D N A are func t i ona l l y

s imi la r to t hose f o r m e d b e t w e e n the e n z y m e a n d

p h a g e D N A s in several respects .

F i r s t , the f o r m a t i o n o f s table p r e - i n i t i a t i o n c o m -

p lexes is s i g m a - d e p e n d e n t ; in the absence o f s igma

fac to r , all t r a n s c r i p t i o n o f A d 2 D N A by co re e n z y m e

is b l o c k e d by r i f ampic in . Second , t he level o f t r an -

s c r ip t ion f r o m these c o m p l e x e s exhib i t s cha rac t e r i s t i c

s i g m o i d a l d e p e n d e n c e o n the t e m p e r a t u r e o f pre-

i n c u b a t i o n (Zi l l ig etal . , 1970); Bau tz , B a u t z a n d

Beck, 1972; M a n g e l a n d C h a m b e r l i n , 1974c) ; fo r

A d 2 D N A , T m ( the t r a n s i t i o n t e m p e r a t u r e ) a n d Tmax

a re 2 6 - 2 8 ° a n d 3 8 - 4 0 ° C , respec t ive ly , Th i rd , the

ra te o f i n i t i a t i on f r o m R S c o m p l e x e s b e t w e e n col i

R N A p o l y m e r a s e a n d A d 2 D N A is c o m p a r a b l e wi th

ra tes o f i n i t i a t i on f r o m c o r r e s p o n d i n g c o m p l e x e s w i t h T7 D N A ( M a n g e l and C h a m b e r l i n , 1974a), P M 2

D N A ( R i c h a r d s o n , 1975), a n d 2 D N A (resul ts r e p o r t e d

here) .

by neutral and alkaline sucrose gradient centrifugation and, in some cases, electron microscopic examination, to be unit length and free of single-strand breaks (about one single-strand break/2 x 107 daltons).

In vitro transcription

Unless otherwise indicated, reaction mixtures contained 30 mM Tris-HC1 (pH 7.9); 10raM MgC12; 50mM KC1; 0.4mM KH2PO4 (pH 7,5); 0.6raM ATP, GTP, and CTP; 0.096ram 3H-UTP (spec. act. 0.4Ci/mmole); 500gg/ml bovine serum albumin (Miles Laboratories, RNAase free); 0.1raM Dithiothreitol; 6gg/ml of purified DNA; and 24 units/ml of glycerol-gradient purified E. coli RNA pelymerase (Burgess, 1969). Polymerase preparations used in these experiments had sigma-to-core ratios of about 0.8 and specific activities of about 300-1 000 Burgess units/mg protein.

For the formation of pre-initiation complexes, enzyme was incubated with DNA for 15 rain at 25 ° C (or other temperatures where indicated) ; RNA synthesis was initiated with the addition of nucleoside triphosphates at time zero and reactions were allowed to proceed at 37 ° C. When indicated, rifampicin was added together with nucleoside triphosphates. Reactions were terminated by the addition of 3 ml of cold 3.5 per cent perchloric acid (PCA). For time course reactions, 50 gl aliquots were withdrawn at indicated times and precipitated with 3 ml of 3.5 per cent PCA. Precipitates were col- lected on glass fiber filters, washed five times with 5 ml of cold 1N HC1 containing 0.1 M Na,P2OT, dried and then assayed for radioactivity in a toluene-based scintillation fluid in a Beckman scinti!lation counter.

