Differential effects of mutations on discrete steps in transcription initiation at the λ PRE promoter

Cell, Vol. 34, 941-949,

October

1983, Copyright

0 1983 by MIT

00928674/83/100941-09

$02.00/O

Differential Effects of Mutations on Discrete Steps in Transcription Initiation at the X PREPromoter Ming-Che Shih and Gary N. Gussin Genetics Ph.D. Program and Department University of Iowa Iowa City, Iowa 52242

of Zoology

Summary The effects of cy mutations on transcription initiation at the X PREpromoter were determined using abortive initiation analysis (McClure, 1980). In the presence of X c/l protein, which activates PRE, three mutations in the -10 region dramatically reduce k2, the forward rate constant for the isomerization of closed to open complexes, but only slightly affect K& the equilibrium constant for the initial recognition by RNA polymerase to form closed complexes. In contrast, five -35 region mutations caused decreases of 30 to 150 times in Ke with much smaller effects on k2. In the absence of c/l protein, the effects of mutations in the -10 region are qualitatively similar to those observed in the presence of c/l protein, although the reductions in k2 are much less dramatic. In contrast, none of the mutants with defects in the -35 region is distinguishable from wildtype PREin the absence of c/l protein. Thus RNA polymerase may recognize different sequences in the -35 region in the absence of cl/ protein than in its presence. Introduction According to current models, there are two major sequence-specific steps in procaryotic promoter site selection (Chamberlin, 1974, 1976): one, binding of RNA polymerase holoenzyme to a promoter to form a closed complex (recognition step), and two, transition of the closed complex to an open complex (isomerization step). The second step is followed by rapid RNA chain initiation in the presence of ribonucleoside triphosphates (NTPs) (Mangel and Chamberlin, 1974a; McClure, 1980). Biochemical and mutational studies have revealed that two regions of E. coli promoters are especially important in transcription initiation (see Rosenberg and Court, 1979; Siebenlist et al., 1980). The -10 region includes the consensus hexanucleotide TATAAT, which is usually located from -6 to -12 (6 to 12 nucleotides preceding the transcription startpoint). The second region is located near -35 and includes the consensus sequence TTGACA. Both regions make close contacts with RNA polymerase holoenzyme, and in many promoters mutations in these two regions have been found to alter promoter function (Siebenlist et al., 1980; Youderian et al., 1982). The role of each region in transcription initiation can be analyzed by investigating the effects of specific mutations on promoter function in vitro. For example, abortive initiation assays (McClure, 1980) can be used to determine KS, the equilibrium constant for the formation of closed com-

plexes (step 1, above), and k2, the forward rate constant for the isomerization reaction (step 2, above). Such methods have been applied successfully to the study of wildtype and mutant derivatives of the promoters PRMand fR of bacteriophage X (Hawley and McClure, 1980, 1982; Shih and Gussin, 1983) as well as several other wild-type promoters (McClure et al., 1982). We apply the abortive initiation analysis to the h PRE promoter, which directs the synthesis of the cl repressor during the establishment of a lysogen and is subject to positive control by the X c/l protein (Reichardt and Kaiser, 1971; Echols and Green, 1971; Schmeissner et al., 1980). Mutations in PRE(Wulff et al., 1980, 1983) are clustered in the -35 and -10 regions, although neither region of fRE matches the corresponding consensus sequence very well (Figure 1). We have reported elsewhere that c/l protein activates PRE by enhancing k0 and Ks approximately 40-fold and 15fold, respectively (Shih and Gussin, submitted). Here we apply the abortive initition analysis to mutations that alter the interaction of RNA polymerase with PRE (Wulff et al., 1980). We find that, in the presence of c/l protein, mutations in the -10 region decrease k2 20 to 30 times while only slightly affecting Ke. In contrast, mutations in the -35 region decrease Ke 30 to 150 times with much smaller effects on k2. Surprisingly, in the absence of c/l protein, the effects of the mutations are much less dramatic. Mutations in the -10 region decrease kZ only 2 to 3 times (with no effect on KB), while mutations in the -35 region have virtually no effect on either k2 or KS. These results are discussed in terms of the mechanism of action of c/l protein and models for promoter recognition by E. coli RNA polymerase.

Results Abortive Initiation at PRE Transcription from PREcan initiate at either of two adjacent purines, A or G, in a ratio of about 6:4 (Shimatake and Rosenberg, 1981; see Figure 1). Analysis of the abortive initiation products synthesized in the presence of only ATP and GTP yielded similar results (Shih and Gussin, submitted). About 55% of the abortive initiation product was pppApGpA (initiation at A) and 45% was pppGpA (initiation at G), both in the presence and absence of c/l protein. We have chosen to limit transcription to initiation at the A startpoint by using the dinucleotide uridylyl-5’-adenosine (UpA) as substrate in place of ATP. In this case, the abortive initiation product is UpApG, with the U corresponding to the T at -1 in the DNA sequence (Figure 1). For wild-type PRE, the kinetics of initiation are the same whether or not initiation is limited in this way (Shih and Gussin, submitted). In the presence of substrates corresponding to the first two nucleotides in the transcript initiated at a particular promoter, RNA polymerase in open complexes catalyzes the repeated synthesis of the corresponding oligonucleotide (in this case, UpApG) without dissociating from the

