INTRODUCTION

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Gene expression is often regulated at transcription initiation. In the absence of gene regulatory proteins, the rate of initiation at a particular promoter depends on the concentration of RNA polymerase and the sequence of the promoter element. In both prokaryotes and eukaryotes, transcription initiation is a multistep process that begins with sequence-specific recognition and proceeds through formation of two or more intermediate RNA polymerase-promoter complexes prior to synthesis of the first phosphodiester bond of the product RNA (31). Subsequently, the initiated complex isomerizes into one that generates full-length RNA products (for exceptions, see references 18 and 34). The fractional occupancy of a promoter by RNA polymerase and the rate at which RNA polymerase proceeds through the initiation pathway determine, in part, the amount of RNA produced from a gene.

The pathway for transcription initiation is best understood in prokaryotes (Fig. 1A). In these organisms, RNA polymerase binds to promoter DNA and initially forms an unstable "closed" complex that dissociates with the addition of polyanionic competitors such as single-stranded DNA or heparin (31). The closed complex isomerizes through a number of intermediate complexes (21, 33) in which the DNA at the transcription start site remains base paired (Fig. 1A). Subsequently, these intermediates isomerize to form one or more competitor-resistant "open" complexes, in which the DNA strands surrounding the transcription start site display increasing separation (4, 36). RNA polymerase initiation complexes form after the DNA in the region between 11 and ca. +5 is fully denatured and the synthesis of small (8- to 12-base) "abortive" oligonucleotide products takes place and/or the polymerase escapes from the promoter, forming an elongation complex competent to synthesize full-length RNA transcripts.

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Transcription pathway and sequence of the starting promoter. (A) Proposed transcription initiation pathway for E. coli RNA polymerase, based on evidence described in the text and reference 31. (B) Sequence of the modified bacteriophage 434 PR promoter that is the starting material for the selection experiments. The 35 and 10 regions in this promoter are underlined (2, 37).

In prokaryotes, both the affinity of RNA polymerase for DNA and the rate of isomerization of the RNA polymerase-DNA complexes to form the various intermediate species depend on the particular sequence of the promoter (12, 14). In addition, the rate of escape of RNA polymerase from the promoter to form full-length RNA transcripts may also be determined by promoter sequence (3, 15). Hence, the overall RNA polymerase occupancy of a promoter and the rate of RNA synthesis from that promoter depends on its sequence. It is not known, however, how promoter sequence determines the efficiency of the individual steps along the initiation pathway. Insight into this question is critical to understanding the interrelationship between promoter strength and the mechanisms by which gene regulatory proteins modulate transcription (19).

The consensus promoter utilized by Escherichia coli RNA polymerase-⁷⁰ is comprised of two conserved, hexameric DNA sequence elements separated by ~17 bp and located ~35 and ~10 bp upstream of the first transcribed nucleotide (11, 13, 23). The consensus sequences of these so-called 35 and 10 elements are TTGACA and TATAAT, respectively. In the RNA polymerase holoenzyme the ⁷⁰ subunit makes direct protein-DNA contacts with double-stranded DNA in the 35 region (7, 9, 35). In open complexes in which the DNA strands near the start point of transcription are separated, another portion of the ⁷⁰ subunit interacts with the bases on the nontemplate strand in the 10 region (25). Recent work (26) has examined the nature of the protein-DNA contacts made in the open promoter complex. Although they most certainly occur (1, 21, 33), the type of RNA polymerase-promoter DNA contacts made in the 10 region in the closed and the transitional intermediate promoter complexes has not yet been established.

The consensus sequence of the E. coli RNA polymerase-⁷⁰ promoter has been deduced from sequence compilations (11, 13, 23), and the importance of these conserved promoter elements has been demonstrated by random (28) and site-directed (9, 17, 27) mutagenesis studies. Many of these studies focused on the effect of promoter sequence changes on transcriptional efficiency. While they provide useful information how sequence contributes to overall promoter efficiency, these methods provide only a composite measure of sequence effects on promoter function. More-recent studies examined the sequence dependence of the binding of single-stranded DNA (ssDNA) corresponding to the nontemplate strand of the 10 region to RNA polymerase (8, 25, 30). However, this type of experiment does not provide information about the relationship between promoter sequences and the efficiency of steps leading up to open-complex formation. Thus, these two types of studies are unable to examine the effects of promoter sequences on individual steps in the transcription initiation pathway.

