Competing promoters in prokaryotic transcription.

Two (or more) bacterial promoters are often found in close proximity, and may compete for the binding of RNA polymerase. These competing promoters have interesting characteristics in vitro and analysis of the competition should be valuable to kinetic studies of more complex transcription systems.

Regulation of open complex formation at the Escherichia coli galactose operon promoters. Simultaneous interaction of RNA polymerase, gal repressor and CAP/cAMP.

The regulation of open complex formation at the Escherichia coli galactose operon promoters by galactose repressor and catabolite activator protein/cyclic AMP (CAP/cAMP) was investigated in DNA-binding and kinetic experiments performed in vitro. We found that gal repressor and CAP/cAMP bind to the gal regulatory region independently, resulting in simultaneous occupancy of the two gal operators and the CAP/cAMP binding site. Both CAP/cAMP and gal repressor altered the partitioning of RNA polymerase between the two overlapping gal promoters. Open complexes formed in the absence of added regulatory proteins were partitioned between gal P1 and P2 with occupancies of 25% and 75%, respectively. CAP/cAMP caused open complexes to be formed nearly exclusively at P1 (98% occupancy). gal repressor caused a co-ordinated, but incomplete, switch in promoter partitioning from P1 to P2 in both the absence and presence of CAP/cAMP. We measured the kinetic constants governing open complex formation and decay at the gal promoters in the absence and presence of gal repressor and CAP/cAMP. CAP/cAMP had the largest effect on the kinetics of open complex formation, resulting in a 30-fold increase in the apparent binding constant. We conclude that the regulation of open complex formation at the gal promoters does not result from competition between gal repressor, CAP/cAMP and RNA polymerase for binding at the gal operon regulatory region, but instead results from the interactions of the three proteins during the formation of a nucleoprotein complex on the gal DNA fragment. Finally, we present a kinetic model for the regulation of open complex formation at the gal operon.

Mechanism of CRP-cAMP activation of lac operon transcription initiation activation of the P1 promoter.

CRP-cAMP was shown to activate transcription initiation at the Escherichia coli lac promoter in vitro as a result of two separate effects. An indirect component of the activation resulted from an enhancement of the fraction of promoters productively bound by RNA polymerase. This effect was due largely to CRP-cAMP repression of RNA polymerase binding to an overlapping site (lac P2) within the promoter region. In addition, a direct enhancement of RNA polymerase binding at the principal lac promoter (lac P1) was found. The combination of indirect and direct activation by CRP-cAMP was suggested to be responsible for the large activation observed in vivo. Promoter strength parameters were also determined for the L8, UV5 and Ps promoters. The effect of CRP-cAMP on these mutant promoters was shown to be consistent with the activation mechanism deduced for the lac wild-type promoter. DNA supercoiling enhanced the promoter strength of the lac wild-type and UV5 promoters. The combination of supercoiling and CRP-cAMP was necessary for optimal promoter strength for the lac wild-type promoter.

Bacteriophage P22 Mnt repressor. DNA binding and effects on transcription in vitro.

We have examined the binding of Mnt repressor to operator DNA in vitro and have determined how this binding affects the level of transcription from two nearby promoters, Pant and Pmnt. Mnt binds to a region of DNA that overlaps the startpoint of transcription of Pant and the -35 region of Pmnt. Mnt represses transcription in vitro from Pant and enhances transcription from Pmnt. Protection and interference experiments show that Mnt binds to a single, 17 base-pair operator site. The operator sequence and the protein-DNA contacts are symmetric. Mnt makes major groove contacts on both faces of the operator DNA. At pH 7.5, 200 mM-KCl, 22 degrees C, the Mnt tetramer binds operator with high affinity (Kd = 2.2 X 10(-11M) and the protein-DNA complex is quite stable (t1/2 = 48 min). Operator binding shows large dependencies on pH, salt concentration, and temperature.

Interaction of the bacteriophage P22 Arc repressor with operator DNA.

Are repressor binds to a single, partially symmetric, 21 base-pair operator site that is centered between the -10 and -35 regions of the Pant promoter. Protection and interference experiments show that Arc makes contacts with the operator on one side of the DNA helix. Although Arc is a small protein (53 residues/subunit), it makes contacts that are farther from the center of the operator than those made by many larger repressors. These extended contacts include the phosphate groups at the ends of the 21 base-pair site. Under standard conditions (pH 7.5, 100 mM-KCl, 3 mM-MgCl2, 22 degrees C) half-maximal operator binding is observed at an Arc concentration of 2.5 X 10(-9) M and the protein-DNA complex is very stable (t1/2 approximately equal to 80 min).

