Abortive Initiation Assays Research Articles

Assays for transcription factor activity.

Transcription factors interact at promoters to modulate the transcription of genes. This chapter describes three in vitro methods that can be used to monitor their activity: transcript assays, abortive initiation assays, and potassium permanganate footprinting. These techniques have been developed using bacterial systems, and can be used to study the kinetics of transcription initiation, and hence to unravel regulatory mechanisms.

Interaction of Escherichia coli RNA Polymerase σ70 Subunit with Promoter Elements in the Context of Free σ70, RNA Polymerase Holoenzyme, and the β′-σ70 Complex

Promoter recognition by RNA polymerase is a key point in gene expression and a target of regulation. Bacterial RNA polymerase binds promoters in the form of the holoenzyme, with the σ specificity subunit being primarily responsible for promoter recognition. Free σ, however, does not recognize promoter DNA, and it has been proposed that the intrinsic DNA binding ability is masked in free σ but becomes unmasked in the holoenzyme. Here, we use a newly developed fluorescent assay to quantitatively study the interactions of free σ(70) from Escherichia coli, the β'-σ complex, and the σ(70) RNA polymerase (RNAP) holoenzyme with non-template strand of the open promoter complex transcription bubble in the context of model non-template oligonucleotides and fork junction templates. We show that σ(70), free or in the context of the holoenzyme, recognizes the -10 promoter element with the same efficiency and specificity. The result implies that there is no need to invoke a conformational change in σ for recognition of the -10 element in the single-stranded form. In the holoenzyme, weak but specific interactions of σ are increased by contacts with DNA downstream of the -10 element. We further show that region 1 of σ(70) is required for stronger interaction with non-template oligonucleotides in the holoenzyme but not in free σ. Finally, we show that binding of the β' RNAP subunit is sufficient to allow specific recognition of the TG motif of the extended -10 promoter element by σ(70). The new fluorescent assay, which we call a protein beacon assay, will be instrumental in quantitative dissection of fine details of RNAP interactions with promoters.

Mechanism of Transcriptional Activation by Pseudomonas aeruginosa ExsA

ExsA is a transcriptional activator of the Pseudomonas aeruginosa type III secretion system (T3SS). The T3SS consists of >40 genes organized within 10 transcriptional units, each of which is controlled by the transcriptional activator ExsA. ExsA-dependent promoters contain two adjacent ExsA binding sites that when occupied protect the -30 to -70 region from DNase I cleavage. The promoters also possess regions bearing strong resemblance to the consensus -10 and -35 regions of sigma(70)-dependent promoters. The spacing distance between the putative -10 and -35 regions of ExsA-dependent promoters, however, is increased by 4 to 5 bp compared to that in typical sigma(70)-dependent promoters. In the present study, we demonstrate that ExsA-dependent transcriptional activation requires a 21- or 22-bp spacer length between the -10 and -35 regions. Despite the atypical spacing in this region, in vitro transcription assays using sigma(70)-saturated RNA polymerase holoenzyme (RNAP-sigma(70)) confirm that ExsA-dependent promoters are indeed sigma(70) dependent. Potassium permanganate footprinting experiments indicate that ExsA facilitates an early step in transcriptional initiation. Although RNAP-sigma(70) binds to the promoters with low affinity in the absence of ExsA, the activator stimulates transcription by enhancing recruitment of RNAP-sigma(70) to the P(exsC) and P(exsD) promoters. Abortive initiation assays confirm that ExsA enhances the equilibrium binding constant for RNAP while having only a modest effect on the isomerization rate constant.

