Free RNA Polymerase Research Articles

Fluorescence detection of nucleic acids and proteins in multi-component crystals

A highly sensitive and specific assay for the rapid detection of nucleic acids in crystals of macromolecular complexes is reported. The assay is based on the observation of crystals under a fluorescence microscope after incubation with a fluorescent dye that specifically binds nucleic acids. The assay allows clear distinction between crystals of free RNA polymerase II and crystals of the polymerase bound to DNA and RNA or RNA alone, even when the nucleic acids account for only 3% of the crystalline material. Fluorescence detection was also used to monitor the diffusion and accumulation of a fluorescence-labelled polymerase-binding protein in preformed polymerase crystals. Additional experiments suggest the general applicability of these fluorescence assays for the characterization of crystal contents.

The involvement of the aspartate triad of the active center in all catalytic activities of multisubunit RNA polymerase.

Three conserved aspartate residues in the largest subunit of multisubunit RNA polymerases (RNAPs) coordinate two Mg2+ ions involved in the catalysis of phosphodiester bond synthesis. A structural model based on the stereochemistry of nucleotidyl transfer reaction as well as recent crystallographic data predict that these Mg2+ ions should also be involved in the reverse reaction of pyrophosphorolysis as well as in the endo- and exonucleolytic cleavage of the nascent RNA. Here, we check these predictions by constructing point substitutions of each of the three Asp residues in the β′ subunit of Escherichia coli RNAP and testing the mutant enzymes' functions. Using artificially assembled elongation complexes, we demonstrate that substitutions of any of the three aspartates dramatically reduce all known RNAP catalytic activities, supporting the model's predictions that same amino acids participate in all RNAP catalytic reactions. We demonstrate that though substitutions in the DFDGD motif decrease Mg2+ binding to free RNAP below detection limits, the apparent affinity to Mg2+ in transcription complexes formed by the mutant and wild-type RNAPs is similar, suggesting that NTP substrates and/or nucleic acids actively contribute to the retention of active center Mg2+.

Control of rRNA synthesis in Escherichia coli: a systems biology approach.

The first part of this review contains an overview of the various contributions and models relating to the control of rRNA synthesis reported over the last 45 years. The second part describes a systems biology approach to identify the factors and effectors that control the interactions between RNA polymerase and rRNA (rrn) promoters of Escherichia coli bacteria during exponential growth in different media. This analysis is based on measurements of absolute rrn promoter activities as transcripts per minute per promoter in bacterial strains either deficient or proficient in the synthesis of the factor Fis and/or the effector ppGpp. These absolute promoter activities are evaluated in terms of rrn promoter strength (V(max)/K(m)) and free RNA polymerase concentrations. Three major conclusions emerge from this evaluation. First, the rrn promoters are not saturated with RNA polymerase. As a consequence, changes in the concentration of free RNA polymerase contribute to changes in rrn promoter activities. Second, rrn P2 promoter strength is not specifically regulated during exponential growth at different rates; its activity changes only when the concentration of free RNA polymerase changes. Third, the effector ppGpp reduces the strength of the rrn P1 promoter both directly and indirectly by reducing synthesis of the stimulating factor Fis. This control of rrn P1 promoter strength forms part of a larger feedback loop that adjusts the synthesis of ribosomes to the availability of amino acids via amino acid-dependent control of ppGpp accumulation.

Direct Stimulation of the λpaQ Promoter by the Transcription Effector Guanosine-3′,5′-(bis)pyrophosphate in a Defined in Vitro System

The bacterial response to nutritional deprivation, called the stringent response, results in the introduction of the specific nucleotide guanosine-3',5'-(bis) pyrophosphate (ppGpp). This nucleotide interacts with RNA polymerase and alters its action so that transcription from certain promoters is inhibited, whereas transcription from others seems to be activated. The exact mechanism of transcriptional stimulation by ppGpp in vivo remains unknown. A passive control model has been proposed according to which transcription inhibition during the stringent response at several very active promoters, like those for rRNA and tRNA genes, makes more free RNA polymerase (RNAP) molecules available for transcription at promoters with weak binding affinities for RNAP, thus leading to their passive activation. Among promoters whose transcription is activated by ppGpp in vivo is the histidine operon promoter (hisGp). However, in vitro it is only possible to demonstrate this effect in a coupled transcription-translation system. Here we demonstrate, using another in vivo ppGpp-stimulated promoter, the phage lambdapaQ promoter, that activation by ppGpp in a defined in vitro system is direct. A systematic study of ppGpp effects on the stimulation of paQ revealed that, as in the case of promoters inhibited by this nucleotide, ppGpp decreases the half-life of paQ open complexes. Our results also indicate that the equilibrium binding affinity of RNA polymerase to paQ seems not to be affected in the presence of ppGpp. Our data indicate that the mechanism underlying ppGpp stimulation of paQ is due to an increased rate of productive open complex formation.

