The influence of rifampicin, streptolydigin, tetracycline and chloramphenicol on phage DNA transport from the T7 virion into the E. coli cell was studied. It has been found that the DNA transport proceeds in at least three stages. During the initial stage the phage injects into the host cell the left approximately 10 per cent of its DNA molecule. The entrance of the next 50 per cent of 17 DNA molecule is blocked by inhibitors which block transcription but not translation. Moreover, the entrance time of this part of the T7 DNA increases in the case of the T7 mutant D111 (which contains a deletion of the A2 and A3 promoters) and decreases in the case of the D53 mutant (which contains a deletion in the region of the early gene transcription terminator). It would appear, that the second stage of the phage DNA transport is tightly coupled with its transcription and that a mechanical function is carried out by RNA polymerase. The translation inhibitors completely block the entrance of the remaining 40 per cent of the 17 DNA molecule (class III genes) into the host cell. It would appear that some class I and (or) II gene product(s) are obligatory components of the final stage of 17 DNA transport. Some probable consequences of this virus DNA transport model as well as its agreement with the functional structure of T7 chromosome and with T7 development are discussed.
A purification procedure described previously resulting in electrophoretically pure Bacillus subtilis ATP-dependent DNAase has now been modified by adding a fractionation stage with Polymin P to permit large-scale isolation of the enzyme. It has been found that the enzyme molecule ( M , = 300000) consists of two large subunits with M , 155000 and 140000. The purified enzyme has three activities: (1) DNAase on linear single-stranded and double-stranded DNAs, (2) DNAunwinding and (3) ATPase. Circular duplex DNAs were not affected by the enzyme. Study of the dependence of these activities on temperature, pH, and ATP and Mg2+ concentrations has revealed two different states of the enzyme. At low ATP concentrations and alkaline pH, it showed chiefly nuclease action, degrading considerable amounts of DNA to small fragments five residues long on average. At higher ATP concentrations and neutral pH (more physiological conditions) it predominantly unwound DNA. Simultaneously it cut preferentially one of the duplex strands to fragments more than 1000 residues in length. The results obtained suggest that the energy of the enzymecleaved ATP is mainly expended on unwinding rather than on degrading DNA molecules.ATP-dependent DNAase has been found in many bacterial species. For some of them (Escherichia coliproof was obtained that the enzyme participates in genetic recombination.Early work on the properties of ATP-dependent DNAase established that under conditions optimal for DNAase action, namely at high pH and low (0.05 mM or less) ATP concentration, it vigorously cleaves linear duplex DNA to acid-soluble oligonucleotides five residues long on average. At the same time the E. coli enzyme does not digest circular DNA duplexes unless these contain gaps, but it nicks singlestranded circles [4]. These properties are difficult to reconcile with current concepts of the mechanism of genetic recombination which apparently involves rather large DNA fragments only. Therefore attempts were made recently to find conditions under which ATP-dependent DNAase does not cause considerable DNA degradation. It was shown that on brief incubation at neutral pH, lowered temperature and high ATP concentration [5,6] or low Mg2+ concentration [7], DNA degradation by E. coli or Haemophilus inpuenzae ATP-dependent DNAase decreases manifoldly. The resulting large single-stranded fragmentsEnzymes. ATP-dependent deoxyribonuclease or exonuclease V (EC 3.1.4.33); alkaline phosphatase (EC 3.1.3.1); lysozyme (EC 3.2.1.17); S1 nuclease (EC 3.1.4.21). __ -~and duplexes with long single-stranded tails could be intermediates of recombination.Earlier we found ATP-dependent DNAase in B. subtilis cells, investigated some properties of the partially purified enzyme [3] and obtained data for its involvement in the recombination process [8]. Recently we were able to overcome the difficulties associated with the lability of B. subtilis ATP-dependent DNAase and to develop a novel procedure for its purification to electrophoretic homogeneity [9]. This has led us to a fur...
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.
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