Studies of the binding of Escherichia coli RNA polymerase to DNA. V. T7 RNA chain initiation by enzyme-DNA complexes

Initiation of T7 RNA chains by Escherichia coli RNA polymerase-T7 DNA complexes has been followed using incorporation of λ-³²P-labeled ATP and GTP to determine the relation between the enzyme binding sites and RNA chain initiation sites on the T7 genome. If the period of RNA synthesis is limited to less than two minutes, the stoichiometry of RNA chain initiation can be measured in the absence of chain termination and re-initiation. About 70% of the RNA polymerase holoenzyme molecules in current enzyme preparations are able to rapidly initiate a T7 RNA chain. The ratio of ATP- to GTP-initiated T7 RNA chains is not altered by variations in the number of enzyme molecules added per DNA, nor by alterations in the ionic conditions employed for RNA synthesis. This suggests that RNA chain initiation sites are chosen randomly through binding of RNA polymerase to tight (class A) binding sites on T7 DNA.     

Nucleotide sequence from the genetic left end of bacteriophage T7 DNA to the beginning of gene 4

The nucleotide sequence running from the genetic left end of bacteriophage T7 DNA to within the coding sequence of gene 4 is given, except for the internal coding sequence for the gene 1 protein, which has been determined elsewhere. The sequence presented contains nucleotides 1 to 3342 and 5654 to 12,100 of the approximately 40,000 base-pairs of T7 DNA. This sequence includes: the three strong early promoters and the termination site for Escherichia coli RNA polymerase: eight promoter sites for T7 RNA polymerase; six RNAase III cleavage sites; the primary origin of replication of T7 DNA; the complete coding sequences for 13 previously known T7 proteins, including the anti-restriction protein, protein kinase, DNA ligase, the gene 2 inhibitor of E. coli RNA polymerase, single-strand DNA binding protein, the gene 3 endonuclease, and lysozyme (which is actually an N-acetylmuramyl-l-alanine amidase); the complete coding sequences for eight potential new T7-coded proteins; and two apparently independent initiation sites that produce overlapping polypeptide chains of gene 4 primase. More than 86% of the first 12,100 base-pairs of T7 DNA appear to be devoted to specifying amino acid sequences for T7 proteins, and the arrangement of coding sequences and other genetic elements is very efficient. There is little overlap between coding sequences for different proteins, but junctions between adjacent coding sequences are typically close, the termination codon for one protein often overlapping the initiation codon for the next. For almost half of the potential T7 proteins, the sequence in the messenger RNA that can interact with 16 S ribosomal RNA in initiation of protein synthesis is part of the coding sequence for the preceding protein. The longest non-coding region, about 900 base-pairs, is at the left end of the DNA. The right half of this region contains the strong early promoters for E. coli RNA polymerase and the first RNAase III cleavage site. The left end contains the terminal repetition (nucleotides 1 to 160), followed by a striking array of repeated sequences (nucleotides 175 to 340) that might have some role in packaging the DNA into phage particles, and an A · T-rich region (nucleotides 356 to 492) that contains a promoter for T7 RNA polymerase, and which might function as a replication origin.     

Letter: The isolation and properties of the specific binding sites for Escherichia coli RNA polymerase on T4 and T7 bacteriophage DNAs.

RNA polymerase of Escherichia coli was allowed to bind to labeled T4 or T7 bacteriophage DNA. The unbound and “weakly” bound polymerase molecules were removed by adding an excess of poly(I) which has a high affinity for the enzyme (Bautz et al., 1972). After the unbound DNA regions were digested with pancreatic DNAase and snake venom phosphodiesterase, the “protected” DNA-RNA polymerase complexes were isolated by Sephadex G200 column chromatography. The protected DNA sites were then isolated by phenol extraction and hydroxylapatite chromatography. Studies of the DNA recognition regions led to the following conclusions. (1) No binding is observed in the absence of the sigma subunit or at low temperatures. (2) The amount of protection ranges from 0·18% to 0·24% of T4 DNA and from 0·25% to 0·34% of T7 DNA. In the absence of poly(I), higher protections are observed and the protected regions display heterogeneity in size and secondary structure. (3) The protected regions are double-stranded, as shown by hydroxylapatite chromatography, base composition analysis, and thermal chromatography. (4) The length of the protected regions comprise about 50 to 55 nucleotide pairs, as suggested by end-group analysis, sucrose density-gradient centrifugation, and polyacrylamide gel electrophoresis. (5) The results suggest the interaction of dimeric polymerase molecules at these sites. On the basis of DNA sizes, there are 7 to 9 such sites on T4 DNA and 2 to 3 on T7 DNA. (6) The protected regions are high in (A + T): 68% for T4 and 62% for T7 DNA. (7) Thermal chromatograms reflect these base compositions and suggest the homogeneity of these regions with respect to size and base composition.     