Cell 942

67 CM-,0 thfst -*I “II” -40 S’-ATCTAAGGAAATACTTACA~~G~TCGTGCAAACAAACOCAACGAGGC~-3’ ACAGTT TAATAT GCTCCGA-5’

Figure 1. Genetic

Map and Nucleotide

Sequence

of the X fRE Region

The nucleotlde sequence of wild-type and mutant derivatives of PRE (Wulff et al.. 1960) is shown in relation to a limited portion of the h genetic map. A 1310 bp Hae Ill restriction fragment (bounded by vertical arrows) containing both frr and fmp (Shimatake and Rosenberg, 1961) was used as template for abortive initiation. The arrow below the sequence indicates the possible startpoints for transcription from PRE; the sequence is numbered relative to the A startpoint. Consensus sequences (Rosenberg and Court, 1979; Siebenlist et al., 1960) In the -10 and -35 regions are shown above the corresponding sequences for P,. The underlined 5’.TTGC-3’ sequences in the lower strand indicate sites of binding of c/l protein (Ho et al.. 1963), while the underlined ATG denotes the startpoint for rightward translation of the c/l gene. wild-type

template DNA. The rate of oligonucleotide synthesis at any time is proportional to the number of open complexes present (Johnston and McClure, 1976). Thus when RNA polymerase holoenzyme, promoter-containing DNA, and substrates for abortive initiation are added simultaneously to a reaction mixture at time zero, there is a noticeable lag period before UpApG synthesis reaches the steady-state rate. The lag period, 7&s, is the average time required for the formation of open complexes (McClure, 1980). For example, at an RNA polymerase concentration of 67 nM, the lag time for wild-type PRE in the presence of c/l protein is about 1.2 min (Figure 2). For the -35 region mutant ~~3075, the lag time is 17 min. On the other hand, if RNA polymerase and the DNA template are preincubated (in the presence of c/l protein) for several minutes to allow open complex formation prior to the addition of substrates, no lqg period is observed (Figure 2). For certain mutants in the presence of c/l protein, and for all mutants and wild-type PRE in the absence of c/l protein, the lag time is so long that it is difficult to measure accurately using the continuous assay illustrated in Figure 2. In these cases, a fixed-time assay (Hawley and McClure, 1982) is used, in which enzyme and DNA are incubated from time zero in the absence of substrates. At various times, aliquots are removed from the reaction mixture, substrates are added, and synthesis is allowed to proceed for a short time interval (6-10 min, as indicated). Synthesis of UpApG during the fixed-time interval provides a measure of the average rate (v) of synthesis during the interval. A plot of In (l-v/V) as a function of time yields a straight line with slope -I/T&* (Hawley and McClure, 1982). (V is the “maximal” rate of UpApG synthesis, measured after

2

4

6

8

10

Time (min) Figure 2. Continuous

Synthesis

of UpApG In the Presence

of c/l Protein

DNA template (1 nM) and purified c/l protein (6 Kg/ml) were incubated for IO min at 37°C prior to the addition of 67 nM RNA polymerase. To measure rob. (open symbols), UpA and o1-=P.GTP were added at the same time as RNA polymerase. In parallel experiments (closed symbols), substrates were added 10 min (wild type) or 90 min (~~3075) after RNA polymerase. At various times after the addition of substrates, allquots were removed and assayed for UpApG. Extrapolation to the abscissa (short dashed lines) indicates values for 7olrs of 1.2 min and 17 min for wild type (lower panel) and ~~3075 (upper panel), respectively.

incubation of enzyme and DNA for periods greater than 4 times Taco.) Examples of fixed-time assays are shown in Figure 3. At an RNA polymerase concentration of 100 nM, in the absence of cl/ protein, values of T,,~%were 75 and 134 min for wild-type PRE and the mutant cy3019, respectively.

Kinetic Parameters Measured in the Presence of c/l Protein To determine which of the steps in transcription initiation contribute to differences in robs, it is necessary to measure lag times at several concentrations of RNA polymerase. Under conditions used in these experiments, T,,~%is related to the initial RNA polymerase concentration (RNAP), by the equation (McClure, 1980):

1 1 ‘Ohs = k, + kzK8 (RNAP) where k2 is the forward rate constant for isomerization of closed to open complexes and KS is the equilibrium con-

Trarscription

Initiation at the h Pee Promoter

50

40

1 40

I 60

I

1

a0

Time (min) Figure 3. Fixed-trme Protein

Assays

of Abortive

Initiation in the Absence

I2 E

530

cy3075(-34,351

2 of c/l

RNA polymerase (100 nM) and DNA template (2 nM) were incubated at 37°C in the absence of substrates. At various times, atiquots were withdrawn, substrates were added and abortive initiation was allowed to proceed for 8 min. Data were plotted to fit the equation In (l-v/V) = -t/ TV. Calculated values of 7obs were 75 and 134 min for wild type (0) and cy3019 (0) respectively.