Materials and Methods

Growth and Purification of Virus

Adenovirus 2 was grown in spinner cultures of KB cells according to procedures described by Green and Pina (1963) and Green et al. (1967). KB cells and Adenovirus 2 were obtained initially from M. Stinski and H. Raskas, respectively. Bacteriophage T7 was grown in 2 - 4 liter liquid cultures after infection of E. coli strain B according to standard procedures (Davis and Hyman, 1971). 2b2 was grown in multiple plate lysates on infection of E. coli C600. Atl phage and viral preparations were ultimately purified by centrifuga, tion in CsC1 density gradients. Ad 2 was obtained in a pure band of CsC1 (average density 1.34 g/ml) after centrifugation for 21 hours at 30,000 rpm in a Beckman 65 angle rotor. T7 and 2 were obtained after centrifugation in CsC1 (average density 1.50 g/ml) in an SW40 and SW50 swinging bucket rotor for 21-24 hours at 35 - 40,000 rpm. Purified adenovirus was stored at 4 ° after dialysis against Buffer I (0.02 M Tris-HC1, pH 8; 0.15 M NaC1; 0.5raM EDTA). Bacteriophages were stored in CsC1 containing either "T7 Buffer" (0.05M Tris, pH 8.5; 1.0M KC1; lmM EDTA) or "2 dil" (0.01 M Tris, pH 7.0; 0.01 M MgSO4).

Purification of Viral DNAs

Purified virus at a concentration of 15 A260 units per ml was dialyzed against Buffer I. Pronase (predigested for 2 hr at 37 ° C) was added to the dialyzed virus suspension to a final concentration of 100 gg/ ml, followed by the addition of SDS to a final concentration of 0.2 per cent, and then EDTA (sodium salt) to a final concentration of 0.01 M. The mixture was incubated for 1 hr at 50 ° C and then was twice extracted with double-distilled phenol saturated immedi- ately before use with 0.1 M sodium borate. The aqueous fractions were combined and twice extracted with chloroform-isoamyl alcohol (25:1), then dialyzed extensively against Buffer I. The DNA was concentrated with Aquacide II (Calbiochem) and stored over chloroform at 4 ° C. DNA used in transcription experiments was shown

Determination of kz and k2/k*

For the determination of kz/k*, reaction mixtures were modified slightly to conform more nearly to the conditions of Mangel and Chamberlin (1974a) and Hinkle et al. (1972) by changing the substrate concentrations to 0.4 mM for all four nucleoside triphosphates. A series of reaction mixtures were pre-incubated for 15 min at 37 ° C, and then incorporation was allowed to proceed for 90 see at 37 ° following simultaneous addition of rifampicin and substrates.

To determine k2, polymerase and DNA were pre-incubated at 37 ° C in a series of incubation mixtures such as those described above. At time zero, rifampicin was added to varying final concentrations either with simultaneous addition of substrates or with substrate addition after varying times of incubation in the presence of the drug. Incorporation was then allowed to proceed for 5 min at 37 ° prior to precipitation with PCA.

Materials

3H-UTP was purchased either from International Chemical Nuclear Corp., Irvine, California, or from Nuclear Dynamics, El Monte, California. In some experiments c~-32p-ATP was used in place of 3H-UTP as radioactive substrate. Rifampicin was purchased from Schwarz-Mann, New York; for the calculation of rifampicin concentrations, the molecular weight was taken to be 823 daltons.

Results

Transcription o f A d 2 D N A by E. coli R N A Polymerase is Sigma-dependent

Burgess et al. (1969) r e p o r t e d t h a t the p r e sence o f

s i gma p r o t e i n in a t r a n s c r i p t i o n r e a c t i o n c a t a l y z e d by E. coli R N A p o l y m e r a s e m a r k e d l y s t i m u l a t e d t r an - s c r ip t i on o f p h a g e D N A s , bu t s t i m u l a t e d t r a n s c r i p t i o n

S.J. Surzycki et al. : Promoters for E. coli RNA Polymerase on Adenovirus 2 DNA 169

Table 1. Dependence of 3H-UMP incorporation on sigma factor

Templa te Incorporation of 3H-UMP (cpm) Ratio" DNA

core enzyme core + sigma

E. coli 126 1,524 12.1 )~b2 1,985 20,082 10.1 Ad 2 21,808 277,512 12.7 T4 660 13,789 20.9 C. reinhardtii 11,888 20,321 1.7

nuclear DNA

Incorporation was allowed to proceed 20min at 37°C in the absence of rifampicin. Each 0.1 ml reaction mix contained 0.3 ~tg of the indicated DNA and 1.2 units of RNA polymerase core enzyme with or without saturating levels of sigma protein.