Nuclear myosin I is necessary for the formation of the first phosphodiester bond during transcription initiation by RNA polymerase II

The nuclear isoform of myosin, Nuclear Myosin I (NMI) is involved in transcription by RNA polymerase I. Previous experiments showing that antibodies to NMI inhibit transcription by RNA polymerase II using HeLa cell nuclear extract (NE) suggested that NMI might be a general transcription factor for RNA polymerases. In this study we used a minimal in vitro transcription system to investigate the involvement of NMI in transcription by RNA polymerase II in detail. We demonstrate that NMI co-purifies with RNA polymerase II and that NMI is necessary for basal transcription by RNA polymerase II because antibodies to NMI inhibit transcription while adding NMI stimulates transcription. Further investigation revealed that NMI is specifically involved in transcription initiation. Finally, by employing an abortive transcription initiation assay, we demonstrate that NMI is crucial for the formation of the first phosphodiester bond during transcription initiation.

Determination of RNA Polymerase Binding and Isomerization Parameters by Measuring Abortive Initiations

Publisher Summary This chapter explores transcription initiation as a multistep process. The first step in initiation is the formation of a closed complex from free promoter and RNA polymerase. This is followed by a series of isomerization steps culminating in an open complex. The final step is promoter clearance. These steps, further divided in substeps observe that the abortive initiation method, developed as a kinetic method, can separate the bimolecular closed complex formation step from the unimolecular isomerization step. The overall forward rate constant, obtained from the classical two-step scheme, probably largely reflects the slowest step in the pathway. Even today, the abortive initiation remains one of the best methods to estimate the rate constant for the isomerization and stability of the closed complex. The chapter briefly discusses the principle of the abortive initiation assay, data processing, and the fluorescence-based abortive initiation assays.

Effect of A(n) tracts within the UP element proximal subsite of a model promoter on kinetics of open complex formation by Escherichia coli RNA polymerase.

In the open transcription complex (RPo), Escherichia coli RNA polymerase sigma(70) and alpha subunits are known to be in contact with each other and with the promoter region overlapping the -35 hexamer and the proximal part of the UP element. To probe the effect of A(n) DNA bending tracts in this region on initiation of transcription, kinetics of the formation of RPo by Escherichia coli RNA polymerase at two groups of synthetic consensus-like promoters bearing single DNA bending tracts (i). A(5 )within the proximal subsite region of the UP element (promoters Pk and Pl) and (ii). A(5)(Pg) or A(8)(Pm) in the region including the downstream end of the proximal UP subsite and the -35 consensus hexamer was studied in vitro using the fluorescence-detected abortive initiation assay. The kinetic data obtained demonstrate that the overall second-order rate constant k(a) of RPo formation is: (i.by almost one order of magnitude larger at Pk and Pl, relative to that at a control unbent promoter, and mainly due to a higher value of the equilibrium constant, K(1), of the initial closed complex; and (ii). several-fold smaller at Pg and Pm owing to a strongly decreased value of K(1). For Pm, the latter parameter was found to be dependent exponentially on four Mg(2+) ions, as compared with the seven ions remaining in equilibrium with the initial closed complex at the parent Pa promoter. This indicates that promoter region bearing a stiff A(8).T(8) fragment of B -DNA forms a smaller number of ionic contacts with the alpha subunit. These findings provide a new insight to and support the present model of interactions between RNA polymerase alpha and sigma(70) subunits with the proximal UP subsite and the -35 region of promoters.

Effect of A(n) tracts within the UP element proximal subsite of a model promoter on kinetics of open complex formation by Escherichia coli RNA polymerase.

In the open transcription complex (RPo), Escherichia coli RNA polymerase sigma(70) and alpha subunits are known to be in contact with each other and with the promoter region overlapping the -35 hexamer and the proximal part of the UP element. To probe the effect of A(n) DNA bending tracts in this region on initiation of transcription, kinetics of the formation of RPo by Escherichia coli RNA polymerase at two groups of synthetic consensus-like promoters bearing single DNA bending tracts (i). A(5 )within the proximal subsite region of the UP element (promoters Pk and Pl) and (ii). A(5)(Pg) or A(8)(Pm) in the region including the downstream end of the proximal UP subsite and the -35 consensus hexamer was studied in vitro using the fluorescence-detected abortive initiation assay. The kinetic data obtained demonstrate that the overall second-order rate constant k(a) of RPo formation is: (i.by almost one order of magnitude larger at Pk and Pl, relative to that at a control unbent promoter, and mainly due to a higher value of the equilibrium constant, K(1), of the initial closed complex; and (ii). several-fold smaller at Pg and Pm owing to a strongly decreased value of K(1). For Pm, the latter parameter was found to be dependent exponentially on four Mg(2+) ions, as compared with the seven ions remaining in equilibrium with the initial closed complex at the parent Pa promoter. This indicates that promoter region bearing a stiff A(8).T(8) fragment of B -DNA forms a smaller number of ionic contacts with the alpha subunit. These findings provide a new insight to and support the present model of interactions between RNA polymerase alpha and sigma(70) subunits with the proximal UP subsite and the -35 region of promoters.