20

10

10

20

30

40

50

[RNAP]-V,W) stant for the initial binding reaction. Thus a plot of Tabs as a function of I/(RNAP) yields a straight line that extrapolates (at infinite polymerase concentration) to T = l/kZ; the slope of the “tau” plot, which equals l/k2KB, is then used to calculate KB.Tau plots for wild type and three mutant derivatives of PRE are presented in Figure 4. The mutants, which were characterized by Wulff et al. (1980) are classified either as -10 region or -35 region mutants. For the experiments reported here, we excluded mutants with defects in the TTGC sequences with which c/l protein makes close contacts (Ho et al., 1983; Figure 1). In the presence of c/l protein, the -10 and -35 region mutants fall into three classes with characteristic defects in open complex formation. First, all three -10 region mutations cause dramatic decreases in the forward rate constant for the isomerization reaction. For example, the mutation ~~3048 (mutation at -7) changes T from 1 .O min (the wild-type value) to 29 min (Figure 4). Although the slope of the tau plot for ~~3048 differs from the wild-type slope, this is primarily due to the fact that kF is altered. The calculated value of Ks for the mutant differs only slightly from the wild-type value (Table 1). Similar tau plots (not shown) were obtained for ~~2001 (mutation at -11) and ~~3019 (mutation at -14); again, the mutations alter 7 (and kZ) dramatically, but only slightly affect KS (Table 1). A second group of mutants, all of which are altered in the -35 region, are defective in both kZ and Ke. A tau plot for one such mutant, ~~3075 (containing a double mutation at -34/-35) reveals a change in T to 4.8 min (Figure 4). Even more dramatic is the effect of the mutation on the slope of the tau plot. This is reflected in the fact that the calculated value of Ke for ~~3075 is only about one 20th the wild-type value (Table 1). Similar results were obtained with three other -35 region mutants, cy844 (-33) and c//31 14 and c//31 09, both of which are altered at -32 (Table 1). (Note that the latter two mutants were isolated originally as cl/- mutants [Wulff et al., 1983; see Figure I]; the data

Frgure 4. Tau Plots for Wild-type Presence of cl/ Protein

and Mutant

Derivatives

of PRE in the

Values of TV obtained in experiments such as those illustrated in Figures 2 and 3 were plotted to fit equation (1). Linear regression analysis was used to determine the intercept (7 = 1 /k2) and slope (1 /k&J of each plot. Data from several experiments are included in each case. Values of 7 are 1.O, 1.6. 4.6, and 29 min for wild type (0) ~~3107 (A), ~~3075 (A), and ~~3046 (0) respectively.

in Table 1 are the first indication that they are also defective in PRE.) A fifth -35 region mutant, ~~3107, exhibits a third phenotype; KS is reduced 150 times but 7 and k2 are essentially unchanged (Figure 4; Table 1). These results are consistent with previous findings that -10 region mutations in other promoters appear to affect only k2, while -35 region mutations affect KB alone or both kS and Ke, but not kS alone (Hawley and McClure, 1980, 1982; Shih and Gussin, 1983).

Effects of PREMutations in the Absence of cl/ Protein Wild-type PRE is a much weaker promoter in the absence of c/l protein than in its presence (Shih and Gussin, submitted). In the absence of c/l protein, T increases from 1 .O to 42 min (Figure 5) corresponding to a value for k2 of 4.1 x lo-“ see-‘. In addition, KS is reduced by about a factor of 15 (Table 1). Representative tau plots for two mutants are also included in Figure 5. Surprisingly, the tau plot for the -35 region mutant, ~~844, is almost identical to that of the wild-type promoter as are the tau plots (not shown) for the four other -35 region mutants listed in Table 1. Thus in comparison to the wild-type promoter, promoters with mutations in the -35 region are not defective in the absence of c/l protein. Furthermore, the effects of mutations in the -10 region are not as dramatic in the absence of c/l protein as in its

Cell 944

Table 1. Kinetic Parameters

for Wild-type

and Mutant Derivatives

of PFS

c/l Protein Absent PIE allele Wild-type cy3048

(-7)

c/l Protein Present

7. min

ka set-’

KB, M-1

T, min

k xc-

KEG M-1

42

4.0 x 10-4

1.0 x IO7

1.0

1.6 x IO-*

1.7 x 108

89

1.9 x 10-4

1.3 x 10’

29

5.7 x 104

5.3 x 10’ 4.1 x 10’

cy2001(-11)

93

1.8 x lo-’

2.1 x 10’

27

6.3 x IO-'

cy3019

(-14)

80

2.1 x 10-4

1.2 x 10'

19

8.8 x IO4

5.0 x 10’

c/131 09 (-32)

36

4.6 x lo-’

0.8 x 10’

7.0

2.4 x 1O-3

5.8 x 10s

c//31 14 (-32)

38

4.4 x IO-4

0.8 x 10'

5.4

3.1 x IO-3

4.8 X 106

CYM

63

42

4.0 x 10’

1.1 x 10’

6.7

2.5 x 1O-3

3.3 x 106

cy3107(-35)

37

4.5 x 104

0.8 x 10’

1.6

1 .o x 10-Z

1.2 x lop

cy3075(-34/35)

43

3.9 x 104

1.1 x 10’

4.8

3.5 x lo-

6.6 x 108

Parameters are defined by equation (1) and are calculated from linear regression analyses of ‘tau” plots similar to those in Figures 4 and 5; data from two or more experiments were combined for each tau plot. Values of k2 are not corrected to take into account the reversibility of the isomerfzatiin reaction (see Table 2). Numbers in parentheses indicate the position of each mutation (see Figure 1).

presence. For the mutant, ~~3048, the value for T is about 90 min, differing by only a factor of two from the wild-type value (Figure 5). Tau plots (not shown) for the other two mutants with defects in the -10 region are virtually the same (see Table 1). These observations raise two important questions. First, are the assumptions of the abortive initiation analysis applicable for promoters that isomerize so slowly, especially those with mutations in the -10 region? Second, are the -35 region mutants defective in the interaction of the mutant promoters with RNA polymerase in the presence of c/l protein, or are they actually defective in c//-binding?