Rate-limiting steps in RNA chain initiation.

Promoter-specific lags in the approach to the steady-state rate of abortive initiation were observed when Escherichia coli RNA polymerase was added to initiate the reaction. The lag times were related to the time required for free enzyme and free promoter to combine and isomerize into a functionally active complex. The lag times measured for several bacteriophage and bacterial promoters differed widely (10 sec to several minutes) and in most cases corresponded to the rate-limiting step in the initiation process. The unique advantage in using the abortive initiation reaction to measure the lags was that the binding and isomerization steps in a simple two-state model could be quantitated separately. The separation of the contributions of both steps was effected by deriving an equation to describe the rate of formation of the active binary complex. Results from experiments based on the theory showed a linear relationship between the observed lag times and the reciprocal enzyme concentration. The slope and intercept of the equation yielded quantitative estimates of the binding and isomerization steps in initiation. The analysis was applied to the bacteriophage T7 A2 and D promoters to show the bases for the differences in in vitro initiation frequency that have been observed for these promoters.

On the mechanism of streptolydigin inhibition of Escherichia coli RNA polymerase.

The mechanism of streptolydigin inhibition of RNA synthesis has been investigated with a combination of steady state kinetics and product analysis by employing the abortive initiation reaction of Escherichia coli RNA polymerase. The pattern of inhibition by streptolydigin on the poly[d(A-T)] . poly[d(A-T)]template (non-competitive versus AMP; competitive versus UTP) was consistent with one inhibitor binding site and with an ordered addition of AMP followed by UTP. The more complicated patterns observed on the poly[d(I-C)] . poly[d(I-C)] template and the bacteriophage T7 A2 promotor (noncompetitive versus CTP) were explained by assuming that streptolydigin could stabilize the translocated ternary complex containing the product dinucleotide. Product analysis of two other abortive initiation reactions showed that the product did not dissociate from the inhibitor-bound translocated ternary complex. Finally, rifampicin and streptolydigin were shown to be functionally independent during initiation on several templates.

In vitro comparison of initiation properties of bacteriophage lambda wild-type PR and x3 mutant promoters.

The in vitro initiation properties of the PR promoter of bacteriophage lambda and of a PR mutant, x3, were compared. Using the abortive initiation reaction, we measured the lags in the approach to a final steady-state rate when dinucleotide synthesis was initiated with RNA polymerase. These lags corresponded to the average times required for the formation of transcriptionally active open complexes. By measuring the lags at different RNA polymerase concentrations, we could separate open complex formation into two steps, based on a simple model in which the initial bimolecular association of free promoter and polymerase in a closed complex is followed by an isomerization to the open complex. The contribution of each step to the overall rate of open complex formation was quantitated for both promoters. We found that the x3 mutation, which is located in the -35 region of PR, resulted in a decrease in the association constant for the initial binding to the closed complex to 5% of its wild-type value and a decrease in the rate of the isomerization to 20%. The lifetimes and abortive initiation characteristics of the mutant and wild-type promoters were similar. We concluded that the main effect of the x3 mutation was to increase the average time of open complex formation and that the functional properties of the open complexes did not differ significantly between the two promoters.

Role of the sigma subunit of Escherichia coli RNA polymerase in initiation. I. Characterization of core enzyme open complexes.

Open complexes of Escherichia coli RNA polymerase core enzyme with a poly[d(A-T)]-poly[d(A-T)]template have been characterized and compared with the previously characterized holoenzyme open complexes on the same template (Hansen, U. M., and McClure, W. R. (1979) J. Biol. Chem. 254, 5713-5717). The open complexes were monitored by the abortive initiation synthesis of the dinucleotide pApU, which is catalyzed by both enzymes. The major differences between the two complexes were: 1) the Michaelis constant for UTP was 60 times higher for core enzyme than for holoenzyme, 2) the intrinsic binding constant of core enzyme to the DNA was 3 orders of magnitude less than that of holoenzyme, and 3) cooperative binding of 2 core units was required for activity. Thus, the presence of the sigma subunit significantly altered the nature and extent of open complex formation. The rate of formation of the open complexes, however, was rapid for both enzymes. Rifampicin is shown to have a slight stimulatory effect on the extent of open complex formation of the core enzyme.