As a first step to overcoming this limitation, we used E. coli RNA polymerase-⁷⁰ holoenzyme to select promoter sequences from a pool of DNAs randomized at the 10 hexamer. Two types of selections were performed. We demanded that the promoter sequences either support rapid formation of heparin-resistant RNA polymerase-promoter complexes or support the formation of such complexes under conditions of very low RNA polymerase concentration. The first selection strategy should identify 10 sequences that allow RNA polymerase to advance rapidly through the isomerization steps that precede heparin-resistant complex formation. The second selection regimen should reveal sequences that allow RNA polymerase to productively bind promoter DNA with high affinity. Hence these selection protocols allow us to dissect the role of the 10 base sequence in determining, respectively, the rate and efficiency with which RNA polymerase forms stable complexes with a promoter. The starting material for these selections is derived from the P_R promoter from the lambdoid bacteriophage 434 (Fig. 1B). This promoter bears a consensus 35 element sequence and is separated from the randomized 10 region by a 17-bp spacer.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Construction of promoter DNA. Bacteriophage 434 P_R promoters bearing random sequences at the 10 region were constructed in two steps. We obtained (Integrated DNA Technologies) two pairs of oligonucleotides complementary to the upper and lower strands of 434 P_R; in each pair one oligonucleotide contained six random bases at the position of the 10 region and the other was complementary to DNA 225 bp upstream or downstream of the 434 O_R region in pJX (37). Two separate amplification reactions, one with each pair of oligonucleotides, resulted in DNA molecules that encoded the upstream and downstream halves, respectively, of the 434 O_R region with a random sequence at the 10 region of P_R. Subsequently, these two DNA molecules were annealed and the intact 434 O_R region bearing a random 10 region was obtained by amplifying the annealed product with the upstream and downstream primers. DNA purified from this amplification reaction was labeled at the 5' ends using T4 polynucleotide kinase and [-³²P]ATP and was used directly in the selections.

In vitro selection. For sequence selections based on affinity, initially 20 nM RNA polymerase was mixed with equimolar DNA in transcription buffer containing 100 mM KCl, 40 mM Tris (pH 7.9), 10 mM MgCl₂, and 10 mM dithiothreitol (DTT) and incubated at 37°C for 15 min. This period is at least three times longer than the measured association and dissociation half-times of the DNA pool, ensuring that the mixture had reached equilibrium. Subsequently, 0.1 mg of heparin/ml was added and the mixture was incubated an additional 5 min at 37°C before the reaction products were fractionated on a 5% polyacrylamide gel at room temperature using TBE (89 mM Tris [pH 8.9], 89 mM boric acid, 1 mM EDTA) as the electrophoresis buffer. The RNA polymerase concentration was lowered to 5 nM at round 5 and to 1 nM at round 15, holding the DNA concentration constant. To select 10 sequences that bind rapidly to RNA polymerase, 20 nM RNA polymerase was mixed with equimolar DNA in transcription buffer and incubated for 15 s. Subsequently, 0.1 mg of heparin/ml was added and the mixture was incubated an additional 5 min at 37°C before the reaction products were fractionated on a 5% polyacrylamide gel.

Measurements of the rate and affinity of RNA polymerase-promoter binding. The 450-bp DNA fragments containing selected promoters were separately isolated from the pEMBL8+ derivatives by cleavage with PvuII and HindIII and were labeled at their 3' ends by incubation with [-³²P]dATP and the Klenow fragment of DNA polymerase I. Binding affinities were determined by mixing increasing concentrations of RNA polymerase with 0.1 pM promoter-containing DNA fragment and incubating at 37°C for 15 min. Subsequently, 0.1 mg of heparin/ml was added and the mixture was incubated an additional 5 min at 37°C before the reaction products were fractionated on a 5% polyacrylamide gel at room temperature. The incubation times were long enough to ensure that the reaction was at equilibrium (see above). Heparin was added to these mixtures to facilitate quantitation by removing RNA polymerase that was bound to DNA in non-sequence-specific complexes. Control experiments indicated that the amount of promoter-specific complex was unaffected by the concentration of heparin added or the length of the incubation time (data not shown). The gels were visualized on a PhosphorImager, and the amounts of complex and free DNA was determined. The apparent K_d was determined by fitting the bound counts/total counts fraction versus RNA polymerase concentration to a hyperbolic binding expression. For the rate measurements, 0.1 nM labeled DNA was separately mixed with 1, 2, 5, and 10 nM RNA polymerase. After incubation at 37°C for varying lengths of time, the reactions were quenched by adding 0.1 mg of heparin/ml before the reaction products were fractionated, visualized, and quantified as described above. The apparent association rate constant (k_a) was determined from the slope of the plots of the pseudo-first-order rate constants determined from the progress curve at each RNA polymerase concentration versus RNA polymerase concentration. The apparent k_a values reported are averages of three determinations.