160 ‘2

5120 0 f

80

Relative Values of k and &+ To address the first question, we investigated the possibility that one of the assumptions on which equation (1) is based is incorrect. The assumption is that k2 B k-2, that is, that the formation of open complexes is essentially irreversible. To test this assumption, we followed the decay of open complexes as a function of time in the presence of heparin, which was added to inactivate closed complexes and unbound RNA polymerase. Open complexes formed in the absence of substrates were incubated with heparin, and at various times aliquots were removed, substrates were added, and the synthesis of UpApG was allowed to proceed for 8 min. Since open complexes are very stable, the amount of UpApG synthesized in each 8 min interval is proportional to the number of open complexes present at the time of addition of substrates. In the presence of 50 pg/ml heparin, open complexes formed at wild-type fRE in the absence of c/l protein are quite stable, though not so stable as complexes formed in the presence of c/l protein (Figure 6). Furthermore, the apparent rate of decay of open complexes formed in the absence of c/l protein is virtually the same for all -35 region mutants as for wild-type PRE. Data for one such mutant, ~~3107, are shown in Figure 6. On the other hand, open

Figure 5. Tau plots for Wild-type Absence of cl/ Protein

and Mutant

Derivatives

of Pm in the

Values of 7ti obtained in experiments such as those illustrated in Figures 2 and 3 were plotted to fit equation (1). Linear regression analysis was used to determine the intercept (T = 1/k2) and slops (l/k.&) of each plot. Data from several experiments are included in each case. Values of z were 42, 42, and 90 min for wild type (0) cy844 (0) and ~~3048 (A), respectively.

complexes formed at mutant promoters with defects in the -10 region are significantly less stable than those formed at wild-type fRE. Data for the representative mutant ~~3048 are included in Figure 6. Decay of open complexes in these experiments arises both from their isomerization to closed complexes and from their slow inactivation by heparin (Pfeffer et al., 1977; Cech and McClure, 1980). Thus first-order rate constants for inactivation of open complexes in the presence of heparin are only apparent rate constants (&,). Values of kWP obtained at several heparin concentrations must be extrapolated linearly to zero heparin concentration to obtain km2 (Cech and McClure, 1980). Values of kep for wild-type

Tra&cription

lnliation at the X P, Promoter

100 g

Table 2. lsomerization Rate Constants the Absence of cl/ Protein

90

Wild-type

for Mutations

in the -10 Region in

RX

CYW (-7)

cy2001 (-11)

cy3019 (-14)

1. T (min)

42

89

93

80

2. k2 (se@)

4.0 x lo-’

1.9 x IO-’

1.8 x IO-’

2.1 x lo-

3. k+ (set-‘)

0.4 x lo4

0.7 x lo-’

0.6 x lOA

0.6 x IO-’

4. kz” (set-‘)

3.6 x lo-’

1.2 x lo-’

1.2 x lo-’

1.5 x lo-’

46

140

140

110

-$ .5 a b

80

$

80

5. 7O (min)

50

k2 and 7 were obtained without correction on the assumption that the intercepts of tau plots equal l/k* (see Figure 5; Table 1); k2” and 7” are corrected values of k2 and 7. which take into account the reversibility of the isomerbation reaction for mutants with defects in the -10 region (see text). In the notation used here, k2’ = k,--kz and 7’ = l/k,“.

70

I

I

1

20

40

80

80

100

Time (mid Figure 6. Measurements

of km2

Enzyme (67 nM) and DNA (2 nM) were incubated for 60 min at 37% in the presence or absence of c/l protein. At time zero, 50 Ag/ml heparin was added. At various times thereafter, substrates were added and transcription was allowed to proceed for 6 min to assay for open complexes. Each experiment is used to detenine kw, the apparent rate constant for isomerization of open to closed complexes. Hatf-times of open complexes at this hepartn concentration in the absence of c/l protein were 120 min for wild type (0) and ~~3107 (A) and 103 min for cy3048 (A). The half-time for wild-type PRE in the presence of c/l protein (0) was about 15-18 hr.

fRE and the three -10 mutants are listed in Table 2 (line 3). While the value of km2 for wild-type PRE in the absence of cl/ protein is about one tenth as great as the observed value of k2, the isomerization reaction clearly cannot be considered irreversible in the case of the mutants (compare lines 2 and 3, Table 2). When k+ is not negligible relative to k2, equation (1) must be modified as follows (Strickland et al., 1975): 1 ‘Ohs = x

1 + (k2 + k&B(RNAP)

(2)

Thus the intercepts of tau plots actually equal l/(k2 + kV2) unless k-z is negligible. Therefore the values of k2 and 7 listed in Table 1 should be corrected to take into account measured values of k-* for the -10 region mutants. The corrected data (Table 2, lines 4 and 5) indicate that, although the effects of the -10 mutations on k2 are not so great in the absence as in the presence of cll protein, they are nonetheless significant. Note that values of KS do not require correction, since KS is determined by dividing the intercept of the tau plot by the slope.

