Escherichia coli mutant which restricts T7 bacteriophage has an altered RNA polymerase.

We have previously described an Escherichia coli K-12 mutant, Y49, which restricts the growth of bacteriophage T7 and causes the accumulation of short DNA molecules and head-related particles during infection. We now show that the basis for these effects is the inability of the T7 gene 2 product to inactivate the Y49 RNA polymerase during infection, similar to what has been shown by DeWyngaert and Hinkle (J. Biol. Chem. 254:11247--11253, 1979) for the BR3 and tsnB strains of E. coli.     

Characteristics of electrostatic interaction of Escherichia coli RNA polymerase with promoters of T4 phage DNA]

A comparative analysis of electrostatic potential distribution for "early" T4 phage promoters was undertaken. The data obtained indicate that there are some particular elements in the patterns of electrostatic potential distribution of promoter DNA specific for promoter groups differing by their functional response to ADP-ribosylation of the alpha-subunit as well as to rpoB403- or rpoB409 mutationals of the beta-subunit of RNA-polymerase.     

Cold sensitive mutant of Escherichia coli with altered RNA polymerase

Summary Cold sensitive (cs) mutants of E. coli unable to grow at 26° C were isolated. Three of about 200 mutants tested have RNA polymerase which is about 5 times less active at low temperatures than the wild type RNA polymerase. One of such mutants cs 1 was studied in more detail. The mutation affects core enzyme and is closely linked to rif-r mutations. By transduction analysis the following order of markers was established: argH — rif-r — cs 1.     

Kinetic measurements of Escherichia coli RNA polymerase association with bacteriophage T7 early promoters

During infection of Escherichia coli by bacteriophage T7, E. coli RNA polymerase utilizes only three promoters (A1, A2, and A3). In vitro, the A promoters predominate at very low polymerase concentration, but at higher polymerase concentration the minor B, C, D, and E promoters are used with equal efficiency. The binding constant for the initial association of polymerase with promoters and the forward rate of isomerization to an "open" complex capable of initiation have been measured for the A1, A3, C, and D promoters using the abortive initiation reaction. At 80 mM KCl, 37 degrees C, both major and minor promoters isomerize rapidly (t1/2 = 10 to 30 s). In contrast, initial binding to the minor promoters (KI = 10(7) ) is at least 10-fold weaker than binding to major promoters KI greater than or equal to 10(8) ), suggesting promoter selectivity in the T7 system occurs at the point of initial binding. Association kinetics of the A1 and C promoters on intact T7 were the same as measured on restriction fragments of length greater than or equal to 500 base pairs. All open complexes dissociated with half-lives longer than 1 h. Overall equilibrium binding constants estimated from kinetic measurements ranged from 10(10) to greater than or equal to 10(11) M-1 for minor and major promoters, respectively. Data on heparin attack and abortive initiation turnover rates indicate open complex polymerase conformation may be different at the A1 and A3 promoters.     

A temperature-sensitive mutant of Escherichia coli with an altered RNA polymerase beta' subunit.

$\text{Penicillium charlesii}$ extracts contain UDP-galactose:NAD⁺ 2-hexosyl oxidoreductase (1). ADP-ribose also serves as a substrate resulting in formation of NADH and an oxidized ADP-ribose derivative. Treatment of the oxidized product with NaBH₄ followed by hydrolysis at pH 2 and 100° releases xylose as well as ribose. We conclude that ADP-D-$\text{glycero}$-D-$\text{glycero}$-3-pentosulose (ADP-3-ketoribose) is the product derived from ADP-ribose.     

RNA polymerase mutant with altered sigma factor in Escherichia coli.

Summary E. gracilis chloroplast DNA Bam fragments E and D, coding for rRNA were cloned separately using the plasmid pBR 322 as vector and E. coli as host. The newly constructed recombinant plasmids EgcKS 8 and EgcKS 11 (containing the Bam HI fragments E and D respectively) were analysed and characterized by gel electrophoresis, electronmicroscopy and analytical ultracentrifugation.Abbreviations Ap Ampicillin - Tc Tetracycline-hydrochloride - Bam HI endonuclease isolated from Bacillus amyloliquefaciens - Eco RI endonuclease isolated from E. coli RY13 - Bgl II endonuclease isolated from Bacillus globiggi - EDTA Ethylene-diamine-tetra-acetic-acid - ctDNA chloroplast DNA An abstract of this work was presented at the 10th annual meeting of the Union Schweizerischer Gesellschaften für Experimentelle Biologie, Davos 19th and 20th Mai, 1978. The recommendations of the Schweizerische Akademie für medizinische Wissenschaften for work with recombinant DNA-molecules were respected throughout this work.     