In vitro transcription. DNA fragments containing the selected promoters were isolated from the pEMBL8+ derivatives by cleavage with PvuII. Approximately 1 nM each fragment was separately incubated with increasing RNA polymerase concentrations for 15 min (for concentration dependence measurements, see Figures 3A and B) or with 10 nM RNA polymerase at increasing times at 37°C (for time dependence measurements, see Fig. 3C and D) in transcription buffer, prior to initiation of runoff transcription reactions by addition of ribonucleotide triphosphates together with 0.1 mg of heparin/ml. Heparin was added to these mixtures to ensure that only a single round of transcription occurred. Control experiments established that heparin addition did not affect the stability of open complexes formed by RNA polymerase on any of the promoters we used under these conditions. Hence, the time and RNA polymerase concentration dependence of transcript formation reveal the differential effects of promoter sequence, not variation in heparin competition. For the concentration dependence measurements, the RNA polymerase concentration was increased in twofold steps starting at 0.6 nM. All transcription reactions were terminated after 10 min by addition of a formamide-containing dye mixture and heating to 90°C for 5 min. Template DNA concentrations were determined by digitally comparing the fluorescent intensities of bands on ethidium bromide-stained gels of DNA samples of known and unknown concentrations. For these measurements, a standard curve was constructed from a DNA sample of known concentration that was identical in length and sequence composition to the unknown input template DNA. DNA concentrations determined in this fashion are reproducible with an error of ±5%.

KMnO₄ footprinting. DNA fragments containing the selected promoters were separately isolated from the pEMBL8+ derivatives by cleavage with PvuII and HindIII and were labeled on the template strand. These DNAs were incubated with 20 nM RNA polymerase for 15 min at 37°C, followed by addition of 15 mM KMnO₄. After 2 min of further incubation at 37°C, the reaction was quenched by ethanol precipitation. The DNA was cleaved by incubation with piperidine at 90°C and was subsequently processed for electrophoresis and electrophoresed on an 8% polyacrylamide gel containing 7 M urea. The gels were visualized on a PhosphorImager, and the relative amount of KMnO₄ reactivity was determined from the intensity of the bands corresponding to the entire 10 region, relative to the total intensity of the individual lane. These experiments were repeated three to five times, and the values obtained are reproducible within the errors of the measurement.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Figure 2 shows that the preferred sequences obtained from the selections for a high rate of open-complex formation or a high-affinity RNA polymerase binding contain at least three (7, 9, and 11) positions that match the consensus sequence. At these positions, RNA polymerase selects only the consensus base pair (Fig. 2). Work of others (30) showed that of positions 7, 9, and 11, only substitutions at the 11 position appear to substantially affect the affinity of ssDNA oligonucleotides encoding the nontemplate strand of the 10 region for RNA polymerase. Similarly, the identities of the bases at 7 and 11 have been shown to be critical for the binding of a "forked junction" template to RNA polymerase (26), a complex that has been proposed to mimic the open promoter complex (10, 32). Hence our findings extend these observations and suggest that in addition to 7 and 11, the identity of the base pair at position 9 is also critical to facilitating binding of the nontemplate DNA strand to RNA polymerase.

View larger version (33K): [in this window] [in a new window]   FIG. 2.   The 10 sequences preferred by 70 RNA polymerase for formation of heparin-resistant complexes. Sequences were selected and determined as described in Materials and Methods. Shown is the frequency distribution of the occurrence of a selected base sequence at each position in the selected promoter, as selected on the basis of high-affinity RNA polymerase binding (A) or rapid open-complex formation (B). The percent occurrence of a base pair at each position was derived from analysis of 14 and 22 cloned sequences for panels A and B, respectively.

The consensus sequences of the selected 10 sequence promoters differ from the consensus 10 sequence (TATAAT) at as many as three (8, 10, and 12) positions (Fig. 2). At these positions, RNA polymerase chooses between only two of the possible four base pairs. This finding suggests that the two base sequences not selected at each of these positions are incompatible with efficient open-complex formation. We find that the degree of preference for a particular base at a given position depends on the selection regimen (Fig. 2). Thus, the particular base sequence at positions 12, 10, and 8 may uniquely affect a particular step of the pathway towards open-complex formation.

To test this idea, we chose to analyze a subset of the promoters isolated during our selections. The promoter sequences were chosen to allow us to best examine the effect of base changes at the polymorphic positions 12, 10, and 8 on promoter function, both within identical sequence contexts and in the background of multiple sequence differences. However, since we obtained only a limited set of sequences in our selections, base changes at a particular site cannot be examined in the context of all base sequences at other positions. Nonetheless, among the sequences we chose is a promoter bearing the TATAAT sequence at its 10 region, facilitating comparisons with this naturally selected consensus sequence.

