Dissection of two hallmarks of the open promoter complex by mutation in an RNA polymerase core subunit

Deletion of 10 evolutionarily conserved amino acids from the beta subunit of Escherichia coli RNA polymerase leads to a mutant enzyme that is unable to efficiently hold onto DNA. Open promoter complexes formed by the mutant enzyme are in rapid equilibrium with closed complexes and, unlike the wild-type complexes, are highly sensitive to the DNA competitor heparin (Martin, E., Sagitov, V., Burova, E., Nikiforov, V., and Goldfarb, A. (1992) J. Biol. Chem. 267, 20175-20180). Here we show that despite this instability, the mutant enzyme forms partially open complexes at temperatures as low as 0 degrees C when the wild-type complex is fully closed. Thus, the two hallmarks of the open promoter complex, the stability toward a challenge with DNA competitors and the sensitivity toward low temperature, can be uncoupled by mutation and may be independent in the wild-type complex. We use the high resolution structure of Thermus aquaticus RNA polymerase core to build a functional model of promoter complex formation that accounts for the observed defects of the E. coli RNA polymerase mutants.     

Regulation of open complex formation at the Escherichia coli galactose operon promoters. Simultaneous interaction of RNA polymerase, gal repressor and CAP/cAMP.

The regulation of open complex formation at the Escherichia coli galactose operon promoters by galactose repressor and catabolite activator protein/cyclic AMP (CAP/cAMP) was investigated in DNA-binding and kinetic experiments performed in vitro. We found that gal repressor and CAP/cAMP bind to the gal regulatory region independently, resulting in simultaneous occupancy of the two gal operators and the CAP/cAMP binding site. Both CAP/cAMP and gal repressor altered the partitioning of RNA polymerase between the two overlapping gal promoters. Open complexes formed in the absence of added regulatory proteins were partitioned between gal P1 and P2 with occupancies of 25% and 75%, respectively. CAP/cAMP caused open complexes to be formed nearly exclusively at P1 (98% occupancy). gal repressor caused a co-ordinated, but incomplete, switch in promoter partitioning from P1 to P2 in both the absence and presence of CAP/cAMP. We measured the kinetic constants governing open complex formation and decay at the gal promoters in the absence and presence of gal repressor and CAP/cAMP. CAP/cAMP had the largest effect on the kinetics of open complex formation, resulting in a 30-fold increase in the apparent binding constant. We conclude that the regulation of open complex formation at the gal promoters does not result from competition between gal repressor, CAP/cAMP and RNA polymerase for binding at the gal operon regulatory region, but instead results from the interactions of the three proteins during the formation of a nucleoprotein complex on the gal DNA fragment. Finally, we present a kinetic model for the regulation of open complex formation at the gal operon.     

Site-directed mutagenesis of the -10 region of the lacUV5 promoter. Introduction of dA4.dT4 tract suppresses open complex formation

Homopolymeric dAn.dTn sequences, where n is 4 or greater, have special properties leading to increased duplex stability and DNA bending. The lacUV5 promoter was used to examine the functional consequences of changing the -10 TATAAT consensus sequence to the sequence TAAAAT. The transversion mutation at the underlined site was accomplished with site-directed mutagenesis using translation termination as the selection procedure. For free DNA, structural differences at the 5' and 3' junction regions of the dA4.dT4 tract can be readily detected by DNase I digestion. However, site binding by Escherichia coli RNA polymerase appeared unaltered by the TAAAAT sequence since identical DNase I footprints were obtained for the lacUV5 and mutant promoters. Binding competition studies under different ionic strengths revealed a significant reduction in mutant promoter open complex formation relative to the lacUV5 promoter. Mutant promoter open complexes also dissociated faster and to a greater extent than the corresponding lacUV5 promoter open complexes when challenged with heparin or a combination of heparin and increased KCl concentration. Consequently, mutant promoter open complexes appear less stable than lacUV5 promoter open complexes.     

Modification of the reactivity of three amino-acid residues in elongation factor 2 during its binding to ribosomes and translocation

The accessibility of three amino acids of EF-2, located within highly conserved regions near the N- and C-terminal extremities of the molecule (the E region and the ADPR region, respectively) to modifying enzymes has been compared within nucleotide-complexed EF-2 and ribosomal complexes that mimic the pre- and posttranslocational ones: the high-affinity complex (EF-2)-nonhydrolysable GTP analog GuoPP[CH2]P ribosome and the low-affinity (EF-2)-GDP-ribosome complex, EF-2 and ribosomes being from rat liver. We studied the reactivity of two highly conserved residues diphthamide-715 and Arg-66, to diphtheria-toxin-dependent ADP-ribosylation and trypsin attack, and of a threonine that probably lies between residues 51 and 60, to phosphorylation by a Ca2+/calmodulin-dependent protein kinase. Diphthamide 715 and this threonine residue were unreactive within the high-affinity complex but seemed fully reactive in the low-affinity complex. Arg-66 was resistant to trypsin in both complexes. The possible involvement of the E and ADPR regions of EF-2 in the interaction with ribosome in the two complexes is discussed.     

Differential anti-proliferative properties of novel hydroxydicarboxylatoplatinum(II) complexes with high or low reactivity with thiols

We have examined the anti-proliferative effect of 13 recently synthesised platinum dicarboxylate complexes, very similar in their chemical, structural and kinetic properties to carboplatin. We used the L5178Y model: two murine lymphoma sublines, which differ in nucleotide excision repair ability and hence, in sensitivity to those platinum complexes that react with DNA. The anti-proliferative effect of the examined compounds mainly depends on the kind of amine ligand. Complexes with the primary amine (ethylenediamine) are more effective than complexes containing the tertiary amine (1-alkylimidazole). The ethylenediaminemalatoplatinum(II) complexes show a differential in vitro anti-proliferative activity in the L5178Y model; hence, it may be expected that they inflict DNA lesions that are repaired by the nucleotide excision system. The cytotoxicity of these complexes is directly correlated with reactivity with glutathione (GSH). The 1-alkylimidazole complexes are of low toxicity and moderate to low reactivity with GSH; in contrast to the ethylenediaminemalatoplatinum(II) complexes, their cytotoxicity is inversely correlated with reactivity with GSH. Two of the 1-alkylimidazole complexes, bis(1-ethylimidazole)(L-malato)platinum(II) and bis(1-propylimidazole (L-malato)platinum(II), show a considerable ability to arrest cells in G2 phase. We expect that the properties of these two groups of platinum complexes may be exploited in combined platinum complex treatment and irradiation.