Dephosphorylation of RNA Polymerase II by CTD-phosphatase FCP1 is Inhibited by Phospho-CTD Associating Proteins

Reversible phosphorylation of the repetitive C-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit plays a key role in the progression of RNAP through the transcription cycle. The level of CTD phosphorylation is determined by multiple CTD kinases and a CTD phosphatase, FCP1. The phosphorylated CTD binds to a variety of proteins including the cis/ trans peptidyl-prolyl isomerase (PPIase) Pin1 and enzymes involved in processing of the primary transcript such as the capping enzyme Hce1 and CA150, a nuclear factor implicated in transcription elongation. Results presented here establish that the dephosphorylation of hyperphosphorylated RNAP II (RNAP IIO) by FCP1 is impaired in the presence of Pin1 or Hce1, whereas CA150 has no influence on FCP1 activity. The inhibition of dephosphorylation is observed with free RNAP IIO generated by different CTD kinases as well as with RNAP IIO engaged in an elongation complex. These findings support the idea that specific phospho-CTD associating proteins can differentially modulate the dephosphorylation of RNAP IIO by steric hindrance and may play an important role in the regulation of gene expression.

Free RNA polymerase and modeling global transcription in Escherichia coli

Growth rate-dependent changes in the cytoplasmic concentration of free functional RNA polymerase, [ R f], affect the activity of all bacterial genes. Since [ R f] is not accessible to direct experimental quantitation, it can only be found indirectly from an evaluation of promoter activity data. Here, a theory has been derived to calculate [ R f] from the concentrations of total RNA polymerase and promoters in a model system with known Michaelis–Menten constants for the polymerase–promoter interactions. The theory takes transcript lengths and elongation rates into account and predicts how [ R f] changes with varying gene dosages. From experimental data on total concentrations of RNA polymerase and kinetic properties of different classes of promoters, the theory was developed into a mathematical model that reproduces the global transcriptional control in Escherichia coli growing at different rates. The model allows an estimation of the concentrations of free and DNA-bound RNA polymerase, as well as the partitioning of RNA polymerase into mRNA and stable RNA synthesizing fractions. According to this model, [ R f] is about 0.4 and 1.2 μM at growth rates corresponding to 1.0 and 2.5 doublings/h, respectively. The model accurately reflects a number of further experimental observations and suggests that the free RNA polymerase concentration increases with increasing growth rate.

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.

Kinetic properties of rrn promoters in Escherichia coli

How do bacteria adapt and optimize their growth in response to different environments? The answer to this question is intimately related to the control of ribosome bio-synthesis. During the last decades numerous proposals have been made to explain this control but none has been definitive. To readdress the problem, we have used measurements of rRNA synthesis rates and rrn gene dosages in E. coli to find the absolute transcription rates of the average rrn operon (transcripts per min per operon) at different growth rates. By combining these rates with lacZ expression data from rRNA promoter- lacZ fusions, the abolute activities of the isolated rrnB P1 and P2 promoters were determined as functions of the growth rate in the presence and absence of Fis and of the effector ppGpp. The promoter activity data were analyzed to obtain the relative concentrations of free RNA polymerase, [ R f], and the ratio of the Michaelis-Menten parameters, V max/ K m (promoter strength), that characterize the promoter-RNA polymerase interaction. The results indicate that changes in the basal concentration of ppGpp can account for all growth-medium dependent regulation of the rrn P1 promoter strength. The P1 promoter strength was maximal when Fis was present and the level of ppGpp was undetectable during growth in rich media or in ppGpp-deficient strains; this maximal strength was 3-fold reduced when Fis was removed and the level of ppGpp remained undetectable. At ppGpp levels above 55 pmol per cell mass unit (OD 460) during growth in poor media, the P1 promoter strength was minimal and not affected by the presence or absence of fis. The half-maximal value occurred at 20 pmol ppGpp/OD 460 and corresponds to an intracellular concentration of about 50 μM. In connection with previously published data, the results suggest that ppGpp reduces the P1 promoter strength directly, by binding RNA polymerase, and indirectly, by inhibiting the synthesis of Fis.

