Binding of Escherichia coli RNA polymerase to T7 DNA. Displacement of holoenzyme from promoter complexes by heparin.

Escherichia coli RNA polymerase holoenzyme bound to promoter sites on T7 DNA is attacked and inactivated by the polyanion heparin. The highly stable RNA polymerase-T7 DNA complex formed at the major T7 A1 promoter can be completely inactivated by treatment with heparin, as shown by monitoring the loss of activity of such complexes, and by gel electrophoresis of the RNA products transcribed. The rate of this inactivation is much faster than the rate of dissociation of RNA polymerase from promoter complexes, and thus represents a direct attack of heparin on the polymerase molecule bound at promoter A1. Experiments employing the nitrocellulose filter binding technique suggest that heparin inactivates E. coli RNA polymerase when bound to T7 DNA by directly displacing the enzyme from the DNA. RNA polymerase bound at a minor T7 promoter (promoter C) is much less sensitive to heparin attack than enzyme bound at promoter A1. Thus, the rate of inactivation of RNA polymerase-T7 DNA complexes by heparin is dependent upon the structure of the promoter involved even though the inhibitor binds to a site on the enzyme molecule.     

Broad host range, regulated expression system utilizing bacteriophage T7 RNA polymerase and promoter

An IPTF-regulated broad host range expression system was constructed using compatible broad host range plasmids, the T7 RNA polymerase, and T7 promoter sequences. The system is implemented by the coexistence of two plasmids. The first contains the T7 RNA polymerase gene under the control of lacl or lacl(q) genes and lacUV5 promoter. The second encodes the T7 promoter upstream of a multicloning site. IncP1 or IncP4 T7 promoter plasmids, and IncP1, IncP4 or IncW T7 RNA polymerase plasmids were constructed. The expression from the IncP1 promoter plasmids in the presence of the IncP4 polymerase plasmids was tested by in vivo lacZ fusions and vivo labeling of proteins. In this combination, the use of lac(q) improves the regulation levels in Escherichia coli, whereas, in Pseudomonas phaseolicola, a 28.5-fold regulation was obtained with lacl, Although the level of lacZ expression from the T7 promoter in P. phaseolicola is low compared with E. coli, it is similar to levels obtained with the pm promoter in Pseudomonas putida when the differences in the copy number of the expression vectors are taken into consideration (c) 1993 Wiley & Sons, Inc.     

Initiation of DNA replication at cloned origins of bacteriophage T7

Bacteriophage T7 DNA replication is initiated at a site 15% of the distance from the genetic left end of the chromosome. This primary origin contains two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by an A + T-rich region. When the primary origin region is deleted replication initiates at secondary origins. We have analyzed the ability of plasmids containing cloned fragments of T7 to replicate after infection of Escherichia coli with bacteriophage T7. All cloned T7 fragments that support plasmid replication contain a T7 promoter but a T7 promoter alone is not sufficient for replication. Replication of plasmids containing the primary origin is dependent on T7 DNA polymerase and gene 4 protein (helicase/primase) and a portion of the A + T-rich region. The other T7 fragments that support plasmid replication after T7 infection are promoter regions phi OR, phi 13 and phi 6.5 (secondary origins). When both the primary and secondary origins are present simultaneously on compatible plasmids, replication of each is temporally regulated. Such regulation may play a role during T7 DNA replication.     

Formation of specific T3-RNA-polymerase complexes with oligoribonucleotides and inhibition of DNA-dependent RNA synthesis

It was shown previously that E. coli RNA polymerase and T7 RNA polymerase being incubated with oligonucleotides of different length derived from RNA endonuclease hydrolysate bind selectively to certain oligonucleotides with the length larger than or equal to 5. The data presented demonstrate that T3 RNA polymerase also binds selectively from the isoplith mixtures certain oligonucleotides starting from pentanucleotides. Adding of excess of T3 RNA polymerase it was possible to exhaustively extract the recognizable oligonucleotides from the isoplith mixture. However, the exhausted by T3 RNA polymerase mixture of pentanucleotides still contained those which are bound selectively by T7 and E. coli RNA polymerases. The data suggest that various RNA-polymerases recognize different oligoribonucleotides. It was shown that T3 DNA inhibits the selective binding of penta-or heptaribonucleotides to T3 RNA polymerase competing obviously for the enzyme. The T3 RNA polymerase bound penta- or heptanucleotides inhibit DNA-dependent RNA synthesis carried out by the enzyme; the isoplith mixtures which do not contain T3 RNA polymerase bound oligonucleotides are deprived of the inhibitory properties. Only those isoplith mixtures contain T3 RNA polymerase bound oligonucleotides which were derived from symmetrically transcribed RNA which have obviously promoter simulating sequences. The data provide evidence that T2 RNA polymerase binds selectively the oligonucleotides mimicking the promotor recognition sites.     