Ratio of cpm incorporated in the presence of sigma to cpm incorporated in the absence of sigma for this experiment.

10 2'0 30 TIM E('min)

Fig. 1. Effect of G on rifampicin-resistant pre-initiation complexes between E. coli RNA polymerase and Ad 2 DNA. Incorporation of 3H-UMP proceeded for the indicated times at 37 ° C in the presence ofrifampicin (7 txg/ml) after pre-incubation for l 5 min at 25 ° C. Transcription was catalyzed by saturating levels of reconstituted E. coli RNA polymerase holoenzyme (o-o-e) or by an equivalent concentration of core enzyme alone (o-©-©)

of calf thymus DNA to a much lesser extent. Similar experiments designed to test the role of sigma in transcription of Ad 2 DNA are summarized in Table 1. In these experiments, the level of transcription of E. coli, 2b2, and Ad 2 DNAs was 1 0 - 1 2 times as great in the presence of sigma as in its absence, and with T4 DNA as template, the level of transcription was stimulated more than 20-fold by the addition of sigma. In contrast, transcription of nuclear DNA from Chla- mydomonas reinhardtii, like that reported for calf thymus DNA (Burgess et al., 1969), was stimulated only two-fold by sigma protein. These results suggest that sigma may be essential for the recognition of initiation sites on Ad2 DNA by the coli enzyme, and that these sites may be similar to those found in phage DNAs.

A better test of the specificity of initiation sites for E. coli R NA polymerase is to restrict transcription to that catalyzed by rifampicin-resistant pre-initiation

complexes (Bautz and Bautz, 1970; Sippel and Hart- mann, 1970). RNA polymerase holoenzyme is allowed to form pre-initiation complexes with D N A during an incubation period in the absence of nucleoside triphosphates. At the end of this incubation period, rifampicin and triphosphates are added simultaneously, permit- ting transcription from those complexes that initiate rapidly enough to escape the action of the drug (Man- gel and Chamberlin, 1974a). Under these conditions transcription is usually restricted to that initiated from known promoters (Wu et al., 1972; Delius et al., 1973; Hinkle et al., 1972).

When Ad 2 D N A and RNA polymerase were pre- incubated for 15 min at 37 ° C prior to the addition of substrates, the ability to initiate transcription in the presence of rifampicin (7 gg/ml) was strictly dependent on the presence of sigma protein (Fig. 1). As has been observed for phage D N A templates, transcription catalyzed by saturating amounts of sigma protein plus core polymerase increased continuously for 1 0 - 1 5 minutes at 37 ° before reaching a plateau (due to the inhibitory effect of rifampicin on re-initiation). In contrast, incorporation of 3H-UMP into RNA was negligible in the absence of sigma protein, never exceeding 5 per cent of the plateau value attained in the presence of sigma. The ability of Ad 2 DNA and E. coli RNA polymerase to form sigma-dependent, rifampicin-resistant pre-initiation complexes is a further indication that transcription was initiated from phage-like promoters on Ad 2 DNA.

Comparison o f Transition Temperatures for A d 2, T7, and 2b2 D N A s

The level of incorporation from rifampicin-resistant pre-initiation complexes between phage DNAs and coli R N A polymerase exhibits sigmoidal dependence on the temperature of pre-incubation (Zillig et al., 1970; Bautz et al., 1972). According to the model of Mangel and Chamberlin (1974c), the temperature-dependent step is the conversion of " I " complexes to " R S " complexes. Variations in the transition temperature (Travers, 1974) that is, in the " Tm" established from a sigmoidal curve, presumably reflect differences in the nucleotide sequences of promoters from which transcription is initiated in the presence of rifampicin.