In vitro transcription of a torsionally constrained template.

RNA polymerase (RNAP) and the DNA template must rotate relative to each other during transcription elongation. In the cell, however, the components of the transcription apparatus may be subject to rotary constraints. For instance, the DNA is divided into topological domains that are delineated by rotary locked boundaries. Furthermore, RNAPs may be located in factories or attached to matrix sites limiting or prohibiting rotation. Indeed, the nascent RNA alone has been implicated in rotary constraining RNAP. Here we have investigated the consequences of rotary constraints during transcription of torsionally constrained DNA by free RNAP. We asked whether or not a newly synthesized RNA chain would limit transcription elongation. For this purpose we developed a method to immobilize covalently closed circular DNA to streptavidin-coated beads via a peptide nucleic acid (PNA)-biotin conjugate in principle mimicking a SAR/MAR attachment. We used this construct as a torsionally constrained template for transcription of the beta-lactamase gene by Escherichia coli RNAP and found that RNA synthesis displays similar characteristics in terms of rate of elongation whether or not the template is torsionally constrained. We conclude that transcription of a natural bacterial gene may proceed with high efficiency despite the fact that newly synthesized RNA is entangled around the template in the narrow confines of torsionally constrained supercoiled DNA.

Induction of σ B-dependent general stress genes by amino acid starvation in a spo0H mutant of Bacillus subtilis

Solely σ B-dependent genes like gsiB and gspA are not significantly induced in amino acid-starved wild-type cells, since amino acid starvation does not trigger activation of σ B. The general stress gene yvyD is subject to the control of both σ B and σ H therefore displaying induction in response to amino acid starvation at the σ H-dependent promoter. Surprisingly, the proteins YvyD, GsiB and GspA were significantly induced in amino acid-starved cells of a strain lacking σ H activity. Transcriptional studies provided evidence that σ B-dependent transcription is indeed induced in a spo0H mutant during amino acid starvation and depends on RsbP but not on RsbU indicating that the stress signal transduction is not required for this induction. A similar phenomenon of σ B activation was observed in amino acid-starved cells of a spo0A deletion mutant. The σ B-dependent transcription in a spo0H mutant further needs an active RelA protein which is responsible for strong repression of house-keeping genes after amino acid starvation (stringent response). Our data indicate that in the absence of σ H and under conditions which provoke the stringent response, RsbP-dependent levels of active σ B can more effectively compete for increased levels of free RNA polymerase core enzyme leading to the induction of the probably strongest σ B-dependent genes.

Induction of sigma(B)-dependent general stress genes by amino acid starvation in a spo0H mutant of Bacillus subtilis.

Solely sigma(B)-dependent genes like gsiB and gspA are not significantly induced in amino acid-starved wild-type cells, since amino acid starvation does not trigger activation of sigma(B). The general stress gene yvyD is subject to the control of both sigma(B) and sigma(H) therefore displaying induction in response to amino acid starvation at the sigma(H)-dependent promoter. Surprisingly, the proteins YvyD, GsiB and GspA were significantly induced in amino acid-starved cells of a strain lacking sigma(H) activity. Transcriptional studies provided evidence that sigma(B)-dependent transcription is indeed induced in a spo0H mutant during amino acid starvation and depends on RsbP but not on RsbU indicating that the stress signal transduction is not required for this induction. A similar phenomenon of sigma(B) activation was observed in amino acid-starved cells of a spo0A deletion mutant. The sigma(B)-dependent transcription in a spo0H mutant further needs an active RelA protein which is responsible for strong repression of house-keeping genes after amino acid starvation (stringent response). Our data indicate that in the absence of sigma(H) and under conditions which provoke the stringent response, RsbP-dependent levels of active sigma(B) can more effectively compete for increased levels of free RNA polymerase core enzyme leading to the induction of the probably strongest sigma(B)-dependent genes.

