No abstract

No abstract

Bacteriophage T7 primase catalyzes the synthesis of the oligoribonucleotides pppACC(C/A) and pppACAC from the single-stranded DNA template sites 3'-d[CTGG(G/T)]-5' and 3'-(CTGTG)-5', respectively. The 3'-terminal deoxycytidine residue is conserved but noncoding. A series of nucleoside analogues have been prepared and incorporated into the conserved 3'-d(CTG)-5' site, and the effects of these analogue templates on T7 primase activity have been examined. The nucleosides employed include a novel pyrimidine derivative, 2-amino-5-(beta-2-deoxy-D-erythro-pentofuranosyl)pyridine (d2APy), whose synthesis is described. Template sites containing d2APy in place of the cryptic dC support oligoribonucleotide synthesis whereas those containing 3-deaza-2'-deoxycytidine (dc(3)C) and 5-methyl-6-oxo-2'-deoxycytidine (dm(5ox)C) substitutions do not, suggesting that the N3 nitrogen of cytidine is used for a critical interaction by the enzyme. Recognition sites containing 4-amino-1-(beta-2-deoxy-D-erythro-pentofuranosyl)-5-methyl-2,6[1H, 3H]-pyrimidione (dm(3)2P) or 2'-deoxyuridine (dU) substitutions for dT support oligoribonucleotide synthesis whereas those containing 5-methyl-4-pyrimidinone 2'-deoxyriboside (d(2H)T) substitutions do not, suggesting the importance of Watson-Crick interactions at this template residue. Template sites containing 7-deaza-2'-deoxyguanosine (dc(7)G) or 2'-deoxyinosine (dI) in place of dG support oligoribonucleotide synthesis. The reduced extent to which dc(7)G is successful within the template suggests a primase-DNA interaction. Inhibition studies suggest that the primase enzyme binds "null" substrates but cannot initiate RNA synthesis.

Starvation induces Bacillus subtilis to initiate a developmental process (sporulation) that includes asymmetric cell division to form the prespore and the mother cell. The integral membrane protein SpoIIE is essential for the prespore-specific activation of the transcription factor F , and it also has a morphogenic activity required for asymmetric division. An increase in the local concentration of SpoIIE at the polar septum of B. subtilis precedes dephosphorylation of the anti-anti-sigma factor SpoIIAA in the prespore. After closure and invagination of the asymmetric septum, phosphatase activity of SpoIIE increases severalfold, but the reason for this dramatic change in activity has not been determined. The central domain of SpoIIE has been seen to self-associate (I. Lucet et al., EMBO J. 19:1467-1475, 2000), suggesting that activation of the C-terminal PP2C-like phosphatase domain might be due to conformational changes brought about by the increased local concentration of SpoIIE in the sporulating septum. Here we report the inclusion of purified SpoIIE protein into a model membrane as a method for studying the effect of local concentration in a lipid bilayer on activity. In vitro assays indicate that the membrane-bound enzyme maintains dephosphorylation rates similar to the highly active micellar state at all molar ratios of protein to lipid. Atomic force microscopy images indicate that increased local concentration does not lead to self-association.Spore formation by the gram-positive soil bacterium Bacillus subtilis has been extensively studied as a simple example of cellular development and differentiation (21, 67). During vegetative growth of B. subtilis, medial division gives rise to two identical daughter cells, but under conditions of nutritional starvation, the cell forms a large mother cell and a smaller prespore, which develops into a tough, dormant spore. Sporulation in prokaryotes is a complex process, involving an ordered multiprotein division apparatus, which initiates an annular invagination of the cell wall and the formation of an asymmetric septal membrane of two bilayers, separated by peptidoglycan. Differentiation in the prespore is initiated by activation of the transcription factor F , the first sporulationspecific sigma factor in the developmental cascade (21,37,44,63). The sigma factor F and its regulatory proteins SpoIIAA, SpoIIAB, and SpoIIE are synthesized before septation (6, 25, 56), but F remains bound to the anti-sigma factor SpoIIAB, and therefore in an inhibited state, prior to closure of the asymmetric septum (3,19,51). In the predivisional cell, the anti-anti-sigma factor SpoIIAA is phosphorylated at a single site (16, 18, 51, 52). The resulting SpoIIAA-P cannot bind SpoIIAB (the protein kinase for SpoIIAA) to release F from the SpoIIAB-F complex (16,19,51). The phosphatase domain of the 91.5-kDa enzyme SpoIIE is responsible for the hydrolysis of SpoIIAA-P (4, 17, 24). Expression of SpoIIE during the early stages of sporulation does not lead to discernible concentrations of no...

A new model to replace the Ogstron and tube reptation models for gel retardation of DNA is proposed that explicitly takes into account screening of the hydrodynamic interactions and polyelectrolyte effects. At short DNA sequence lengths, significant anomalous migration is predicted whose onset is dependent on the size of polyacrylamide gel pores. Thus, a 2-residue fragment has the same electrophoretic mobility as a 12-residue fragment for a polyacrylamide gel with a mesh size of 60Å. The oligonucleotide length at which anomalous migration is observed also depends on pore size. Experimental measurement of gel mobility for DNA fragments of the form N(pN)(n), where n = 1-11, 14 and 19 substantiate this phenomenon.