Response of Mutant Promoters to cl/ Protein It has been shown that c/l protein binds at two TTGC sequences (Figure 1) that flank the PRE -35 region (Ho et al., 1983). As was mentioned previously, the fact that all five -35 region mutants examined here have the same kinetic properties as wild-type fRE in the absence of c/l protein raises the possibility that these mutants may actually be defective in c//-binding, rather than in their interaction with RNA polymerase. Two types of experiments rule out this possibility. First,

even when the concentration of c/l protein is doubled, values of K2 and KS for all five mutants are unchanged (data not shown). Second, it is possible to use fixed-time assays (Hawley and McClure, 1982) to distinguish different states of the same promoter. If the population of promoters in a reaction mixture exists in only one state-that is, either all or none of the DNA molecules in the reaction mixture are bound by an activator-then the plot of In (1 -v/V) as a function of time (see Figure 3) should be monophasic with slope -l/~~~. On the other hand, if the activator is present at suboptimal concentrations, only a portion of the DNA molecules should be able to form open complexes rapidly, and a plot of the fixed-time data should be biphasic (Shih and Gussin, 1983). Plots of fixed-time assays for three -35 region mutants, ~~3075, ~~844, and ~~3107 in the presence of cll protein are clearly monophasic (Figure 7) indicating that c/l protein at 6 pg/ml is sufficient to bind to all the DNA molecules in the reaction mixture. Note that if none of the DNA molecules had bound cl/ protein, Values of Tabs would have been very much greater (compare tau plots in Figures 4 and 5); that is, they would have equalled values obtained in the complete absence of c/l protein. Similar results were obtained for the other -35 region mutants, indicating that the mutants are indeed defective in the interaction of RNA polymerase with PRE, but only in the presence of c/l protein. Similar studies of the -10 region mutants produced the same results (not shown). Thus these mutants apparently are defective in open complex formation in both the presence and absence of cl/ protein, but bind c/l protein normally.

Dual Startpoints for PRE Carpousis et al. (1982) have found that mutations in the LacP promoter significantly affect the relative frequencies of initiation at each of four purines that can be used as startpoints for the Lac transcript. We have investigated whether any of the mutations in PRE influences the choice of startpoint (see Experimental Procedures). In the presence of cll protein, the A startpoint was used 56% of the

Cell 946

100 80 80

10

20

40

80

80

Time (mid Figure 7. Fixed-time Protein

Assays

of Abortive

Initiation in the Presence

of cl/

Assays were the same as those described in the legend to Figure 3, except that c/l protein (6 pg/ml) was added to DNA 10 min prior to the addition of RNA polymerase, and the polymerase concentration was 67 nM. Calculated values of ~~ were 18.530, and 49 min for ~~3075 (0) ~~3107 (0) and cy844 (A), respectively. In the case of ~644, the experiment was actually carried out to 90 min, at which time 1-v/V was 25%. and the plot of the data was still linear

time when wild-type template was used, and 53%-59% of the time for seven of the eight mutant templates. In the absence of c/l protein, the corresponding frequencies were 53% for wild type and 46%-59% for the same seven mutants. Thus neither c/l protein nor mutation significantly affects the choice of startpoint. The eighth mutant, ~~3048, deviated the most from the others, with values of 48% and 42% for the frequency of initiation at A in the presence and absence of c/l protein, respectively, but these differences are not statistically significant. (These results do not exclude the possibility that additional startpoints are used with low efficiency in the case of the mutant promoters.)

Discussion Partitioning of Information Required for Open Complex Formation In the presence of c/l protein, we find that mutations in the -10 region cause a decrease of 20 to 30 times in kz but only slightly affect Ke. However, mutations in the -35 region cause decreases of 30 to 150 times in KB with much smaller effects on k2. These results are consistent with previous reports of the effects of mutations in the PRMand PR promoters of lambda and the /acP promoter (Hawley and McClure, 1980, 1982; McClure et al., 1982; Shih and Gussin, 1983). In general, mutations in -10 regions affect only k2, while mutations in -35 regions affect KS alone, or both KB and kz (with a much smaller effect on k$ Thus information in physically separate regions of the promoter differentially influences distinct steps in open complex formation. Physical and kinetic measurements indicate that the formation of open complexes is accompanied by denatur-

ation of about 8-10 bp of promoter DNA (Mangel and Chamberlin, 1974b; Hsieh and Wang, 1978). In fact, T7 A3 promoter DNA in open complexes contains a denatured region extending from -8 to +3 (Siebenlist, 1979). The fact that all five mutations in the -10 regions of three different promoters affect only the isomerization rate suggests that nucleotide sequences in the -10 region primarily encode information essential for DNA strand separation. It is also clear that the -35 region contains information necessary for the initial binding of RNA polymerase (recognition), since nine mutations in -35 regions of PR, PRM, and PRE have been shown to affect KS. However, the fact that many of these mutations also affect kz suggests that the -35 region may also influence the isomerization of closed to open complexes. However, there is an alternative model for open complex formation, in which mutations in the -35 region might alter measured values of both Ke and k2, even though they affected only the formation of closed complexes (Hawley and McClure, 1982). In terms of this “sequential recognition” model, two types of closed complexes, Cl and C2, are in rapid equilibrium, and only C2 can isomerize irreversibly to form open complexes. Thus three parameters would characterize the formation of open complexes: K,, the equilibrium constant for formation of Cl ; KB, the equilibrium constant for isomerization of Cl to C2; and kp, rate constant for isomerization of C2 to open complexes. If this model were correct, mutations that affected K, would affect only KS, while mutations that affected KB would appear to affect both KS and kz (Hawley and McClure, 1982). Although there is no direct evidence in favor of the model, additional steps in open complex formation have been proposed for other reasons (Chamberlin et al., 1982). (It is possible to calculate theoretical values of K,, KB, and k2 for the wild-type and mutant promoters if one assumes that mutations affecting both KS and kz actually alter only KB (Hawley and McClure, 1982). Experimental data for a particular mutant promoter can be used in conjunction with the corresponding wild-type data to determine k2, K,, KB, and K;I (the mutant value of KB). In principle, calculations based on experimental parameters for different mutants should yield the same values of k2, K,, and K,. For the four -35 region mutations in PRE that affect both k2 and Ke (see Table I), the calculated values of K, ranged from 2.8 to 5.2 X 106 M-‘; KB ranged from 30 to 60; and k2 ranged from 1.8 to 1.9 X lo-’ set-‘. Considering possible sources of error in these calculations, the variation in the calculated values is small enough to be consistent with the sequential recognition model.) The possibility that a single mutation affects more than one step in transcription initiation is not precluded by the kinetic models. Indeed, comparison of values of KB and k2 for the double mutant ~~3075 with the corresponding values for ~~3107 (Table 1) suggests that the single base pair difference (at -34) between the two mutants must affect two separate steps in open complex formation. Otherwise, it would be difficult to imagine how the difference in sequence could cause KS to increase 5-fold and