Position 8. Although the preference of RNA polymerase for a C · G base pair at position 8 is stronger for the rate-based selection than for the affinity-based selection (80% versus 65%), the strong bias of RNA polymerase for this base pair over the consensus A · T, independent of the selection regimen (Fig. 2), is particularly striking. Compilations of E. coli RNA polymerase ⁷⁰ promoter sequences show that the consensus A · T pair is found in 55 to 60% (depending on spacer length) of known promoters (23). However, only 18 to 20% of known promoters contain C · G base pairs at this position, a frequency that is similar to that of the nonconsensus T · A base pairs at this position. Nonetheless, the preference of RNA polymerase for C · G base pairs at position 8 of the test promoter mirrors the positive effect that the presence of this base pair has on the function of several promoters (16, 27). Similarly, 50% of promoters that have been selected for high transcription levels have a C · G base pair at position 8 (28), and the consensus A · T base pair is not selected. Thus, a C · G base pair at position 8 contributes favorably to transcriptional efficiency of strong promoters. In their in vitro selection experiments, Gourse and colleagues find that the highest-affinity promoters for the E. coli RNA polymerase-^s holoenzyme have either a C · G or an A · T base pair at this position (R. Gourse, personal communication). Thus, C · G pairs are well tolerated at position 8 in the high-affinity promoters of at least the related ⁷⁰ and ^s-RNA polymerase holoenzymes.

View this table: [in this window] [in a new window]   TABLE 1.   Characterization of rate- and affinity-selected 10 sequences

Position 10. When promoters are selected solely on the basis of rapid open-complex formation, RNA polymerase prefers a C · G base pair at position 10, whereas if the selection is performed at a low enzyme concentration, RNA polymerase prefers T · A base pairs at this position (Fig. 2). The effect of selection regimen on position 10 base preference is not as large as that at position 8. The consensus T · A base pair is found at position 10 in ~55% of known promoters. By contrast, a C · G base pair is found at position 10 in only 10 to 15% of naturally occurring promoters, a frequency at which the other nonconsensus base pairs are found at this position.

Position 12. Under conditions where RNA polymerase must rapidly form open complexes, the enzyme exhibits a strong preference for the consensus T · A base at position 12. If the selection is performed at a low enzyme concentration, RNA polymerase prefers the nonconsensus G · C base pair at this position. Consistent with the results of the selection, RNA polymerase binds to promoters bearing G · C base pairs at position 12 with twofold-higher affinity than it does to promoters bearing T · A base pairs at this position. Also consistent with the selection regimen, the apparent k_a of RNA polymerase with T · A-containing promoters is ~4-fold higher than that with G · C-containing promoters.

Effect of 10 sequence on promoter function. Previous studies have shown that increasing the correspondence of a promoter's sequence with the consensus does not always increase the rate or amount of mRNA product synthesized from the promoter (16). Since RNA transcript formation is the product of a sequential multistep reaction, the amount of active RNA polymerase-promoter complexes and the rate of RNA synthesis from these complexes can be limited by the affinity of RNA polymerase for the promoter as well as by the rates and efficiencies of steps that occur subsequent to binary complex formation. In an effort to determine whether our selected promoters efficiently mediate transcription initiation and whether the various promoter sequences differentially affect transcript formation, we measured amounts of runoff transcripts formed from promoters bearing changes at individual positions in time- and RNA polymerase concentration-dependent single-round runoff transcription assays. We also measured the extent of open-complex formation using KMnO₄.

View larger version (40K): [in this window] [in a new window]   FIG. 3.   Runoff transcription directed by the rate- and affinity-selected promoters. Concentration-dependent (A and B) and time-dependent (C and D) runoff transcription directed by the rate- and affinity-selected promoters was determined in single-round runoff transcription assays, performed as described in Materials and Methods. The products of a typical transcription reaction are shown (A and C) as visualized by a PhosphorImager. Data from three experiments were quantified (B and D). Error bars, standard deviations. The amount of transcript is expressed in PhosphorImager units per microgram of input DNA (p.u.). Symbols in panels B and D represent promoters containing the 10 sequences.

View larger version (39K): [in this window] [in a new window]   FIG. 4.   KMnO4 reactivity of selected promoters in the presence of saturating RNA polymerase concentrations. Labeled DNA fragments were incubated with RNA polymerase containing the indicated promoters and treated as described in Materials and Methods. The positions of the reactive thymines present on the template strand in the 10 region in all the selected promoters are indicated by the bracketed numbers. The relative KMnO4 reactivity of the 10 region is indicated below the gel and was determined as described in Materials and Methods.