Role of the sigma subunit of Escherichia coli RNA polymerase in initiation. II. Release of sigma from ternary complexes.

The sigma subunit of Escherichia coli RNA polymerase was released from a transcribing polymerase-poly[d(A-T)]-poly[d(A-T)]-RNA complex when the nascent RNA reached a length of either eight or nine nucleotides. The dissociated sigma was separated from other reaction components by gel filtration, and was assayed using an activity assay previously described (Hansen, U. M., and McClure, W. R. (1979) J. Biol. Chem. 254, 5713-5717). The extent of RNA synthesis was limited by incorporation of 3'-dATP into the RNA chains; a series of distributions of product lengths was achieved by varying the concentration of 3'-dATP. A correlation of the number of moles of dissociated sigma versus the number of moles of RNA products of varying lengths allowed the determination of the point of release of the sigma subunit. Models to explain the cause of sigma release are discussed.

Analysis of the occurrence of promoter-sites in DNA.

We show that the occurrence and homology score (1) of promoter-sites in DNA depends upon the base composition of the DNA. We used simple probability theory to calculate the mean homology score expected for all promoter-sites that had a specific match in the canonical hexamers. By using the square root of this mean score as a measure of significance, we objectively classify all promoter-sites which are reported. We tested the theoretical approach in two ways. First, we used the program (PROMSEARCH) to analyze approximately 150,000 base pairs of random sequence DNA with different base compositions and we found excellent agreement with the theoretical predictions. Our second test was the analysis of a number of sequences drawn from the GENBANK DNA sequence database. We have analyzed 20 bacterial and bacteriophage sequences, which consisted of at least one operon, for promoter-sites. We found no absolute preference for promoter-sites within noncoding regions. We show the results of analyzing the phages lambda, T7 and fd, and the E. coli lac operon. The major known promoters in these sequences were all found correctly. We discuss the question of the location of a number of minor promoter-sites and show how PROMSEARCH can be used to help identify the correct location of the promoter. This approach can be applied to the search for any DNA site and should allow greater objectivity when comparing DNA sequences for meaningful subsequences.

Kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter. Evidence for a sequential mechanism involving three steps.

The forward and reverse kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter have been studied in the temperature range of 15-42 degrees C. The standard two-step model, involving the formation of a closed intermediate, RPc, followed by an isomerization that leads to the active complex RPo, could not account for the present data. The promoter-enzyme lifetime measurements showed an inverse temperature dependence (apparent activation energy, -35 kcal/mol). A third step, which is very temperature dependent and which is very rapid at 37 degrees C, was postulated to involve the unstacking of DNA base pairs that immediately precedes open complex formation. Evidence for incorporating a new binary complex, RPi, in the pathway was provided by experiments that distinguished between stably bound species and active promoter after temperature-jump perturbations. These experiments allowed measurement of the rate of reequilibration between the stably bound species and determination of the corresponding equilibrium constant. They indicated that the third step became rate limiting below 20 degrees C; this prediction was checked by an analysis of the forward kinetics. A quantitative evaluation of the parameters involved in this three-step model is provided. Similar experiments were performed on a negatively supercoiled template: in this case the third equilibrium was driven toward formation of the open complex even at low temperature, and the corresponding step was not rate limiting.

Antisense RNA control of gene expression in bacteriophage P22. I. Structures of sar RNA and its target, ant mRNA.

Sar RNA is an antisense RNA that is partly responsible for the negative regulation of antirepressor synthesis during development of bacteriophage P22 (Liao SM et al., 1987, Genes & Dev 1:197-203; Wu Th, Liao SM, McClure WR, Susskind MM, Genes & Dev 1:204-212). The structures of sar RNA and its target, ant mRNA, were probed using limited RNase digestion as a function of Mg2+ concentration. Sar RNA forms two hairpins that are present at all Mg2+ concentrations (Mg2+-independent hairpins). One of the hairpins contains three tandem U x U base pairs. Ant RNA forms three Mg2+-independent hairpins and one Mg2+-dependent hairpin. In addition, many nucleotides in sar RNA and ant RNA appear to be involved in tertiary interactions. The effects of RNA structure on the pairing reaction are considered in the accompanying paper (Schaefer KL, McClure WR, 1997, RNA 3:157-174).

Antisense RNA control of gene expression in bacteriophage P22. II. Kinetic mechanism and cation specificity of the pairing reaction.