T&;yiption

lnrtiation at the h PRE Promoter

at the same time cause k2 to decrease 3 times (see Table 1).

Effects of Mutations in the Absence of cl/ Protein The extreme weakness of fRE in the absence of c/l protein (Shimatake and Rosenberg, 1981; Shih and Gussin, submitted) is not surprising as neither the -10 region nor the -35 region shows strong homology with the corresponding consensus sequence (Rosenberg and Court, 1979; Siebenlist et al., 1980; Figure 1). Nevertheless, there are three strongly conserved nucleotides (at -7, -8, and -11) in PRE, as well as a weakly conserved nucleotide at -14 (Rosenberg and Court, 1979). The importance of the conserved nucleotides is reflected in the fact that mutations at three of the four sites (~~3048, ~~2001, and ~~3019) cause a significant decrease in kn for fRE even in the absence of c/l protein. On the other hand, in the absence of c/l protein, tau plots for all five mutants with changes in the -35 region are virtually identical to the tau plot for wild-type PRE. This is a surprising observation in view of the dramatic effects of the mutations on PRE function in the presence of cl/ protein. An obvious explanation for these results is that the mutations cause defects in c//-binding to PRE. Three lines of evidence suggest that this is not the case. First, the kinetic parameters do not change if the concentration of added c/l protein is doubled. Second, fixed-time assays in the presence of levels of c/l protein sufficient to saturate wild-type PRE indicated that the population of mutant DNA molecules was completely saturated with c/l protein (see Figure 7). When similar experiments were performed with a X operator mutant defective in activation of PRM by repressor, biphasic plots of the fixed-time data were obtained (Shih and Gussin, 1983). Finally, Ho et al. (1983) have presented evidence from DNAase I protection experiments that the -35 region mutation cy844 has little or no effect on binding of cl/ protein to PRE. In spite of the fact that c/l protein binds to mutant DNA, it is conceivable that the mutations alter interactions between bound c/l protein and RNA polymerase. Values of k2 for all eight mutants are increased by c/l protein, but the increases are not so great as the increase in the wildtype value of kp. Furthermore, values of Ke are actually reduced by c/l protein in many cases. For ~~3107, Ke is about 6-7 times as great in the absence of c/l protein as in its presence. This suggests that c/l protein can inhibit the initial binding reaction, but that the inhibition is obscured in the case of wild-type I%. In terms of the sequential recognition model, c/l protein might increase K. and/or k2, while decreasing K,. An alternative interpretation of results obtained with mutations in the -35 region in the absence of c/l protein is that RNA polymerase recognizes different sequences in PREin the presence of c/l protein than it does in its absence. For example, the contacts made by polymerase in the -35 region of PRE might be very weak (or nonexistent) in the absence of c/l protein, but bound c/l protein may force (or assist) RNA polymerase to make specific close contacts

with DNA sequences in the interval between the two TTGC sequences. The 6 bp in this interval (from -30 to -35) are not homologous to the -35 consensus sequence, but are positioned 17 bp (the consensus distance) from the consensus sequence at -10 (Rosenberg and Court, 1979; Stefano and Gralla, 1982). Thus one function of c/l protein might be to constrain bound RNA polymerase in such a way that its contacts with -35 region sequences are similar to those made at stronger promoters, in spite of the fact that the PRE sequence in this region cannot be easily aligned to the consensus sequence. In contrast to mutations in PRE, two mutations in the -35 region of Pa,’ exhibit the same phenotypes (relative to wild type) in the presence and absence of repressor. This suggests that repressor does not change the way that RNA polymerase interacts with the -35 region of PRM. Experimental

Procedures

Phage Strains DNA templates were isolated from XSam7 derivatives containing cy (PR) mutations (Wulff et al., 1980, 1983). The mutants ~644, cy3019, ~~3048, and ~~2001 were obtained from D. Wulff via A. Oppenheim as O- or P derivatives; Sam7 derivatives of these mutants were constructed by standard genetic methods. The mutants cy3017Sam7. cy3075Sam7, c113114Sam7, and c113107Sam7 were obtained directly from D. Wulff. Nucleotide changes associated with each mutatron are indicated in Figure hc1857Sam7

Enzymes

was used as the source of P, wild-type

DNA

and DNA

RNA polymerase holoenzyme was purified by the method of Burgess and Jendrisak (1975). Purified enzyme was fudged to be about 95% pure and 90% sigma-saturated based on SDS polyacrylamrde gel electrophoresrs. About 40% of the purified enzyme is active based on the assay of Cech and McClure (1980). The procedure used to purify c/l protein has been described previously (Shih and Gussin, submitted); c/l protein obtained in this way is >95% pure based on SDS polyacrylamide gel electrophoresis. The DNA template was a 1310 bp Hae III restriction fragment containing P&, which produces a truncated transcript 192 nucleotides long: this fragment also contains the oop promoter, which produces a 77 base transcript (Shimatake and Rosenberg, 1981). Wild-type and mutant DNAs were Isolated from Sam7 derivatives according to procedures described by Rosen et al. (1980).