A comparison of the phage T4 gene 32 protein and Escherichia coli RNA polymerase binding sites on hamster papovavirus DNA

Phage T4 gene 32 protein and Escherichia coli RNA polymerase were bound to hamster papovavirus DNA. The binding regions were identified by electron microscopy employing a protein-free spreading technique. After gene 32 protein treatment four denaturation regions could be mapped, at 0.04–0.12, 0.30–0.36, 0.50–0.60 and 0.75–0.90 DNA map units, respectively, using the unique BamHI cleavage site as zero point. Eight RNA polymerase binding sites can be found which are localized at positions 0.05; 0.11; 0.18; 0.31; 0.57; 0.66; 0.76 and 0.82. A comparison of the RNA polymerase binding sites with the gene 32 protein denaturation pattern reveals a correspondence of six of eight polymerase binding sites with (A + T)-rich regions within the hamster papovavirus genome.     

The effect of low substrate concentrations on the extent of productive RNA chain initiation from T7 promoters A1 and A2 by Escherichia coli RNA polymerase

The extent of productive RNA chain initiation in vitro by Escherichia coli RNA polymerase holoenzyme from the bacteriophage T7 A1 and A2 promoters on purified T7 DNA templates has been investigated at very low concentrations of the ribonucleoside triphosphate substrates. As the concentration of ribonucleoside triphosphates in the reaction is decreased from 10 to 1 micro M, the extent of productive RNA chain initiation at these promoter sites drops precipitously at about 3 micro M. At 1 micro M substrate concentration, productive chain initiation from the A1 promoter does not occur even after extended incubation although the dinucleoside tetraphosphate pppApU is produced at a significant rate under these conditions. The reason for the inability of RNA polymerase to carry out productive RNA chain initiation at 1 micro M substrate concentration is not yet understood. The phenomenon is not due to substrate consumption, enzyme inactivation, or a requirement for a nucleoside triphosphatase activity in the reaction. The possibility is raised that there are additional nucleoside triphosphate binding sites on E. coli RNA polymerase which play some role in the process of productive RNA chain initiation.     

Physical mapping of the transfer RNA genes on lambda-h80dglytsu+36

The three Escherichia coli transfer RNA genes of the DNA of the transducing phage λ80cI857S^?t68dglyTsu⁺₃₆tyrTthrT (abbreviated λh80T), which specify the structures of tRNA^Gly₂(su⁺₃₆), tRNA^Tyr₂ and tRNA^Thr₃, have been mapped by hybridizing ferritin-labeled E. coli tRNA to heteroduplexes of λh80T DNA with the DNA of the parental phage (λh80cI857S^?t68) and examining the product in the electron microscope. The DNA of λh80T contains a piece of bacterial DNA of length 0·43 λ unit³ that replaces a piece of phage DNA of length 0·46 λ unit, proceeding left from B · P′ (the junction of bacterial DNA and phage DNA) (i.e. att⁸⁰). A cluster of three ferritin binding sites, and thus of tRNA genes, is seen at a position of 0·24 λ unit (1·1 × 10⁴ nucleotides) to the left of B· P′. The three tRNA genes of the cluster are separated by the unequal spacings of 260 (±30) and 140 (± 30) nucleotides, proceeding left from B·P′. The specific map positions have been identified by hybridization competition between ferritin-labeled whole E. coli tRNA with unlabeled purified tRNA^Tyr₂ and with unlabeled partially purified tRNA^Gly₂. The central gene of the cluster is tRNA^Tyr₂. The tRNA^Gly₂gene is probably the one furthest from B·P′. Thus, the gene order and spacings, proceeding left from B·P′, are: tRNA^Thr₃, 260 nucleotides, tRNA^Try₂, 140 nucleotides, tRNA^Gly₂.     

Transcription of polyoma virus DNA in vitro. III. Localization of calf thymus RNA polymerase II binding sites.

The binding sites of calf thymus RNA polymerase II on polyoma DNA were monitored by electron microscopy. Six discrete binding sites were located at positions 0.06, 0.25, 0.57, 0.66, 0.85 and 0.98 on the physical map of polyoma DNA. Although most of these sites are located in easily denaturable regions of the DNA, the strongest binding sites do not overlap with the major A + T-rich regions. In addition, the same binding sites were observed on superhelical or linear polyoma DNA. These results suggest that the eucaryotic RNA polymerase II can recognize specific sequences on double-stranded DNA and not only easily denaturable regions. At least five of these sites correspond to the binding and initiation sites mapped previously for the Escherichia coli RNA polymerase (Lescure et al., 1976).Stable initiation complexes can be formed with both E. coli and calf thymus RNA polymerases in the presence of a single dinucleotide (GpU) and a specific ribotriphosphate (CTP). Under these conditions, the binding of both enzymes to the sites in positions 0.06 and 0.57 is stimulated whereas the binding in positions 0.65 and 0.84 is partially suppressed. Both eucaryotic and procaryotic RNA polymerases may recognize similar sequences of the viral DNA in vitro.     

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.