Association of DNA primase with the beta/gamma subunits of DNA polymerase alpha from Drosophila melanogaster embryos

The DNA polymerase and primase activities of the intact DNA polymerase alpha from early embryos of Drosophila melanogaster co-sediment in native glycerol gradients. However, the activities are separated in glycerol gradients containing 2.8 M urea after treatment of the enzyme with 3.4 M urea. The 182,000-dalton alpha subunit which is required for DNA polymerase activity (Kaguni, L.S., Rossignol, J.-M., Conaway, R. C., and Lehman, I.R. (1983) Proc. Natl. Acad. Sci. U. S.A. 80, 2221-2225) is not required for DNA primase activity. Instead, primase activity resides in the 60,000-dalton (beta) and/or the 50,000-dalton (gamma) subunit. Neither polymerase nor primase has been found in association with the 73,000-dalton polypeptide which co-purifies with the intact enzyme.     

Purification and partial characterization of the DNA-dependent RNA polymerase from Rickettsia prowazekii

The DNA-dependent RNA polymerase was purified from Rickettsia prowazekii, an obligate intracellular bacterial parasite. Because of limitation of available rickettsiae, the classical methods for isolation of the enzyme from other procaryotes were modified to purify RNA polymerase from small quantities of cells (25 mg of protein). The subunit composition of the rickettsial RNA polymerase was typical of a eubacterial RNA polymerase. R. prowazekii had beta' (148,000 daltons), beta (142,000 daltons), sigma (85,000 daltons), and alpha (34,500 daltons) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The appropriate subunits of the rickettsial RNA polymerase bound to polyclonal antisera against Escherichia coli core polymerase and E. coli sigma 70 subunit in Western blots (immunoblots). The enzyme activity was dependent on all four ribonucleoside triphosphates, Mg2+, and a DNA template. Optimal activity occurred in the presence of 10 mM MgCl2 and 50 mM NaCl. Interestingly, in striking contrast to E. coli, approximately 74% of the rickettsial RNA polymerase activity was associated with the rickettsial cell membrane at a low salt concentration (50 mM NaCl) and dissociated from the membrane at a high salt concentration (600 mM NaCl).     

A single amino acid substitution in sigma E affects its ability to bind core RNA polymerase.

We have examined the role of the most highly conserved region of bacterial RNA polymerase sigma factors by analyzing the effect of amino acid substitutions and small deletions in sigma E from Bacillus subtilis. sigma E is required for the production of endospores in B. subtilis but not for vegetative growth. Strains expressing each of several mutant forms of sigE were found to be deficient in their ability to form endospores. Single amino acid substitutions at positions 68 and 94 resulted in sigma factors that bind with less affinity to the core subunits of RNA polymerase. The substitution at position 68 did not affect the stability of the protein in B. subtilis; therefore, this substitution probably did not have large effects on the overall structure of the sigma factor. The substitution at position 68 probably defines a position in sigma E that closely contacts a subunit of RNA polymerase, while the substitution at position 94 may define a position that is important for protein stability or for binding to core RNA polymerase.