Role of curved DNA in binding of Escherichia coli RNA polymerase to promoters.

The ability of curved DNA upstream of the -35 region to affect the interaction of Escherichia coli RNA polymerase and promoter DNA was examined through the use of hybrid promoters. These promoters were constructed by substituting the curved DNA from two Bacillus subtilis bacteriophage SP82 promoters for the comparable DNA of the bacteriophage lambda promoters lambda pR and lambda pL. The SP82 promoters possessed intrinsic DNA curvature upstream of their -35 regions, as characterized by runs of adenines in phase with the helical repeat. In vitro, the relative affinities of purified sigma 70-RNA polymerase for the promoters were determined in a competition binding assay. Hybrid promoters derived from lambda pR that contained curved DNA were bound by E. coli RNA polymerase more efficiently than was the original lambda pR. Binding of E. coli RNA polymerase to these hybrid promoters was favored on superhelical DNA templates according to gel retardation analysis. Both the supercoiled and relaxed forms of the hybrid lambda pL series were better competitors for E. coli RNA polymerase binding than was the original lambda pL. The results of DNase I footprinting analysis provided evidence for the wrapping of the upstream curved DNA of the hybrid lambda pR promoters around the E. coli RNA polymerase in a tight, nucleosomal-like fashion. The tight wrapping of the upstream DNA around the polymerase may facilitate the subsequent steps of DNA untwisting and strand separation.     

Conditional change of DNA replication control in an RNA polymerase mutant of Escherichia coli

The infectivity of the soybean symbiont Rhizobium japonicum changed two- to fivefold with culture age for strains 110 ARS, 138 Str Spc, and 123 Spc, whereas culture age had relatively little effect on the infectivity of strains 83 Str and 61A76 Str. Infectivity was measured by determining the number of nodules which developed on soybean primary roots in the zone which contained developing and preemergent root hairs at the time of inoculation. Root cells in this region of the host root are susceptible to Rhizobium infection, but this susceptibility is lost during acropetal development and maturation of the root cells within a period of 4 to 6 h (T. V. Bhuvaneswari, B. G. Turgeon, and W. D. Bauer, Plant Physiol. 66:1027-1031, 1980). Profiles of nodulation frequency at different locations on the root were not affected by the age of the R. japonicum cultures, indicating that culture age affected the efficiency of Rhizobium infection rather than how soon infections were initiated after inoculation. Inoculum dose-response experiments also indicated that culture age affected the efficiency of infection. Two strains, 61A76 Str and 83 Str, were relatively inefficient at all culture ages, particularly at low inoculum doses. Changes in infectivity with culture age were reasonably well correlated with changes in the proportion of cells in a culture capable of binding soybean lectin. Suspensions of R. japonicum in water were found to retain their viability and infectivity.     

RNA polymerase synthesis in vitro directed by T7 phage DNA

Summary T7 RNA polymerase is synthesized in vitro, dependent on T7 DNA. The in vitro synthesized T7 polymerase has the characteristic properties: resistance to rifampicin and streptolydigin and the typical template specificity.     

A mutant Escherichia coli sigma 70 subunit of RNA polymerase with altered promoter specificity

A mutation is described that alters the promoter specificity of sigma 70, the primary sigma factor of Escherichia coli RNA polymerase. In strains carrying both the mutant and wild-type sigma gene (rpoD), the mutant sigma causes a large increase in the activity of mutant P22 ant promoters with A.T or C.G instead of the wild-type, consensus G.C base-pair at position -33, the third position of the consensus -35 hexamer 5'-TTGACA-3'. There is little or no effect on the activities of the wild-type and 23 other mutant ant promoters, including one with T.A at -33. The mutant sigma also activates E. coli lac promoters with A.T or C.G, but not T.A, at the corresponding position. The rpoD mutation (rpoD-RH588) changes a CGT codon to CAT. The corresponding change in sigma 70 is Arg588----His. This residue is in a region that is conserved among most sigma factors, a region that is also homologous with the helix-turn-helix motif of DNA-binding proteins. These results suggest that this region of sigma 70 is directly involved in recognition of the -35 hexamer.