A stressed intermediate in the formation of stably initiated RNA chains at the Escherichia coli lac UV5 promoter.

We report experiments designed to elucidate the mechanism by which RNA polymerase advances from the open complex to synthesis of a stably bound RNA chain during transcription initiation. Techniques used include deoxyribonuclease I footprinting, methylation protection, and exonuclease III digestion through upstream domains, each applied to the open, abortive and productive transcription complexes of Escherichia coli RNA polymerase with the lac promoter. The results show a slight loss of upstream open complex contacts during abortive transcription of a 6-mer and 8-mer, but a large loss of these contacts upon escape from abortive cycling into productive transcription at the 11-mer. We propose a model for early initiation in which competition between open complex polymerase-DNA contacts on one hand and initiated complex polymerase-DNA-RNA interactions on the other produces a "stressed intermediate" during formation of a short RNA-DNA duplex. The strain energy is relieved either by ejecting the short RNA, resulting in aborted initiation, or by eliminating the sigma subunit and breaking the open complex contacts, thereby escaping abortive cycling into productive transcription. Further evidence for this model is based on the observation that destabilization of interactions specific for either open complex or initiated complex has the predicted effect on the amount of abortive cycling. The model predicts a complicated relationship between overall promoter strength and DNA sequence changes that alter polymerase-DNA interactions.

Comparison of the open complexes formed by RNA polymerase at the Escherichia coli lac UV5 promoter.

In transcription initiation at the lac UV5 promoter, Escherichia coli RNA polymerase forms two open complexes, called Ou and O1, which can be separated by electrophoresis on native polyacrylamide gels. We have compared the properties of these two open complexes, with the objective of rationalizing the functional difference previously reported between the two forms: the complex which is dominant at high temperature (Ou) is better able to escape abortive transcriptional cycling into productive mRNA elongation. Methylation protection and binding domain probing with exonuclease III were used to investigate differences in polymerase binding strength to particular DNA domains. Also, we examined the difference in the extent and temperature dependence of promoter unwinding in the two complexes, as probed by methylation of unpaired cytosines and cleavage by phage T7 endonuclease. We find that O1 has stronger promoter interactions in the DNA domain whose upstream edge is defined by an exonuclease III stop at -24. These -24 domain interactions, which presumably aid in promoter binding and nucleation of DNA unwinding, are inferred to be strong enough to hinder escape of the polymerase from the open complex contacts that are maintained during abortive initiation. The Ou complex has weaker binding to the -24 domain, partially compensated by better upstream interactions and a better ability to accommodate extensive DNA unwinding. It thus escapes abortive initiation more readily because of weaker critical open complex contacts that must be lost when stable initiation occurs from the corresponding stressed intermediates.

Synergy between Escherichia coli CAP protein and RNA polymerase in the lac promoter open complex.

Characterization of ternary complexes containing an Escherichia coli lac promoter DNA fragment, CAP protein and RNA polymerase, separated on non-denaturing polyacrylamide gels and footprinted in the gel slice, reveals a striking stabilization of CAP against dissociation in the open complex, compared to the CAP-DNA complex lacking RNA polymerase. The stabilization is lost when half a helical turn of DNA is inserted between CAP and polymerase sites, but is partially restored with an 11 base-pair insert; stimulation of transcription parallels the stabilization effect. This behavior suggests a direct protein-protein interaction. Comparison of initiation kinetics for wild-type and a mutant in which the P2 promoter has been inactivated shows that CAP both strengthens binding in the closed complex and accelerates isomerization to the open complex; the latter effect accounts for the bulk of the observed transcriptional activation.

Characterization of the PDA1 promoter of Nectria haematococca and identification of a region that binds a pisatin-responsive DNA binding factor.

