Rabbit skeletal muscle F-actin has been selectively labeled at a cysteine residue with the environmentally sensitive fluorophore 6-acryloyl-2-(dimethylamino)naphthalene. The fluorescent actin conjugate behaves similarly to native actin with respect to the polymerization kinetics, critical monomer concentration, and ability to form F-actin paracrystals. Upon polymerization to F-actin, the absorption of the actin conjugate is red-shifted, whereas the fluorescence emission is blue-shifted 740 wavenumbers and is accompanied by a decrease in the fluorescence bandwidth of 470 wavenumbers. These large shifts in the spectral properties of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) in actin provide a simple method for obtaining a spectral discrimination between the G- and F-actin populations during the polymerization reaction. Steady-state fluorescence techniques were used to study the environment of the fluorophore in the monomeric and polymeric forms of actin. Fluorescence emission spectral analysis and quenching and polarization studies of G-actin-Prodan indicated that the fluorophore lies immobile on the protein surface but with one of its faces in full contact with the solvent. In F-actin, the fluorophore has a limited exposure to the solvent and is located in a dielectric environment similar to those seen for Prodan in polar, aprotic solvents or buried within a protein matrix [Macgregor, R. B., Jr., & Weber, G. (1986) Nature (London) 318, 70-73]. Additionally, our results demonstrate that the Prodan molecule conjugated to F-actin is completely immobile during its fluorescence lifetime, exhibits an increase in the resonance energy transfer (RET) from tryptophan residues compared to that observed in G-actin, and shows evidence of homologous RET within the polymer.
Formamide dissociates the G‐actin · DNAse‐I complex and is therefore suitable as an alternative to the eluant containing 3 M guanidinium hydrochloride, suggested originally by Lazarides and Lindberg [Proc. Natl Acad. Sci. USA, 71, 4742–4746 (1974)], to elute actin from immobilized DNAse‐I–agarose. Formamide provides the advantage of being a much weaker denaturant than guanidinium hydrochloride and being a nonionic substance. In the concentration necessary for the dissociation of the G‐actin · DNAse‐I complex (approximately 10 M) formamide denatures actin only slowly (half‐time approximately 150 min at 2°C) and thus allows the recovery of a large fraction of actin in a polymerization‐competent form from the affinity column. Based on these findings a rapid two‐step procedure for the isolation of non‐muscle G‐actin from cultured cells is described. The actin is obtained in high yield and purity (greater than 90%) and can readily polymerize to F‐actin.
The stable initiation complexes formed by interaction of DNA-dependent RNA polymerase from Escherichia coli containing initiation factor CT with native DNAs of bacteriophages T4, T5 and T7 have been differentiated into a "labile-starter" and a "stable-starter'' class. The former exhibits a half-life of 1 min with T5-DNA and of 10 min with T4-DNA under standard conditions, the latter a half-life of more than 100 min for the first-order decay in the presence of the strong template-competitor heparin. The labile class is competed out by core enzyme, the stable is not. The relative amounts of both classes of complexes depend on ionic strength.The stable-starter class has been subdivided into a "delayed-starter'' complex formed rapidly and already possessing high stability in the presence of heparin but unable to directly initiate and an "immediate-starter'' complex as a first-order transition product of the delayed-starter state. From the immediate-starter complex initiation can proceed immediately. The time for transition of half of the former into the latter complex is 3.5 min with TB-DNA. The temperature dependence of the equilibrium concentration of the stable-starter complex reveals a sigmoid shape indicating a highly cooperative event, probably a conformational change in the transition from delayed to immediate starter.From the length-average chain length of the transcripts of T5-DNA in vitro and the length of the transcribed fraction of the DNA an approximate number of 14 promoters per DNA molecule was estimated. For T4-DNA, calculation yields about 6 . I n excess of RNA polymerase about 27 copies of transcript per T5-"promoter" were produced from the stable complex under conditions excluding consecutive initiation by free enzyme and reinitiation. The corresponding value for T4-DNA is between 1 and 4.On the basis of these results, a model for the initiation of transcription is postulated, in which an initiation site on the DNA (promoter) is subdivided into an clement for primary recognition, the entry site R, an element for the storage of enzyme for immediate initiation, termed storage stretch 8, which furnishes space for the stable alignment of a certain number of polymerase molecules, and an element for actual initiation, termed initiator I.Pleiotropic control of the gene products of an operon involves a genetically defined element termed promoter, p, which has been considered the initiation site for transcription of the polycistronic message [l]. The repeatedly proved extensive identity of transcripts in vivo and in vitro (the latter by RNA polymerase containing initiation factor 0) indicates highly specific promoter recognition also in vitro (e.g. P-41).Abbreviations. T4-, T5-, T7-DNA = DNA of bacteriophage T4, T5 and T7. I = ionic strength = (l/2 2 c&) where ci represents the concentration and zi the charges of the individual ions.Enzyme. DNA-dependent RNA polymerase or nucleoside triphosphate RNA nucleotidyl transferase (EC 2.7.7.6).The investigation of a lag phase in the initiation of transcription in ...
The high mobility group (HMG) proteins of the AT-hook family (HMGA) lie downstream in regulatory networks with protein kinase C, Cdc2 kinase, MAP kinase, and casein kinase 2 (CK2) as final effectors. In the cells of the midge Chironomus, almost all of the HMGA protein (cHMGA) is phosphorylated by CK2 at two adjacent sites. 40% of the protein population is additionally modified by MAP kinase. Using spectroscopic and protein footprinting techniques, we analyzed how individual and consecutive steps of phosphorylation change the conformation of an HMGA protein and affect its contacts with poly(dAdT)⅐poly(dA-dT) and a fragment of the interferon- promoter. We demonstrate that phosphorylation of cHMGA by CK2 alters its conformation and modulates its DNA binding properties such that a subsequent phosphorylation by Cdc2 kinase changes the organization of the protein-DNA complex. In contrast, consecutive phosphorylation by MAP kinase, which results in a dramatic change in cHMGA conformation, has no direct effect on the complex. Because the phosphorylation of the HMGA proteins attenuates binding affinity and reduces the extent of contacts between the DNA and protein, it is likely that this process mirrors the dynamics and diversity of regulatory processes in chromatin.
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