Role of Activating Region 1 of Escherichia coliFNR Protein in Transcription Activation at Class II Promoters

FNR is an Escherichia coli transcription factor that activates gene expression in response to anaerobiosis at a large number of promoters by making direct contacts with RNA polymerase. At class II FNR-dependent promoters, where the DNA site for FNR overlaps the -35 element, activating region 1 of FNR is proposed to interact with the C-terminal domain of the RNA polymerase alpha-subunit. Using a model class II FNR-dependent promoter, FF(-41.5), we have performed in vivo and in vitro experiments to investigate the role of this interaction. Our results show that FNR, carrying substitutions in activating region 1, is compromised in its ability to promote open complex formation and thus to activate transcription. Abortive initiation assays were used to assess the contribution of activating region 1 of FNR to open complex formation. A new method for the purification of the FNR protein is also described.

Changes in the 17 bp spacer in the PR promoter of bacteriophage λ affect steps in open complex formation that precede DNA strand separation

Tau plots and temperature-shift experiments were used to determine which step in the formation of transcriptionally-competent open complexes is affected by changing the length of the 17 bp spacer separating the −10 and −35 consensus regions of the PR promoter of bacteriophage λ. Abortive initiation assays at 37 °C indicate that the primary effect of insertion of a base-pair, thereby increasing spacer length to 18 bp, is a decrease in kf, the rate constant for conversion from closed (RPc) to open (RPo) complexes, by approximately a factor of 4. The mutation did not significantly affect KB, the equilibrium constant for formation of closed complexes, and decreased KBkf by a factor of 3. Deletion of a bp to create a 16 bp spacer had a much greater effect, decreasing the measured value of kf by a factor of about 25 to 30, and KBkf by a factor of 7 to 8. When the values of the parameters for the deletion mutant were corrected for incomplete occupancy of RPo at equilibrium, the effects of the deletion were even greater. In particular, the corrected value of KBkf was about 15 times lower than the corresponding value for two promoters with wild-type spacing.Based on temperature shift experiments, the changes in spacer length did not affect the equilibrium at 20 °C between RPi, a stable intermediate in which DNA strands are not separated, and RPo. Although differential sensitivity of single-stranded bases to KMnO4 indicated that in about 20 % of the open complexes at 20 °C the DNA strands are not fully separated (RPo1), the distribution between these complexes and RPo2 (DNA strands fully separated) was also not affected significantly by changes in spacer length. Thus, changes in spacer length primarily affect k2, the rate constant for conversion of RPc to RPi, which corresponds to a nucleation of DNA strand-separation.Application of published data and/or algorithms for determining effects of nucleotide sequence on twist angle or rise at individual bp steps does not provide a simple explanation of the difference in promoter strength between PR derivatives with 16 bp spacing and those with 18 bp spacing.