Cytoplasmic RNA Polymerase in Escherichia coli.

To obtain an estimate for the concentration of free functional RNA polymerase in the bacterial cytoplasm, the content of RNA polymerase beta and beta' subunits in DNA-free minicells from the minicell-producing Escherichia coli strain chi925 was determined. In bacteria grown in Luria-Bertani medium at 2.5 doublings/h, 1.0% of the total protein was RNA polymerase. The concentration of cytoplasmic RNA polymerase beta and beta' subunits in minicells produced by this strain corresponded to about 17% (or 2.5 microM) of the value found in whole cells. Literature data suggest that a similar portion of cytoplasmic RNA polymerase subunits is in RNA polymerase assembly intermediates and imply that free functional RNA polymerase can form a small percentage of the total functional enzyme in the cell. On infection with bacteriophage T7, 20% of the minicells produced progeny phage, whereas infection in 80% of the cells was abortive. RNA polymerase subunits in lysozyme-freeze-thaw lysates of minicells were associated with minicell envelopes and were without detectable activity in an in vitro transcription assay. Together, these results suggest that most functional RNA polymerase is associated with the DNA and that little if any segregates into DNA-free minicells.

Age-related changes of protein- and RNA-synthetic processes in experimental hyper- and hypothyroidism.

The rate of liver and plasma protein synthesis and the activity of liver RNA polymerases 1 and 2 were investigated in rats of various age under experimental hyper- and hypothyroidism. The rate of plasma protein synthesis decreased with age more dramatically than that of liver proteins. Hyper- and hypothyroidism exerted opposite effects on protein synthesis in rats: stimulation and inhibition, respectively. The manifestation of these effects was age related. The thyroid status of animals also influenced the balance of protein synthesis. Thyroxin administration caused preferential incorporation of a label into blood plasma proteins. Changes of thyroid status of old animals insignificantly affected the absolute values of the label incorporation into proteins and the ratio of the label incorporation into local and secreted liver proteins. Age-related decrease of total hepatic nuclear RNA-polymerase activity was due to reduction of the template-bound functionally active forms of RNA-polymerases 1 and 2. Administration of thyroxin caused initial redistribution of the enzyme activity between template-bound and free fractions accompanied by the increase of template bound RNA-polymerases. Prolonged hormonal stimulus also caused an increase of free RNA-polymerases, which reflects the increased synthesis of these enzymes. Mecrazolyl administration reduced the activity of RNA-polymerase 1 and 2. All age groups were characterized by preferential reduction of the bound form. RNA-polymerase 2 activity decreased to a greater extent than that of RNA-polymerase 1. The data suggest age-determined reactions of the body to altered thyroid status.

Mediator of transcriptional regulation.

Three lines of evidence have converged on a multiprotein Mediator complex as a conserved interface between gene-specific regulatory proteins and the general transcription apparatus of eukaryotes. Mediator was discovered as an activity required for transcriptional activation in a reconstituted system from yeast. Upon resolution to homogeneity, the activity proved to reside in a 20-protein complex, which could exist in a free state or in a complex with RNA polymerase II, termed holoenzyme. A second line of evidence came from screens in yeast for mutations affecting transcription. Two-thirds of Mediator subunits are encoded by genes revealed by these screens. Five of the genetically defined subunits, termed Srbs, were characterized as interacting with the C-terminal domain of RNA polymerase II in vivo, and were shown to bind polymerase in vitro. A third line of evidence has come recently from studies in mammalian transcription systems. Mammalian counterparts of yeast Mediator were shown to interact with transcriptional activator proteins and to play an essential role in transcriptional regulation. Mediator evidently integrates and transduces positive and negative regulatory information from enhancers and operators to promoters. It functions directly through RNA polymerase II, modulating its activity in promoter-dependent transcription. Details of the Mediator mechanism remain obscure. Additional outstanding questions include the patterns of promoter-specificity of the various Mediator subunits, the possible cell-type-specificity of Mediator subunit composition, and the full structures of both free Mediator and RNA polymerase II holoenzyme.