The role of the beta-subunit of RNA-polymerase in specific recognition of promoters

The complex formation of T7 DNA with RNA polymerase from E. coli B/r WU-36-10-11-12 (E. coli W12) and its rifampicin resistant mutant with highly pleiotropic effect--rpoB409 was studied. As shown earlier rpoB409 RNA polymerase differs from the normal enzyme by the selection of RNA synthesis from early promoters of DNA from T7 and T4 phages. The change in the RNA specificity synthesis due to rpoB409 mutation was shown to occur at the stage of RNA polymerase interaction with DNA before open promoter complex formation. The data obtained together with the fact of highly pleiotropic effect of the rpoB409 mutation indicate that RNA polymerase beta-subunit takes part in specific recognition of promoters.     

Periodicity in contacts of RNA-polymerase with promotors]

Periodicities in the position of E.coli RNA polymerase promoter contacts on several promoters (lacUV5, T7 A3, tetR, lambda cin, lambda c17, RNA1, and trp S.t.) were found by means of Fourier analysis. The comparison of the Fourier spectrum of core RNA polymerase contacts on the lacUV5 promoter and that of holoenzyme revealed a more prominent 7-periodicity in the Fourier spectrum of holoenzyme contacts. 6-, 7-, and 8-periodicities were found in the primary structure of the majority of E.coli promoters. It is shown that RNA polymerase recognizes specific periodic patterns in the promoter structure.     

Enhancing recombinant protein yields in Escherichia coli using the T7 system under the control of heat inducible lambda PL promoter

A recombinant plasmid containing the complete lacZ gene downstream of the T7 promoter was used to transform Escherichia coli containing another plasmid which had the T7 RNA polymerase gene under the control of heat inducible lambda PL promoter. This recombinant E. coli containing the two plasmids was studied in order to enhance beta-galactosidase expression. The heat shock time which effectively regulates the T7 RNA polymerase was optimized and best expression of beta-galactosidase was obtained with 2 min heat shock. Substrate feeding increased the duration of log phase and allowed induction at a higher cell density without affecting the specific activity. A high cell density (7 g l-1) and high specific activity (approximately 20,000 U) were achieved which effectively increased the product concentration 18-fold.     

Sequence and analysis of the gene for bacteriophage T3 RNA polymerase.

The RNA polymerases encoded by bacteriophages T3 and T7 have similar structures, but exhibit nearly exclusive template specificities. We have determined the nucleotide sequence of the region of T3 DNA that encodes the T3 RNA polymerase (the gene 1.0 region), and have compared this sequence with the corresponding region of T7 DNA. The predicted amino acid sequence of the T3 RNA polymerase exhibits very few changes when compared to the T7 enzyme (82% of the residues are identical). Significant differences appear to cluster in three distinct regions in the amino-terminal half of the protein. Analysis of the data from both enzymes suggests features that may be important for polymerase function. In particular, a region that differs between the T3 and T7 enzymes exhibits significant homology to the bi-helical domain that is common to many sequence-specific DNA binding proteins. The region that flanks the structural gene contains a number of regulatory elements including: a promoter for the E. coli RNA polymerase, a potential processing site for RNase III and a promoter for the T3 polymerase. The promoter for the T3 RNA polymerase is located only 12 base pairs distal to the stop codon for the structural gene.     

Soluble expression of cloned phage K11 RNA polymerase gene in Escherichia coli at a low temperature.

The gene 1 of the Klebsiella phage K11 encoding the phage RNA polymerase was amplified using the polymerase chain reaction of the Pfu DNA polymerase, cloned and expressed under the control of tac promoter in Escherichia coli. Although the gene was efficiently expressed in E. coli BL21 cells at 37 degrees C, most of the K11 RNA polymerase produced was insoluble, in contrast to soluble expression of the cloned T7 RNA polymerase gene. Coexpression of the bacterial chaperone GroES and GroEL genes together did not help solubilize the K11 RNA polymerase. When the temperature of cell growth was lowered, however, solubility of the K11 RNA polymerase was increased substantially. It was found much more soluble when expressed at 25 degrees C than at 30 and 37 degrees C. Thus, the cloned K11 RNA polymerase gene was expressed in E. coli mostly to the soluble form at 25 degrees C. The protein was purified to homogeneity by chromatography using DEAE-Sephacel and Affigel-blue columns and was found to be active in vitro with the K11 genome or a K11 promoter. The purified K11 RNA polymerase showed highly stringent specificity for the K11 promoter. Low-level cross-reactivity was shown with the SP6 and T7 consensus promoters, while no activity shown with the T3 consensus promoter at all.