Fig. 2 illustrates the sigmoidal dependence of incorporation on temperature for Ad 2, 2b2, and T7 DNAs. In several such experiments, the observed transition temperatures ranged from 2 2 - 2 3 ° C, 2 6 - 2 8 °, and 30 - 32 °, for T7, Ad 2, and 2b2, respectively. These data demonstrate a significant difference between transition temperatures for individual promoters on T7 and 2b2 DNAs. Presumably, the intermediate transi-

100

I

80

e .a

240 o

Z

~2


3'0 ' 40 10 20 TEMPERATURE

Fig. 2. Effect of temperature of pre-incubation on transcription from rifampicin-resistant pre-initiation complexes. After pre-incu- bating DNA and saturating levels of RNA polymerase holoenzyme in the absence ofnucleoside triphosphates for 15 rain at the indicated temperatures, incorporation was allowed to proceed at 37 ° C for 45 minutes in the presence of rifampicin (7 lag/ml). T7 DNA ( • - - • ) ; Ad2 DNA ( • - - o ) ; 2b2 DNA (o - -o )

tion temperature for Ad 2 DNA actually represents an average of the transition temperatures of several promoters; in fact, experiments described in the accompanying paper (Surzycki et al., 1975) indicate that Ad 2 DNA possesses several promoters for E. coli RNA polymerase, 2 - 3 which have low transition temperatures characteristic of T7 DNA, and 2 - 3 which have higher transition temperatures characteristic of ;~ DNA.

Rate of Initiation from Rapid-starting Pre-initiation Complexes

Mangel and Chamberlin (1974a) demonstrated that, although stable pre-initiation complexes are 100-fold less sensitive to rifampicin than are free polymerase molecules or polymerase molecules bound at unstable sites, I and RS complexes are nevertheless sensitive to the drug. However, once initiation of RNA chain synthesis has occurred, transcription complexes are completely insensitive to rifampicin. Therefore, it is possible to monitor initiation of transcription in the presence of rifampicin to determine the half-time for initiation of RNA chains.

Experimentally, it is necessary to determine k2, the second-order rate constant for the binding of rifampicin to a stable pre-initiation complex, and k2/k*, in which k* is an apparent first-order rate constant

for RNA chain initiation at a particular substrate concentration (Mangel and Chamberlin, 1974a). The expression k2/k* is a measure of the competition between the binding of rifampicin to an RS complex and the formation of the first phosphodiester bond in newly synthesized RNA chains. The relationship between in vitro transcription and rifampicin concentration when triphosphates and rifampicin are added simultaneously to an incubation mixture containing RS complexes has been derived by Mangel and Chamberlin (1974a) and has been shown to satisfy the following equation:

Co k2 C = k * ( R ) + I (1)

where (R) is the rifampicin concentration, Co is the concentration of polymerase molecules that initiate in the absence of rifampicin, and C is the concentration of polymerase molecules that initiate at a particular concentration of the drug.

Thus, to determine k2/k*, rifampicin at varying concentrations and ribonucleoside triphosphates are added simultaneously to pre-initiation complexes at zero time and then incorporation is allowed to proceed for 90 sec at 37 ° C. k2/k* is the slope of the plot of relative incorporation (cpm incorporated in the absence of rifampicin divided by cpm incorporated in the presence of rifampicin) as a function of rifampicin concentration 2. Representative curves for the inhibition of incorporation when Ad 2, T7, and 2b2 DNAs are used as template are illustrated in Fig. 3, and average values for the slopes determined in three experiments for each DNA are indicated in Table 2. Although the determined values are similar: 3.7, 3.8, and 1.9 x 103 M- 1 for Ad 2, T7, and 2b2 DNAs, respectively, the observed values for Ad 2 and T7 DNAs differ reproducibly from that determined for )~b2 DNA.