Transcription activation by the Escherichia coli cyclic AMP receptor protein: determinants within activating region 3

At Class II CRP-dependent promoters, the Escherichia coli cyclic AMP receptor protein (CRP) activates transcription by making multiple interactions with RNA polymerase (RNAP). Two discrete surfaces of CRP, known as Activating Region 1 (AR1) and Activating Region 2 (AR2), interact with the C-terminal and N-terminal domains, respectively, of the α subunit of RNAP. Activating Region 3 (AR3) is a third separate surface of CRP, which is thought to interact with a target in the C-terminal region of the RNAP σ70 subunit. We have used a CRP mutant that functions primarily via AR3, CRP HL159 KE101 KN52, as a tool to identify residues within AR3 that are important for activation. This was achieved by screening a random mutant library of the gene encoding CRP HL159 KE101 KN52 for positive control mutants at Class II CRP-dependent promoters, and also by performing alanine scanning mutagenesis. Using both in vivo reporter assays and in vitro transcription assays, we measured the effects of key substitutions within AR3 on transcription activation in both CRP HL159 KE101 KN52 and wild-type CRP. We show that a cluster of negatively charged surface-exposed residues at positions 53, 54, 55 and 58 is required for optimal activation at a Class II, but not at a Class I, CRP-dependent promoter. We conclude that these residues in AR3 of CRP form an activatory determinant for Class II transcription activation. Abortive initiation assays were used to show that this activatory determinant accelerates the rate of isomerisation from the closed to open complex at a Class II CRP-dependent promoter. AR3 of CRP also contains an inhibitory determinant: the lysine residue at position 52 of CRP is inhibitory to maximal levels of transcription activation from Class II promoters. We show that the negative effects of K52 are not simply due to “masking” of the negatively charged residues at positions 53, 54, 55 and 58. Our results suggest that, during activation by wild-type CRP, the activatory and inhibitory determinants of AR3 balance each other. Thus, activation is predominantly determined by AR1 and AR2.

Stopped-flow kinetic analysis of the interaction of Escherichia coli RNA polymerase with the bacteriophage T7 A1 promoter

We have conducted a detailed kinetic and thermodynamic analysis of open complex formation between Escherichia coli RNA polymerase and the A1 promoter from bacteriophage T7 by monitoring alterations in the intrinsic protein fluorescence of RNA polymerase in stopped-flow kinetic studies. The stopped-flow kinetic data are consistent with a minimal model involving four steps for the formation of the open complex. Arrhenius plots for both the association and dissociation reactions for the equilibrium binding step leading to the formation of the closed complex were linear. With a positive van’t Hoff enthalpy (ΔHobs = 18(±3) kcal mol−1) and a positive entropy (ΔSobs = 94(±15) e.u.) change for the equilibrium binding process, formation of the closed complex is entropy driven. The value of the apparent association rate constant for this binding step was approximately three orders of magnitude less than that expected for facilitated binding. Thus, a minimum of two steps is required to describe the formation of the closed complex. A fast facilitated binding step appears to be followed by a conformational change in RNA polymerase which leads to the formation of the closed complex. A non-linear Arrhenius plot obtained for the isomerization step in the conversion of the closed complex to an open one indicates that there are at least two steps in the conversion of the closed complex to an open one. We have assigned the apparent activation energy of 9.1(±1.9) kcal mol−1 to the step involving a conformational change in the protein and nucleation of strand separation and the apparent activation energy of 46(±12) kcal mol−1 to the step involving strand separation. At 37°C, the value of the macroscopic isomerization rate constant (0.26(±0.02) s−1) in the conversion of the closed complex to an open one was an order of magnitude greater than the value reported in abortive initiation assays. This suggests that open complex formation is not the rate-determining step in the initiation of transcription in the case of the A1 promoter. To gain greater insight into the mechanism of initiation at the A1 promoter, we investigated the process of abortive product formation (pppApU) under conditions of non-saturating concentrations of the initiating nucleotide. A comparison of the lag times in the approach to the steady-state rate of abortive product formation when the reaction was initiated by the addition of UTP, ATP, the enzyme and the A1 promoter, respectively, indicates that the initiating nucleotide plays a key regulatory role in the initiation of transcription in the case of the A1 promoter.

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.

Multiple ATP-dependent steps in RNA polymerase II promoter melting and initiation.

