The DNA sequence of 168 promoter regions (-50 to +10) for Escherichia coli RNA polymerase were compiled. The complete listing was divided into two groups depending upon whether or not the promoter had been defined by genetic (promoter mutations) or biochemical (5' end determination) criteria. A consensus promoter sequence based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations. In addition, we have tabulated 98 promoter mutations. Nearly all of the altered base pairs in the mutants conform to the following general rule: down-mutations decrease homology and up-mutations increase homology to the consensus sequence.

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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.

We describe a simple algorithm for computing a homology score for Escherichia coli promoters based on DNA sequence alone. The homology score was related to 31 values, measured in vitro, of RNA polymerase selectivity, which we define as the product KBk2, the apparent second order rate constant for open complex formation. We found that promoter strength could be predicted to within a factor of +/-4.1 in KBk2 over a range of 10(4) in the same parameter. The quantitative evaluation was linked to an automated (Apple II) procedure for searching and evaluating possible promoters in DNA sequence files.

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 ratelimiting 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 17 A2 and D promoters to show the bases for the differences in in vitro initiation frequency that have been observed for these promoters. Transcription in Eacherichia coli is catalyzed byDNA-dependent RNA polymerase (nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). The initiation of RNA synthesis from bacterial and bacteriophage promoters is an important control point for gene expression. The frequencies of initiation vary considerably both in vvo and in vitro. Although activator proteins and repressor proteins provide important on/off switches for some operons, many initiation frequencies are determined solely by the interaction of RNA polymerase with the DNA in a promoter region. The sequences of about 50 such promoters have been determined (1, 2). Although striking sequence homologies are found in the DNA, general rules for initiation frequency based on DNA sequence have not emerged. The reason is that until recently there was no quantitative assay for in vitro chain initiation frequency that was also generally applicable to many promoters.A minimal model for RNA chain initiation has existed for many years (3). The enzyme is thought to bind the DNA in the first step and to unwind the DNA in the second step. Subsequent triphosphate binding and phosphodiester bond formation would then-lead to an elongating ternary complex. Chamberlin has focused attention on the two binary complexes and has referred to them as "closed" and "open" (4). For many promoters the formation of the open complex is likely to be rate determining in initiation because triphosphate binding and elongation have been shown to be very rapid (5,6). Results obtained with different promoters and with various techniques have been interpreted as demonstrating that the binding step (7,8) or the isomerization step (9, 10) was uniquely rate limiting. These results are not ne...