2 Assumptions required to derive Eq. (1) as well as assumptions on which the experimental determination of ka/k* is based are dis- cussed in detail by Mangel and Chamberlin (1974a). Among these assumptions are (a) that the binding of nucleoside triphosphates and of rifampicin to RS complexes are irreversible processes, and (b) that total incorporation in 90 sec at 37 ° is a reliable measure of initiation frequency. Electron microscopic observations of rates of transcription by individual transcription complexes (Dodds, Sur- zycki and Gussin, 1975) suggest that the second assumption is valid for Ad 2. Graphs in Figure 3 differ in two ways from the theoretical formulation. First, relative incorporation does not extrapolate to 1.0 at zero rifampicin concentration, and second, an initial steep slope is observed at very low rifampicin concentrations (some initial points were omitted from Fig. 3b and 3c for clarity). These effects were noted by Mangel and Chamberlin (1974a). The first is probably due to initiation by core enzyme alone in the absence of rifampicin, but not in its presence. The second effect may be due to re-initiation of transcription in the control reaction (no rifampicin), which would produce an inflated estimate of the number of initiations that poten- tially could occur from RS complexes in the presence of rifampicin.


3.8

3.4

3.0

2.6

2.2

1.8

3.0

2.6

2.2

" ~ t .8 '

2.6

2.2

1.8

14 I

0 ' 120' ' 240 ' 360 ' 4gO Rifampiein eoneentration ( ~ g )

Fig. 3. Determination of k2/k*. Relative RNA polymerase activity (cpm 3H-UMP incorporated in the absence of rifampicin divided by cpm incorporated at the indicated rifampicin concentration) is plotted as a function of the rifampicin concentration. Slopes were determined analytically by linear regression; in each case, points obtained at rifampicin concentrations less than 10 pg/ml were omitted from the regression analysis because of their obvious deviation from linearity (see Footnote 2). Except for Fig. 3a, above, these points were also omitted from the Figures, for clarity. For the particular experiments shown, the following values of k2/Ic* were obtained: (a) Ad 2 DNA, kz/k*=3.24x 103M 1. (b) 2b2 DNA, kz/k*=2.07x 103 M -1. (c) T7 DNA, k2/k*=3.27x 103 M 1

Table 2. Values of k2/k* determined for Ad 2, )~b2, and T7 DNA

Template kz/k* (x 10 -3) (k2/k*)ave." ( x 10 3) DNA

2.07 M - 1 262 1.88 M -1 1.87_+0.17 M 1

1.7I M 1

3.27 M - 1 T7 3.98 M -1 3.83_+0.50 M 1

4.24 M 1

3.24 M 1 Ad 2 3.79 M -~ 3.71 20.44 M - t

4 .10M -1

a Average of three experiments _+S.D.

(k2/k*) was determined at saturating levels of enzyme as described in Materials and Methods.

In order to determine k2, RNA polymerase and DNA are incubated in a series of reaction mixtures for 15 rain at 37 ° and then rifampicin is added at time zero. The addition of ribonucleoside triphosphates at varying times thereafter permits transcription to be initiated from those complexes with which the drug has not yet interacted (Hinkle et al., 1972). Fig. 4 illustrates the results of one such experiment in which Ad 2 DNA was used as template. The slope of each plot of log [cpm incorporated] vs. time of addition of substrates was determined by linear regression analysis, and used to calculate observed constants (kobs) for each concentration of rifampicin, according to the equation

kob s =lnel0 x (slope).

Valus Ofkob s were then plotted as a function of rifampicin concentration to determine kz as is shown in Fig. 5. The values of k2 determined in this way are 2.8 x 103 M- lsec- 1 forAd 2 DNA, 2.0 x 103 M- as•c- 1 for 2b2 DNA, and 1.6 x 103 M- tsec- 1 for T7 DNA.