Permanganate probing and abortive initiation assays were used to investigate the role of ATP in several successive stages of transcription initiation at the activated adeno E4 and mouse DHFR promoters. Removal of ATP at several points along the multi-step pathway blocked further progress towards its completion. Most strikingly, even if the DNA transcription start site is opened using ATP, the subsequent removal of ATP disallows formation of the first phosphodiester bond of the RNA. After ATP-dependent formation of a short RNA, a new transcription complex forms, which is more stable and has a longer open region. Both RNA and ATP appear to play roles in the formation of this complex. The need for ATP throughout this multi-step initiation pathway leads to new and unexpected possibilities for the use of energy and ATPases in transcription initiation.

Changing the mechanism of transcriptional activation by phage lambda repressor.

The first steps of transcription initiation include binding of RNA polymerase to a promoter to form an inactive, unstable, closed complex (described by an equilibrium constant, K(B)) and isomerization of the closed complex to an active, stable, open complex (described by a forward rate constant, k(f)). lambda cI protein activates the PRM promoter by specifically increasing k(f). A positive control mutant, cI-pc2, is defective for activation because it fails to raise k(f). An Arg to His change in the sigma70 subunit of RNA polymerase was previously obtained as an allele-specific suppressor of cI-pc2. To elucidate how the mutant polymerase restores the activation function of the mutant activator, abortive initiation assays were performed, using purified cI proteins and RNA polymerase holoenzymes. The change in sigma does not significantly alter K(B) or k(f) in the absence of cI protein. As expected, cI-pc2 activates the mutant polymerase in the same way that wild-type cI activates the wild-type polymerase, by increasing k(f). An unexpected and novel finding is that the wild-type activator stimulates the mutant polymerase, but not wild-type polymerase, by increasing K(B).

Transcription activation at class II CRP-dependent promoters: the role of different activating regions.

Transcription activation by the Escherichia coli cyclic AMP receptor protein (CRP) at Class II promoters is dependent on direct interactions between two surface-exposed activating regions (AR1 and AR2) and two contact sites in RNA polymerase. The effects on transcription activation of disrupting either AR1 or AR2 have been measured at different Class II promoters. AR2 but not AR1 is essential for activation at all the Class II promoters that were tested. The effects of single positive control substitutions in AR1 and AR2 vary from one promoter to another: the effects of the different substitutions are contingent on the -35 hexamer sequence. Abortive initiation assays have been used to quantify the effects of positive control substitutions in each activating region on the kinetics of transcription initiation at the Class II CRP- dependent promoter pmelRcon. At this promoter, the HL159 substitution in AR1 results in a defect in the initial binding of RNA polymerase whilst the KE101 substitution in AR2 reduces the rate of isomerization from the closed to the open complex.

The Transcription Initiation Pathway of Sigma 54 Mutants That Bypass the Enhancer Protein Requirement: IMPLICATIONS FOR THE MECHANISM OF ACTIVATION

In vitro transcription, DNase I footprinting, and abortive initiation assays were used to characterize transcription using mutant forms of sigma 54 shown previously to bypass certain enhancer requirements in vitro. The holoenzymes containing these sigma mutants produce low levels of open complexes at both the glnAp2 and glnHp2 promoters. The open complexes are unusual in that they are destroyed by heparin. Enhancer protein and ATP convert them into a stable heparin-resistant state. The enhancer response occurs over a similar range of NtrC concentration as occurs with the wild-type holoenzyme, indicating that the activation determinants have been largely preserved within these mutants. One-round transcription assays show that the mutant holoenzymes can be driven to transcribe both promoters without NtrC. The unstable opening induced by these mutations apparently serves as a conduit that can shuttle templates into transcriptionally competent complexes. The results lead to a model in which activation occurs in two steps. First, the enhancer complex overcomes an inhibitory effect of the sigma 54 leucine patch and unlocks the melting activity of the holoenzyme. Second, different sigma 54 determinants are used to drive stabilization of the open complexes, allowing the full transcription potential to be realized.

DNA-dependent protein kinase: a potent inhibitor of transcription by RNA polymerase I.