Abortive

Initiation

Assays

RNA polymerase and DNA were incubated at 37°C in buffer containing 0.04 M Tns (pH 8.0) 0.05 M KCI. 0.01 M MgCl,, and 901 M dithiothreitol (DTT). 0.05 mM a-32P-GTP (spec. act. 0.4-0.8 Ci/mmole) and 0.5 mM undylyl-5’.adenosine (UpA). DNA concentrations were 0.5-2.0 nM, as indicated. For c/l activation reactions, DNA and c/l protern were preincubated at 37°C for 10 min prior to the addrtion of RNA polymerase. The oligonucleotide product (UpApG) was assayed following separation by ascending paper chromatography on Whatman 3 MM paper in WASP solvent (saturated (NH&SO,, H20, and isopropanol in the ratio 80:18:2), as described by McClure (1980).

Determination

of T&

The average time required mined by three methods.

Continuous

for open complex

formation

(T&

was deter-

Synthesis

Enzyme, DNA and substrates were added simultaneously at time zero, and the amount of UpApG synthesized was plotted as a function of time. The steady-state rate of UpApG synthesis (at “infinite” time) was taken to be the slope in the period corresponding to 3-5 times TV. Starting with this estimated value of the slope and the corresponding estimate of T-, a least square computer analysis was used to find the best fit to the equation:

Cell 948

N = Vt-VT&

(l-e”““-)

(3)

where N = total (UpApG). V = steady-state rate of UpApG t = time of incubation (Hawley and McClure, 1982).

Fixed-time

In (1 -VP/)

SteMIll

and

Assays

Enzyme and DNA were added together at time zero substrates. At various times, 20 ~1 afiquots were removed mixture and incubated with substrates for 5-10 min, products were then assayed chromatographically for enzyme concentration used, the data were plotted to fit

which is average midpoint obtained

synthesis,

= -t/flT*

in the absence of from the reaction as indicated. The UpApG. For each the equation: (4)

obtained by differentiating and rearranging equation (3); v is the rate of UpApG synthesis during each fixed-time interval, t is the of the interval, and V is the average rate of UpApG synthesis at t greater than 4 times lti.

of Open Complexes

Enzyme and DNA were incubated at 37°C for 60 min. At time zero, heparin was added to a final concentration of IO, 20 or 50 pg/ml. At various times, afiquots were removed and incubated with abortive initiation substrates for 6 min, and then assayed chromatographically for UpApG.

Distinction

between

Two Startpoints

Chamberlin, M. J. (1974). them. 43, 721-775.

The selectivity

of transcription.

Ann. Rev. Bio

Chamberlin, M. J. (1976). Interaction of RNA polymerase with the DNA template. In RNA Pdymerase, R. Losick and M. J. Chamberfin, eds. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratwy), pp. 159-191. Chamberlin. M. J., Rosenberg, S., and Kadesch. T. (1982). Studies of the interaction of E. co/i RNA polymerase hdoenzyme with bacteriophage T7 promoter Al: analysis of kinetics and equilibria of template selection and open-promoter complex formation. In Promoters: Structure and Function, R. L. Rodriguez and M. J. Chambertin, eds. (New York: Praeger Press), pp. 34-53. Echols, H., and Green, L. (1971). Establishment and maintenance of repression by bacteriophage lambda: the role of the cl, cfl, and clll proteins. Proc. Nat. Acad. Sci. USA 76, 2190-2194. Hawfey, D. K., and McClure, W. R. (lg80). In vitro comparison of initiation properties of bacteriophage A wild-type PR and x3 mutant promoters. Proc. Nat. Acad. Sci. USA 77,6381-6385. Hawley, D. K.. and McClure, W. R. (1982). Mechanism of activation of transcription initiation from the A PRM promoter. J. Mol. Biol. 757, 493-525. Ho, Y.-S., Wulff, D. L., and Rosenberg, (cll) binds a -35 region repeat sequence 304,703-708.

M. (1983). Transcription activator on one face of the DNA. Nature,

Enzyme, DNA, and substrates (0.1 mM ATP. 0.1 mM a-=-GTP, spec. act. 1 Ci/mmole) were incubated in standard buffer at 37’C for 60 min. The reaction mixture was then divided into two parts. One part was assayed directly for pppApGpA and pppGpA, both of which should be labeled with =P on the 5’ side of G. The second part was treated with bacterial alkaline phosphatase (1.25 U/100 ~1 reaction) at 65°C in the presence of .05 M ZnCI, and 1 M Tris (pH 8.0). At various times aliquots were removed and assayed for radioactively labelled oligonucleotides. In this case, 5’ triphosphates should be removed, and the plateau level of radioactive oligonucleotides should be due entirely to =P remaining in ApGpA. The ratio of ApGpA (second assay) to pppApGpA + pppGpA (first assay) is the proportion of transcripts initiated at the A startpoint (see Figure 1).

Ho, Y.-S., Lewis, M., and Rosenberg, M. (1982). Purification and properties of a transcriptional activator. J. Biol. Chem. 257, 9128-9134.