Isolates of Nectria haematococca (anamorph: Fusarium solani) are able to detoxify the pea phytoalexin pisatin through expression of pisatin demethylase (pda). This enzyme is a substrate-inducible cytochrome P450 monooxyenase that is encoded by the PDA gene family. In the current study, PDA1, a highly inducible PDA gene, was cloned and the 5' untranslated region was sequenced. The PDA mRNA levels were measured in pisatin-treated mycelium and found to increase by 20-fold over untreated control. Gel shift assays identified a 35-bp region, -514 to -480 bp relative to the first mRNA start site, that binds a factor found in extracts of pisatin-treated mycelium and absent in untreated mycelium. The function of the binding site in pisatin regulation of the PDA1 gene was tested in an in vivo competition assay by introduction of multiple ectopic copies of the binding site into N. haematococca through transformation. In such transformants, induction of pda activity by pisatin was delayed and reduced, consistent with the titration of a trans-acting factor which responds to pisatin. These results suggest the 35-bp region is functioning as a pisatin-responsive activator binding site for PDA1. Additional controls were characterized that act on PDA1 expression. Induction of pda by pisatin was suppressed by the addition of 0.8% Casamino Acids or 5% glucose to the suspended mycelium. A unique DNA binding factor was detected only in extracts from mycelia treated with the Casamino Acids that bind to the same 35-bp region of the PDA1 gene as the pisatin-responsive factor.

PCR-based construction of promoter/G-free templates for in vitro transcription analysis allows selection of plasmids with optimal activity in homologous extracts.

In vitro transcription has been used for dissecting transcriptional controls in many eukaryotic systems. One modification which greatly reduces background non-specific transcription is the placement of a guanosine-free (G-free) region of DNA immediately downstream from a promoter [Sawadogo and Roeder, Proc. Natl. Acad. Sci. USA 82 (1985) 4394-4398]; transcription in the presence of RNase T1 and 3' O-Me-GTP eliminates non-specific transcripts, and produces the G-free transcripts initiated at the promoter. Restriction site-based fusion of a G-free cassette downstream from promoters is complicated by the requirement for G nucleotides to be excluded from the coding strand downstream from the transcription start points. We present an approach to add a G-free template onto a eukaryotic promoter by combining PCR-based termini construction and terminal deoxynucleotidyl transferase extension. The pisatin demethylase promoter (PDA1p) of the filamentous fungus Nectria haematococca was used as the test promoter. Three PDA1p/G- free constructs were tested in heterologous Drosophila melanogaster and HeLa and homologous N. haematococca transcription extracts. Each extract produced a PDA1p-specific transcript from each construct, but the relative level of transcription between constructs varied, particularly in the homologous extract. Since the choice of G-free sequence influences transcription differently among systems, this method for producing multiple G-free constructs should be useful for constructing and selecting optimal promoter/G-free templates for in vitro transcription in other homologous systems.

In vitro transcription from the Nectria haematococca PDA1 promoter in an homologous extract reflects in vivo pisatin-responsive regulation.

The PDA1 gene of Nectria haematococca MP VI (anamorph: Fusarium solani) encodes pisatin demethylase. This enzyme detoxifies the isoflavanoid phytoalexin pisatin produced by the plant on which this fungus is pathogenic. Expression of pisatin demethylase activity is induced in a mycelium by pretreatment with pisatin. We have developed homologous in vitro system which accurately initiates transcription from the PDA1 promoter. Transcription levels in vitro reflect the same pisatin-responsive stimulation as measured for PDA1 mRNA in vivo, and are dependent upon sequences in the 5' upstream region of PDA1. Pisatin-responsive transcription from the PDA1 promoter indicates that initiation of transcription is a major regulatory step in the pisatin induction of pisatin demethylase expression.

Effect of drug-DNA interactions upon transcription initiation at the lac promoter.