Fig. 4. Kinetics of inactivation of RNA polymerase holoenzyme- A d 2 DNA complexes by rifampicin. For each concentration of rifampicin, the per cent incorporation during five minutes at 37 ° C is plotted logarithmically as a function of time between addition of rifampicin and the addition of nucleoside triphosphates. Lines drawn in the figure are only visual approximations to the best fit, with 100 per cent defined as the experimental value obtained at zero time for each rifampicin concentration. Actual values for the slopes of the lines were determined analytically by linear regression, to yield values of ]Cobs, and the y-intercept obtained analytically frequently differed from 100 per cent. The rifampicin concentrations used were: ( • - • ) 0.6 pM ; ( © - ©) 1.2 ~tM; (A - zx) 4.9 gM ; ( • - • ) 9 .7pM; ( D - [ ] ) 12.1 ~tM

100

50 O

o 2 0

z

I 3O

I _ 1 60 90

T ime (see)

[

120 150

172 s.J. Surzycki et al. : Promoters for E. coli RNA Polymerase on Adenovirus 2 DNA

Values for k* can be obtained by dividing k 2 by k2/k*, and Eq. (2)

lne2/k* : initiat&n half- t ime (2)

30 a o

20

I I I

b ~ / ~

11]

5 10 1'5 Rifampicin concentration (IJM)

10 O m

o

2~

20

Fig. 5. Second order rate constant (k2) for rifampicin attack on RS complexes. Values for kob s determined as demonstrated in Fig. 4 were plotted as a function of rifampicin concentration; the slopes and intercepts of each line were determined by linear regression analyses, with the slope equal to k2. Each analysis combined points from two separate experiments (open and closed symbols). Symbols in brackets indicate that data were insufficient to determine kob s with precision. (a) Ad2 DNA; (b) 2b2 DNA (© or o); T7 DNA (A or *)

Table 3. Determination of half-times for initiation of RNA chains

Tern- k2/k* k2 a k* RNA ini- plate (x 10 -3) (x 10 3) tiation DNA half-time

2b2 1.87 M 1 2.0 M 1 sec-1 1.1 sec -1 0.65 sec T7 3.83 M- 1 1.6 M- 1 sec- i 0.42 sec- 1 1.6 sec Ad2 3 . 7 1 M - 1 2.8 M -1 sec -a 0.75sec 1 0.92sec

a Values of k2 were determined at saturating levels of enzyme as indicated in Materials and Methods. Standard errors of the regression coefficients, determined from the data illustrated in Fig. 5, were used to calculate 96% confidence limits for values of k2, which were : )~b2, _+0.85; T7, _+0.82; Ad2, ±0.46.

can then be used to calculate the half-time for initiation of R N A chains from promoters on each of the three D N A templates. The values obta ined-0 .6 , 0.9, and 1.6 sec for 2b2, Ad 2, and T7, respectively (Table 3 ) - indicate that, on the average, promoters for E. coli R N A polymerase on Ad 2 D N A are about as efficient at initiating R N A chains from RS complexes as are phage promoters.

For reasons mentioned in the Discussion, the experiments summarized in Table 3 were performed under conditions of enzyme saturation (1.2 units of enzyme, 0.3 btg of D N A per 50 btl reaction m i x t u r e - a molar ratio of 2 0 0 - 4 0 0 enzyme molecules per D N A molecule depending on the polymerase preparation). On the other hand, Mangel and Chamberlin (1974a) determined the initiation half-time for T7 D N A at an enzyme-to-DNA ratio of about 3 : 1, and obtained a value for the half-life of 0.23 sec. To assure ourselves that the discrepancy between this value and that which we had determined for T7 D N A was merely a result of the difference in enzyme-to-DNA ratio used, we repeated the T7 experiments at a lower ratio of enzyme- to -DNA (0.08 units of enzyme, 5 i~g of T7 D N A in 50 gl - a molar ratio of2.4:1 for the particular enzyme preparation used). Under these conditions, k2 was 4.3 x 10 3 M - 1 sec- 1, k 2 / k , was 2.0 x 10 3 M - 1, and the calculated half-time for initiation was about 0.3 sec, which agrees with the value reported by Man- gel and Chamberlin. Possible reasons for the difference in initiation half-time at different ratios of enzyme-to- D N A will be considered in the Discussion.

