DNA sequence for the T7 RNA polymerase promoter for T7 RNA species II

The DNA sequence for the T7 late region class III promoter for T7 RNA species II has been determined. I have found that the DNA sequence for this promoter presented in an earlier report (Oakley et al., 1979) is incorrect and that this class III promoter contains a 23 base-pair sequence identical to those present in all other T7 class III promoters (Rosa, 1979). The T7 RNA species II promoter has been located at 68% on the T7 genome.     

Specific fragmentation of T7 phage DNA at low-melting sites.

A method has been developed for selective fragmentation of T7 DNA at AT-rich regions. The molecules have been subjected to complete digestion with single-strand-specific SI endonuclease after fixation of DNA AT-rich regions in the denatured state by glyoxal. The treatment resulted in three fragments having molecular weights of 13.6 +/- 0.4, 8.2 +/- 0.4 and 3.5 +/- 0.16 megadaltons as determined by electron microscopy. The position of these fragments along the T7 DNA molecule has been determined by means of analysis of the intermediates during SI-cleavage.     

On the promoter complex formation rate of E. coli RNA polymerases with T7 phage DNA.

Influence of ionic strength on the kinetics of the promoter complex formation between E. coli RNA polymerase and T7 phage DNA was investigated using a membrane filter assay. The enzyme-promoter association rate constant was determined. It varies from 10(9) to 3 x 10(7) M-1 sec-1 when the ionic strength is changed from zero to 0.15 M NaCl. Basing on the theoretical analysis of experimental data obtained the model for the promoter site selection assuming the enzyme sliding along the DNA is discussed.     

DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Effect of pyrophosphorolysis and metal ions

Pyrophosphorolysis by bacteriophage T7 DNA polymerase leads to the degradation of specific dideoxynucleotide-terminated fragments on DNA sequencing gels. This reaction can be prevented by pyrophosphatase. It is also inhibited by a high concentration of dNTPs; only the dNTP complementary to the next base in the template is an effective inhibitor, suggesting the formation of a stable polymerase-primer-template-nucleotide complex despite the absence of a 3' hydroxyl group on the primer. The use of pyrophosphatase, a genetically modified T7 DNA polymerase that lacks exonuclease activity, and Mn2+ rather than Mg2+ to eliminate discrimination between dideoxynucleotides and deoxynucleotides (Tabor, S., and Richardson, C. C. (1989) Proc. Nat. Acad. Sci. U. S. A. 86, 4076-4080) generates bands of uniform intensity on a DNA sequencing gel. Uniform band intensities simplify the analysis of a DNA sequence, particularly with automated procedures. For example, when genomic DNA is sequenced directly, heterozygotic sequences are readily detected because their bands have half the intensity of homozygotic sequences. A procedure for automated DNA sequencing is described that exploits the uniformity. A single reaction with a single labeled primer is carried out using four different ratios of dideoxynucleotides to deoxynucleotides; after gel electrophoresis in a single lane, the sequence is determined by the relative intensity of each band.     

The degradation of T7 DNA in converging flow.

The flow-induced degradation of T7 DNA (Molecular Size = 40 Kbp) was studied in a flow device that generates converging flow rather than simple shear flow. We discovered that the sizes of the degradation products were very broadly distributed, covering the range from 10 Kbp to 36 Kbp. An explanation for the broadness of the distribution is given based on a computer simulation of the experiment. The significance of converging flow to the routine handling of large DNA is emphasized.     

T7 RNA polymerase interacts with its promoter from one side of the DNA helix

The interactions of T7 RNA polymerase with its promoter DNA have been previously probed in footprinting experiments with either DNase I or (methidiumpropyl-EDTA)-Fe(II) to cleave unprotected DNA [Basu, S., & Maitra, U. (1986) J. Mol. Biol. 190, 425-437. Ikeda, R. A., & Richardson, C. C. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3614-3618]. Both of these reagents have drawbacks; DNase I is a bulky reagent and so provides low resolution, and (methidiumpropyl-EDTA)-Fe(II) intercalates into DNA and is therefore biased toward cleavage of double-stranded DNA. In this study, the interaction between the polymerase and the promoter has been probed with Fe(II)-EDTA. This reagent generates reactive hydroxyl radicals free in solution, which produces a more detailed picture of the polymerase-promoter complex. Two protected regions are observed on each of the two promoter DNA strands: from position -17 to position -13 and from position -7 to position -1 on the coding strand and from position -14 to position -9 and from position -3 to position +2 on the noncoding strand. From this pattern it is clear that if recognition occurs via double-stranded B-form DNA, then the protected regions lie on one face of the DNA helix, and therefore the enzyme must interact predominantly from one side of the DNA helix. Digestion of the DNA in a polymerase-promoter complex with a single-strand-specific endonuclease shows that a small region of the noncoding strand near position -5 is susceptible to cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)