DNA-dependent protein kinase (DNA-PK) comprises a catalytic subunit of approximately 350 kD (p350) and a DNA-binding component termed Ku. Although DNA-PK can phosphorylate many transcription factors, no function for this enzyme in transcription has been reported thus far. Here, we show that DNA-PK strongly represses transcription by RNA polymerase I (Pol I). Transcriptional repression by DNA-PK requires ATP hydrolysis, and DNA-PK must be colocalized on the same DNA molecule as the Pol I transcription machinery. Consistent with DNA-PK requiring DNA ends for activity, transcriptional inhibition only occurs effectively on linearized templates. Mechanistic studies including single-round transcriptions, abortive initiation assays, and factor-independent transcription on a tailed template demonstrate that DNA-PK inhibits initiation (i.e., the formation of the first phosphodiester bonds) but does not affect transcription elongation. Repression of transcription involves phosphorylation of the transcription initiation complex, and rescue experiments reveal that the inactivated factor remains bound to the promoter and thus prevents initiation complex formation. We discuss the possible relevance of these findings in regard to the control of rRNA synthesis in vivo.

Mechanistic aspects of tagetitoxin inhibition of RNA polymerase from Escherichia coli.

Tagetitoxin inhibits RNA synthesis directed by bacterial RNA polymerase, and the current study explores several mechanistic aspects of this inhibition. Tagetitoxin inhibition of in vitro RNA synthesis directed by Escherichia coli RNA polymerase is independent of the template DNA concentration. The toxin can block Escherichia coli RNA polymerase during elongation of a nascent RNA chain. In abortive initiation assays, the rate of dinucleotide formation is inhibited by tagetitoxin when initiated with ATP or CpA but not when AMP is used to initiate. Formation of longer oligonucleotides is inhibited by the toxin regardless of the initiating nucleotide. These abortive initiation studies indicate that tagetitoxin does not affect nucleotide substrate binding or phosphodiester bond formation and suggest that the toxin may interfere with a subsequent step. It is suggested that tagetitoxin affects the stability of nascent oligonucleotide binding and/or the translocation of the catalytic center with respect to the 3'-OH of nascent oligonucleotides.

Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II

Using a defined RNA polymerase II (pol II) transcription system, we have investigated the roles of basal factors at discrete stages during the transcription cycle (e.g., initiation, promoter clearance, and transcript elongation). Abortive initiation assays revealed that TATA-binding protein, transcription factors TFIIB and TFIIF, and pol II were necessary and sufficient to form functional initiation complexes on both linear and supercoiled templates. By contrast, TFIIE, TFIIH, and ATP hydrolysis were additionally required during promoter clearance from linear templates, while negative supercoiling obviated the need for these auxiliary factors. Furthermore, TFIIE, TFIIH, and supercoiling were not required during elongation. Our results suggest a role for TFIIH-associated helicase activity or supercoiling during promoter clearance rather than open complex formation. These results establish abortive initiation as a useful assay for studying functional initiation complex formation in defined eukaryotic transcription systems and provide a framework for investigating regulation at different stages of the eukaryotic transcription cycle.

Kinetics of activation of the P4 promoter of pBR322 by the Escherichia coli cyclic AMP receptor protein.

The activation of transcription initiation from the P4 promoter of pBR322 by the Escherichia coli cyclic AMP receptor protein (CRP) has been investigated using a fluorescence abortive initiation assay. The effect of the cyclic-AMP/CRP complex on the linear P4 promoter was to increase the initial binding (KB) of RNA polymerase to the promoter by about a factor of 10, but the rate of isomerization of closed to open complex (kf) was unaffected. One molecule of CRP per promoter was required for activation, and the concentration of cyclic AMP producing half-maximal stimulation was about 7-8 microM. Supercoiling caused a 2-3-fold increase in the rate of isomerization of the CRP-activated promoter, but weakened the initial binding of polymerase by about one order of magnitude. The unactivated supercoiled promoter was too weak to allow reliable assessment of kinetic parameters against the high background rate originating from the rest of the plasmid.