Discussion

Our results demonstrate that E. coli R N A polymerase can form stable, rifampicin-resistant pre-initiation complexes with a well-defined eucaryotic template, the D N A of human Adenovirus 2. Characterization of the in vitro transcription reaction has shown that pre- initiation complexes between the coli enzyme and Ad 2 D N A are remarkably similar to complexes formed between the enzyme and phage DNAs. The formation of such complexes is sigma-dependent (Fig. 1), the frequency of initiation in the presence of rifampicin depends on the temperature of pre-incubation (Fig. 2), with a transition temperature within the range of transition temperatures observed for phage DNAs, and the average half-time for formation of the first phosphodiester bond in new R N A chains is similar to half- times determined under identical conditions using 2b2 and T7 D N A as template (Table 3).

In order to determine the average half-time for the initiation of R N A chains, it was necessary to calculate k2, the second-order rate constant for the binding


of rifampicin to the polymerase-DNA pre-initiation complex, and k2/k*, and expression reflecting the competition between initiation of RNA chains and rifampicin binding to pre-initiation complexes. Our methods were essentially the same as those used by Mangel and Chamberlin (1974a) with one important difference. In their experiments, Mangel and Chamberlin used an enzyme-to-DNA ratio of about three, a value corresponding to the number of stable (Type A) binding sites for E. coli RNA polymerase holoenzyme on T7 DNA (Hinkle et al., 1972). At this ratio of enzyme to DNA, the average half-time for initiation is weighted to reflect the half-time(s) for initiation from the strongest binding (and possibly most rapidly initiating) promoters.

In the experiments summarized in Table 3, k2 and lc2/k* were determined at saturating enzyme-to-DNA ratios. This method has the virtue that an experimentally-determined half-time should be an unweighted average of half-times for all stable pre-initiation complexes formed, including those formed at weaker binding sites. Experiments at saturating enzyme levels also require much less DNA, an important consideration in the case of Ad2 DNA. However, complications may arise at these high enzyme-to-DNA ratios. First, at high enzyme concentrations, several polymerase molecules might bind and initiate sequentially from a single promoter. Multiple initiations necessarily would increase the average half-time determined experimentally since such initiations could not occur simultaneously. In addition, T7 DNA contains three closely- spaced promoters at one end of the molecule (Minkley and Pribnow, 1973). It is conceivable that polymerase molecules bound at these promoters could also inter- fere with each other and delay initiation. Finally, the existence of a large number of weak (Type B) binding sites (Hinkle et al., 1972) whether for core enzyme or for holoenzyme could also lead to an increased estima- tion of the average half-time; for example, if a statistically small fraction of polymerase molecules bound to Type B sites escaped inhibition by rifampicin they could contribute significantly to the average half-time.

Thus, it is likely that half-times determined at low enzyme-to-DNA ratios represent half-times for only the strongest binding promoters, while half-times determined at high enzyme-to-DNA ratios represent upper limits for the average half-time for all promoters on a particular DNA molecule. Previously, half-times for initiation of RNA chains by E. coli RNA polymerase have been reported only for T7 DNA (by Mangel and Chamberlin), and for Pseudomonas phage PM 2 DNA. At an enzyme-to-DNA ratio of about 2:1, Richardson (1975) obtained a half-time of 1.2 sec for PM 2 DNA in either the superhelical or the relaxed form. If this value is corrected to take into account

the low concentration of nucleoside triphosphates and lower salt concentration used (see Mangel and Cham- berlin, 1974b), then the initiation half-time would be about 0 . 3 - 0.4 sec (Richardson, pers. comm.). There- fore, for both PM 2 and T7, initiation half-times at low enzyme-to-DNA ratios were lower than those we have obtained (Table 3) for three different DNAs at saturating enzyme levels.