Matefial5 Heparin and UpA were obtained from Sigma Chemical Co. (St. Louis, MO); Hae III restriction enzyme and bacterial alkaline phosphatase were purchased from New England Biolabs (Beverly, MA) and Worthington Biochemiced Corp. (Freehold, NJ), respectively: u-=P-GTP and UTP were obtained either from Amersham/Searie (Chicago, IL) or New England Nuclear Corp. (Boston, MA).

Mangel, W., and Chamberfin, M. J. (1974b). Studies of ribonucleic acid chain initiation by Eschefichia co/i ribonucleic acid polymerase bound to T7 deoxyribonucleic acid. III. The effect of temperature on rfbonucleic acid chain initiation and on the conformation of binary compiexes. J. Biol. Chem. 249,3007-3012.

We thank W. McClure and D. Hawley for helpful advice, and A. Dppenheim and D. Wulff for providing mutant phage strains. K. Mat.? and M. Wyckoff provided technical assistance. This work was supported by grant #All 7608 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

June 15. 1983; revised July 28. 1983

References Burgess, R. Ft., and Jendrisak. J. J. (1975). A procedure for the rapid, large scale purification of E. co/i DNA-dependent RNA pdymerase involving polymin P precipitation and DNA-cellulose chromatography. Biochemistry 14,2440-2447. Carpousis, A. J., Stefano, J. E., and Grafla. J. D. (1982). 5’ nucleotide heterogeneity and altered initiation of transcription at mutant lac promoters. J. Mol. Biol. 757, 619-634. Cech, C. L., and McClure, W. R. (1980). Characterization of RNA polymerase-T7 promoter binary complexes. Biochemistry i9, 2440-2447.

Hsieh, T., and Wang, J. C. (1978). Physiochemical studies on interactions between DNA and RNA polymerase. Nucl. Acids Res., 5, 33373345. Johnston, D. E., and McClure, W. R. (1976). Abortive initiation of in vitro RNA synthesis on bacteriophage A DNA. In RNA Polymerase. R. Losick and M. J. Chambertin. eds. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory), pp. 413-427. Mangel, W. F., and Chamberfin, M. J. (1974a). Studies of rtbonucleic acid chain initiation by Escherichia co/i ribonucleii acid polymerase bound to T7 deoxyribonucleic acid. I. An assay for the rate and extent of ribonucleic acid chain initiation. J. Biol. Chem. 249, 2995-3601.

McClure, W. R. (1980). Rate limiting steps Nat. Acad. Sci. USA 77, 5634-5638.

in RNA chain initiation. Proc.

McClure, W. R.. Hawley, D. K., and Malan, T. K. (1982). The mechanism of RNA polymerase activation of the A Pm and La0 promoters. In Promoters: Structure and Function, R. L. Rodriguez and M. J. Chambertin. eds. (New York: F’raeger Press). pp. 11 l-120. Pfeffer, S. R.. Stahl, S. J.. and Chamberlin, M. J. (1977). Binding of fscherichia co/i RNA polymerase to T7 DNA: displacement of holoenzyme from promoter complexes by heparin. J. Biol. Chem. 252, 54035407. Reichardt, L. F., and Kaiser, A. D. (1971). Control of A repressor Proc. Nat. Acad. Sci. USA 68,2185-2189.

synthesis.

Rosen, E. D., Hartley, J. L., Matz, K., Nichols, 9. P.. Young, K. M., Donelson, J. E., and Gussin. G. N. (1980). DNA sequence analysis of prm- mutations of coliphage A. Gene 17, 197-205. Rosenberg, M., and Court, D. (1979). Regulatory sequences involved in the promotion and termination of RNA transcription. Ann. Rev. Genet. 73, 319-353. Schmeissner, U., Court, D., Shimatake, H.. and Rosenberg, M. (1980). The promoter for the establishment of repressor synthesis in bacteriophage A. Proc. Nat. Acad. Sci. USA 77, 3191-3195. Shih, M.-C., and Gussin. G. N. (1983). Mutations affecting two different steps in transcription initiation at the phage A Pm promoter. Proc. Nat. Acad. SCI. USA 80.496500. Shimatake, H., and Rosenberg, M. (1981). Purified A regulatory protein ctl positively activates promoters for lysogenic development. Nature 292, 128132.

Trr;scription

Initiation at the h PRE Promoter

Siebenlist, U. (1979). RNA polymerase unwinds of a 77 promoter. Nature 279, 651-65.2.

an 11 base pair segment

Siebenlist, U., Simpson, R. B., and Gilbert, W. (1980) E. coli RNA pofymerase interacts homologously with two diierent promoters. Cell 20, 269-281. Stefano, J. E., and Gralla. J. D. (1982). Spacer mutations promoter. Proc. Nat. Acad. Sci. USA 79, 1069-1072.

in the lac p*

Strickland, S., Palmer, G., and Massey, V. (1975). Determination of dissociation constants and specific rate constants of enzyme-substrate (or protein-lgand) interactions from rapid reaction kinetic data. J. Biol. Chem. 250.4048-4052. Wulff, D. L., Beher, M., Izumi, S., Beck, J., Mahoney, M., Shimatake, H., Brady, C., Court, D.. and Rosenberg, M. (1980). Structure and function of the cy control region of bacteriophage A. J. Mol. Biol. 138. 209-230. Wulff, D. L., Mahoney, M.. Shatzman, A., and Rosenberg, M. (1983). Mutational analysis of the P, promoter and the N-terminal region of the cll gene of bacteriophage lambda. Proc. Nat. Acad. Sci. USA, in press. Youdertan, P., Bouvier, S., and Susskind, M. M. (1982). minants of promoter activity. Cell 30, 843-853.

Sequence

deter-