The bacteriophage P22 sar RNA-ant mRNA pairing reaction was characterized kinetically. The pairing reaction proceeds by a three-step pathway. First, reversible base pairs form between complementary hairpin loops in sar RNA and ant RNA (Kd = 270 nM). Next, stable duplex formation initiates between single-stranded nucleotides in sar RNA and ant RNA; the ant RNA nucleotides are at the bottom of a hairpin stem that is partially accessible. Concomitant unwinding of one sar RNA hairpin and the complementary ant RNA hairpin then occurs, to form a partially paired intermediate (k2 = 12 min(-1). Finally, a complete duplex forms after unwinding of the other sar RNA hairpin and the complementary ant RNA hairpin (k3 = 7 min(-1). Experiments with sar RNA sequence and length variants demonstrate that the precise structures of both sar RNA hairpins affect the kinetic parameters. The pairing reaction is Mg2+-dependent, and shows high specificity for the required cation. Maximal pairing rates are achieved when more than one Mg2+ ion is bound. The cation-dependence and specificity indicate a requirement for Mg2+-dependent tertiary structure.

Selective binding of Escherichia coli RNA polymerase to topoisomers of minicircles carrying the TAC16 and TAC17 promoters.

We have studied the effect of DNA supercoiling on open complex formation by Escherichia coli RNA polymerase at the TAC16 and TAC17 promoters. A two-dimensional gel retardation assay was used to measure the relative rate of association between RNA polymerase and the TAC promoters on individual topoisomers. Plasmid DNAs usually have several promoters that complicate the analysis of RNA polymerase binding to only one of them. We avoided this problem by using minicircles of DNA carrying only a single promoter. These were generated in vivo by the site-specific recombination system of bacteriophage lambda. Both the TAC16 and TAC17 promoters exhibited a biphasic response to negative supercoiling. Between superhelical densities of 0 and -0.04, increases in the negative linking difference favored binding. Beyond -0.04, either no effect on binding (TAC16) or a slight inhibition of binding (TAC17) was observed for increases in the negative linking difference. The unwinding at the rate-limiting step was calculated for both TAC promoters at moderate superhelical densities. This unwinding was found to be a fraction (about 20-30%) of the total unwinding measured by topoisomerase I relaxation (about one turn). In addition, the 1-base pair difference in spacer length between the TAC16 and TAC17 promoters resulted in different extents of activation and inhibition by negative supercoiling.

Characterization of the closed complex intermediate formed during transcription initiation by Escherichia coli RNA polymerase.

We have carried out detailed DNase I footprinting studies of the closed complex formed on the phage lambda prmup-1 Delta265 promoter under reaction conditions such that the contribution of the open complex to the footprint was negligible. Detailed quantification shows that the closed complex detected has the same binding constant as that determined in kinetic studies. The footprinting pattern of the closed complex shows major differences from that of the open complex. Not only is it about 20 base pairs shorter, there are also many fewer positions being protected around and upstream of the -35 region. We have derived potential contact regions in the closed and open complexes based on the DNase I footprinting patterns, and confirmed the contact region for the open complex by hydroxyl radical footprinting. One important finding is that most of the essential contacts with the phosphate groups in the -35 region are formed during the isomerization step, a conclusion consistent with our kinetic data showing that this step is salt dependent on this promoter. In addition, we found that the derived contact regions for the closed and open complexes are offset by about three base pairs in the -35 region, which suggests a shift of the contact during isomerization. Finally, we found that the footprinting pattern of the complex formed at 4 degreesC has some similarities to as well as differences from the closed complex formed under standard transcription conditions.

Stimulation of open complex formation by nicks and apurinic sites suggests a role for nucleation of DNA melting in Escherichia coli promoter function.

We report the effects of depurination and prenicking at various positions of the phage lambda prmup-1Delta265 promoter DNA on the rate of open complex formation. We have found that depurination and prenicking at positions around the -10 region strongly stimulated the rate of open complex formation. Since nicking and depurination are known to destabilize DNA helical structure, our observations indicate that the instability of the -10 region is important for open complex formation. We further infer that (i) the nucleation of DNA melting, which occurs during the isomerization from the closed complex into the open complex, contributes to the rate of open complex formation; (ii) the nucleation of melting occurs around the -10 region; and (iii) the propagation of DNA melting from the nucleation region is not rate-limiting. In addition, we have found that depurination at several positions inhibited open complex formation. We used dimethyl sulfate modification protection studies to show that most of the guanine bases that are among these positions are in contact with RNA polymerase in the open complex.