The Sensitivity of RNA Polymerase II in Elongation Complexes to C-terminal Domain Phosphatase

The phosphorylation state of the carboxyl-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit plays an important role in the regulation of transcript elongation. This report examines the sensitivity of RNAP II to dephosphorylation by CTD phosphatase (CTDP) and addresses factors that regulate its sensitivity. The CTDP sensitivity of RNAP IIO in paused elongation complexes on a dC-tailed template does not significantly differ from that of free RNAP IIO. RNAP IIO contained in elongation complexes that initiate transcription from the adenovirus-2 major late promoter in the presence of a nuclear extract is relatively resistant to dephosphorylation. Complexes treated with 1% Sarkosyl remain elongation-competent but demonstrate a 5-fold increase in CTDP sensitivity. Furthermore, the sensitivity of RNAP IIO in both control and Sarkosyl-treated elongation complexes is dependent on their position relative to the start site of transcription. Elongation complexes 11-24 nucleotides downstream are more sensitive to dephosphorylation than complexes 50-150 nucleotides downstream. The incubation of Sarkosyl-treated elongation complexes with nuclear extract restores the original resistance to dephosphorylation. These results suggest that a conformational change occurs in RNAP II as it clears the promoter, which results in an increased resistance to dephosphorylation. Furthermore, the sensitivity to dephosphorylation can be modulated by a factor(s) present in the nuclear extract.

Activities of constitutive promoters in Escherichia coli

The in vivo activities of seven constitutive promoters in Escherichia coli have been determined as functions of growth rate in wild-type relA+spoT+strains with normal levels of guanosine tetraphosphate (ppGpp) and in ppGpp-deficient ΔrelAΔspoT derivatives. The promoters include (i) the spc ribosomal protein operon promotor Pspc; (ii) the β-lactamase gene promotor Pblaof plasmid pBR322; (iii) the PLpromoter of phage λ; (iv) and (v) the replication control promoters PRNAIand PRNAIIof plasmid pBR322; and (vi) and (vii) the P1 and P2 promoters of the rrnB ribosomal RNA operon. Each strain carried an operon fusion consisting of one of the respective promoter regions linked to lacZ and recombined into the chromosome at the mal locus of alac deletion strain. The amount of 5′-terminal lacZ mRNA and of β-galactosidase activity expressed from these promoters were determined by standard hybridization or enzyme activity assays, respectively. In addition, DNA, RNA and protein measurements were used to obtain information about gene dosage, rRNA synthesis and translation rates. By combininglacZ mRNA hybridization data with gene dosage and rRNA synthesis data, the absolute activity of the different promoters, in transcripts/minute per promoter, was determined. In ppGpp-proficient (relA+spoT+) strains, the respective activities of rrnB P1 and P2 increased 40 and fivefold with increasing growth rate between 0.7 and 3.0 doublings/hour. The activities of Pspc, PL, Pbla, and PRNAIincreased two- to threefold and reached a maximum at growth rates above 2.0 doublings/hour. In contrast, PRNAIIactivity decreased threefold over this range of growth rates. In ppGpp-deficient (ΔrelAΔspoT) bacterial strains, the activities of rrnB P1 and P2 promoters both increased about twofold between 1.6 and 3.0 doublings/hour, whereas the activities of Pspc, PL, Pbla, and PRNAI, and PRNAIIwere about constant. To explain these observations, we suggest that the cellular concentration of free RNA polymerase increases with increasing growth rate; for saturation the P1 and P2 rRNA promoters require a high RNA polymerase concentration that is approached only at the highest growth rates, whereas the other promoters are saturated at lower polymerase concentrations achieved at intermediate growth rates. In addition, the data indicate that the respective rrnB P1 and PRNAIIpromoters were under negative and positive control by ppGpp. This caused a reduced activity ofrrnB P1 and an increased activity of PRNAIIduring slow growth in wild-type (relA+spoT+) relative to ppGpp-deficient (ΔrelAΔspoT) bacterial strains.

Repression and activation of promoter-bound RNA polymerase activity by gal repressor

By binding to the DNA site OE at position −60.5 in the gal operon, the GalR protein activates transcription from the P2 promoter located on the opposite face of DNA (position −5) and represses transcription from the P1 promoter located on the same face (position +1). GalR increases RNA polymerase binding at P2 and inhibits isomerization at P1 by forming a GalR-DNA-RNA polymerase ternary complex in each case. The specific effect of GalR at one promoter is independent of the presence of the other promoter. The enhancement or repression is also not the intrinsic property of a promoter; the regulation can be reversed by switching the angular orientation of the promoters relative to OE. Both enhancement and repression appear to require the same interaction between RNA polymerase α-subunit and GalR and/or the same interaction between RNA polymerase α-subunit and DNA in the ternary complexes. We have discussed how GalR might exert opposite effects in the steps involved in the formation of the open complex from free RNA polymerase and DNA.