Although these considerations do not affect our basic conclusion- that Ad 2 DNA contains promoters for E. coli RNA polymerase that permit very rapid initiation of RNA chains (initiation half-time of 0.9 sec or less), it is important to consider the extent to which half-times for 2b2 and Ad 2 DNA might be inflated. 2b2 DNA was used as template because it contains a 13 per cent deletion that removes initiation sites of unknown specificity contained in the wild-type b2 region (Roberts, 1969). Thus transcription of 2b2 DNA should be initiated primarily at the right and left promoters, PR and PL (Roberts, 1969 ; Wu et al., 1972; Blattner and Dahlberg, 1972). There is one re- port that multiple, sequential initiations may occur at PL (Willmund and Kneser, 1973), but sequence determination and polymerase binding studies (Maniatis, Jeffrey and Kleid, 1975; Pirrotta, 1975; Walz and Pirrotta, 1975) indicate that there are not multiple RNA polymerase binding sites within either PL orpR. In the case of Ad 2, there is no direct evidence. However, in the accompanying paper (Surzycki et al., 1975), we discuss suggestive evidence that transcription of Ad 2 DNA is initiated at several discrete sites each of which binds a single RNA polymerase molecule. If these conclusions are correct, then the half-times determined for 2b2 and Ad 2 DNA may be much less affected by differences in enzyme-to-DNA ratios than is the half-time for TT. (The effect could only be 2 - 3 fold in any case).

A priori, it may have been assumed that k2 should be affected neither by the enzyme-to-DNA ratio nor by the nature of the template DNA. However, our values of k2 for T7 DNA are appreciably different at the two enzyme-to-DNA ratios u s e d - l . 6 and 4.3 x 103 M -1 sec -1 at high and low ratios, respectively. Although it is clear that the distribution of sites from which initiation occurs at the two enzyme-to- DNA ratios may differ, it is difficult to see how this" could affect k2 so dramatically.

We can also consider whether the differences in the values of k2 determined for T7, )~b2, and Ad 2 DNAs are significant. Based on standard errors of the regression coefficients for the lines drawn in Fig. 5, values of k2 for 2b2 and T7 DNA are not statistically distinguishable, but both values differ significantly (p < 0.05) from that obtained for Ad 2 DNA. Richard- son (pers. comm.) also finds a reproducible difference


between k 2 for PM2 DNA (Richardson, 1975) and his unpublished values for T7 DNA. Apparently the rate constant for binding of rifampicin to an RS complex can vary depending on the nature of the complex and perhaps on the nucleotide sequence of the promoter involved.

Though data for k2 obtained in separate experiments with T7 and Ad 2 DNA agree quite well, consis- tently accurate data for ,~b2 D N A have been difficult to obtain. Note particularly that in the case of )~b2 DNA (Fig. 5b), the y-intercept is significantly different from zero, indicating that even in the absence of rifampicin, there is inactivation of RS complexes during incubation at 37 ° C. We have no explanation for this result.

In spite of these complications, it is clear that Ad 2 DNA possesses strong promoters for transcription by E. coli RNA polymerase-promoters that are functionally equivalent to those found in phage DNAs. Previously, Allet et al. (1974) demonstrated that at least one promoter for E. coli RNA polymerase on Ad 2 DNA may contain a hexanucleotide sequence in common with known promoters on coliphage DNAs. It is attractive to speculate that this possible sequence similarity and the functional similarity that we have demonstrated are not accidental. Rather, promoters for E. coli RNA polymerase may be found in Ad 2 DNA because they are actually closely related or identical to promoters for mammalian RNA polymerases.

Acknowledgements. We are grateful to H. Raskas and M, Stinski for gifts ofAdenovirus 2 and KB cells, respectively, to J.P. Richard- son for a gift of T7 and for comments on the manuscript, and to J. Dodds who helped with some experiments. C. Newlon and J. Menninger also provided comments on the manuscript and J. Hegmann provided statistical advice. This work was supported by funds from the NIAID and from the Damon Runyon Memorial Fund.

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Communicated by E. Bautz

Received August 12, 1975

in vitro transcription of adenovirus 2 dna

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