We have examined the effects of six DNA binding drugs upon initiation at the lac UV5 promoter by Escherichia coli RNA polymerase. Experiments were directed at determining the influence of added drug on open complex formation, open complex stability, initiation from the open complex, and stability of the resulting initiated complex. The narrow groove binding drugs distamycin and 4',6-diamidino-2-phenylindole dihydrochloride were more effective in inhibiting initiation through their effect on the first three of these factors than were the intercalators ethidium bromide, daunomycin, and actinomycin. The bisintercalator bis(daunomycin) inhibited open complex formation better than its parent daunomycin. With the possible exception of actinomycin, the drugs tested were not able to disrupt preformed initiated complex, in contrast to their destabilizing effect upon the open complex. Combined with other results, the data suggest that the antitumor activity of daunomycin is unlikely to result from its effect on transcription. We compare the relative effectiveness of the drugs with the known physical properties of the corresponding drug-DNA interactions. The rate of open complex formation seems to be influenced by both the on and off rates of the drug, probably due to the relative slowness of open complex formation. This is in contrast to elongation, a much quicker process, which seems to be limited by the drug off rate alone; these considerations may possibly rationalize the difference in relative effect of particular drugs upon initiation and elongation. All drugs were able actively to disrupt open complex, although to substantially different extents; some possible mechanisms for this disruption, and the insensitivity of the initiated complex, are discussed.

Identification of elements in the PDA1 promoter of Nectria haematococca necessary for a high level of transcription in vitro.

Expression of the PDA1 gene in the ascomycete Nectria haematococca MPVI (anamorph: Fusarium solani) is induced by exposure of mycelium to pisatin, an isoflavonoid phytoalexin produced by its host plant, garden pea. The PDA1 gene encodes a cytochrome P-450 monooxygenase which detoxifies pisatin. Regulatory elements controlling transcription from the PDA1 promoter were identified using a homologous Nectria in vitro transcription system through analysis of 5' deletions, specific oligonucleotide competition, and fusion of upstream segments to a heterologous promoter. A promoter-distal element which provided transcriptional activation was localized to a 35-bp region positioned -514 to -483 upstream of the transcriptional start site. This 35-bp region binds a previously characterized pisatin-responsive DNA-binding factor (PRF) and thus may provide pisatin-responsive control of transcription. A second promoter-proximal positive-acting region was found to be necessary for promoter transcription in both homologous and heterologous extracts, and so is likely to bind less genespecific transcription activator(s). A negative-acting element located between these two positive regions may act to make the positive-acting elements interdependent. The identification of an activator responding to pisatin provides a model for the control of a number of genes and processes controlled by host-specific signals, particularly the flavonoids.

Intermediates in transcription initiation from the E. coli lac UV5 promoter.

We have used nondenaturing polyacrylamide gel electrophoresis to separate intermediates in transcription initiation that result from action of E. coli RNA polymerase on the lac UV5 promoter. The resolved gel complexes are characterized by DNAase I footprinting, protein subunit content, RNA content, and transcription ability. There are two "open" complexes, whose equilibrium ratio is a function of temperature; they differ in their ability to escape abortive cycling, but not in their DNAase I footprints. We find three "initiated" complexes, containing RNA chains at least 11 nucleotides long, and lacking the sigma subunit of RNA polymerase. These experiments provide a detailed view of the early initiation steps and their thermal regulation at the E. coli lac promoter.

Peptide synthetase gene in Trichoderma virens.

Trichoderma virens (synonym, Gliocladium virens), a deuteromycete fungus, suppresses soilborne plant diseases caused by a number of fungi and is used as a biocontrol agent. Several traits that may contribute to the antagonistic interactions of T. virens with disease-causing fungi involve the production of peptide metabolites (e.g., the antibiotic gliotoxin and siderophores used for iron acquisition). We cloned a 5,056-bp partial cDNA encoding a putative peptide synthetase (Psy1) from T. virens using conserved motifs found within the adenylate domain of peptide synthetases. Sequence similarities with conserved motifs of the adenylation domain, acyl transfer, and two condensation domains support identification of the Psy1 gene as a gene that encodes a peptide synthetase. Disruption of the native Psy1 gene through gene replacement was used to identify the function of this gene. Psy1 disruptants produced normal amounts of gliotoxin but grew poorly under low-iron conditions, suggesting that Psy1 plays a role in siderophore production. Psy1 disruptants cannot produce the major T. virens siderophore dimerum acid, a dipetide of acylated N(delta)-hydroxyornithine. Biocontrol activity against damping-off diseases caused by Pythium ultimum and Rhizoctonia solani was not reduced by the Psy1 disruption, suggesting that iron competition through dimerum acid production does not contribute significantly to disease suppression activity under the conditions used.