Reversible Phosphorylation of the C-terminal Domain of RNA Polymerase II

Reversible Phosphorylation of the C-terminal Domain of RNA Polymerase II

Control of the Escherichia coli rrnB P1 Promoter Strength by ppGpp

Fusions of the rrnB P1 and P2 promoters, and of the tandem P1-P2 combination, to a wild-type lacZ gene were constructed on plasmids and recombined into the mal region of the bacterial chromosome, close to the normal location and in the normal orientation of rrnB. The upstream activator region (Fis-binding sites) was always present with the P1 promoter, and all constructs contained the box A antitermination site of rRNA genes. Using these constructs, beta-galactosidase specific activities were measured in Escherichia coli strains carrying either both ppGpp synthetases, PSI and PSII (relA+ spoT+), or only PSII (delta relA spoT+), or neither (delta relA delta spoT), using different media supporting growth rates between 0.6 and 2.8 doublings/h at 37 degrees C. The beta-galactosidase activities were used to estimate the relative strength of the rrnB P1 promoter in comparison to the isolated rrnB P2 promoter. Promoter strength (transcripts initiated per min per promoter per free RNA polymerase concentration) was distinguished from promoter activity (transcripts initiated per min per promoter). In ppGpp-synthesizing (wild-type) bacteria, the relative strength of the rrnB P1 promoter increased nearly 10-fold with increasing growth rate from 0.17 to 1.5, but in the ppGpp-less double mutants it decreased by 20% from 1.7 to 1.5. Thus, at low or zero levels of ppGpp, the P1 promoter was 1.5-1.7 times stronger than the isolated P2 promoter. These results indicate that the normal growth rate control of the rrnB P1 promoter strength requires ppGpp, and that the strength is reduced at basal levels of ppGpp found during exponential growth. No additional ppGpp-independent control of the rrnB P1 promoter strength was evident. From the beta-galactosidase data and previously determined values of rRNA gene activities, the activities of the isolated rrnB P1 and P2 promoters, and of the P2 promoter in the tandem combination, were estimated. With increasing growth rate, the activity of the isolated P2 promoter increased 6-fold from 6 to 33 initiations/min, while the activity of the isolated P1 promoter increased 24-fold from 2 to 54 initiations/min. The increasing activity of the isolated P2 promoter is assumed to reflect the increasing RNA polymerase concentration at constant promoter strength, whereas the steeper increase in P1 promoter activity reflects increases in both polymerase concentration and promoter strength. When in tandem with P1, the P2 promoter activity is inferred to decrease as the P1 promoter activity increases.

Epitope mapping and functional characterization of monoclonal antibodies specific for the alpha subunit of Escherichia coli RNA polymerase.

The epitopes have been localized for a set of monoclonal antibodies specific for the alpha subunit of the Escherichia coli RNA polymerase. The antibodies are classified into three groups based on their epitopic assignments. Group 1, mAb 123C2, maps in the N terminus of alpha between amino acids 1 and 23; Group 2 antibodies (mAb 129C4, mAb 124D1 and mAb 121C5) map in the central region between amino acids 190 and 210; Group 3 antibodies (mAb 130B1 and mAb 125C6) map in the C terminus between amino acids 310 and 320. mAb 130C2 is anomalous since it maps to the N terminus between amino acids 1 and 23 as well as to the C terminus between amino acids 320 and 329. The antibodies were used to investigate the role of alpha in transcription activation with cAMP receptor protein-dependent promoters. Three antibodies (130C2, 121C5, and 125C6) inhibited cAMP receptor protein-dependent initiation with lac P+ but not with lac UV5 or gal P+. Inhibition was observed with free RNA polymerase and the closed promoter complex; the preformed open promoter complex was insensitive. Only lac P+ was sensitive to these anti-alpha antibodies supporting the concept that the mode of interaction of RNA polymerase with cAMP receptor protein differs between lac P+ and gal P+.