Interaction of Escherichia coli RNA Polymerase with the Eukaryotic TATA-Binding Protein

Interaction with eukaryotic TATA-binding protein (TBP) was analyzed for natural Escherichia coli RNA polymerase or the recombinant holoenzyme, minimal enzyme, or its subunit. Upon preincubation of full-sized RNA polymerase with TBP and further incubation with a constant amount of a ³²P-labeled phosph-amide derivative of a TATA-containing oligodeoxyribonucleotide, the yield of the holoenzyme–oligonucleotide covalent complex decreased with increasing TBP concentration. This was considered as indirect evidence for complexing of RNA polymerase with TBP. In gel retardation assays, the holoenzyme, but neither the minimal enzyme nor the subunit, interacted with TPB, since the labeled probe formed complexes with both proteins in the reaction mixture combining TBP with the minimal enzyme or the subunit. It was assumed that E. coli RNA polymerase is functionally similar to eukaryotic RNA polymerase II, and that the complete ensemble of all subunits is essential for the specific function of the holoenzyme.     

Highly efficient labeling of DNA polymerases by a binary system of photoaffinity reagents

A binary system of photoaffinity reagents was proposed earlier for highly efficient labeling of DNA polymerases by 5"-[³²P]DNA primers. In the present study we demonstrate the feasibility of this approach to increase the efficiency of DNA polymerase labeling. A photoactive 2,3,5,6-tetrafluoro-4-azidobenzoyl (FAB) group was incorporated at the 3"-end of 5"-[³²P]DNA primers synthesized by DNA polymerase or Tte in the presence of one of the dTTP analogs—FAB-4-dUTP, FAB-9-dUTP, or FAB-4-ddUTP. The reaction mixture was irradiated by light with wavelength of 334-365 nm (direct labeling) or 365-450 nm in the presence of photosensitizer, one of dTTP analogs containing a pyrene moiety, Pyr-6-dUTP or Pyr-8-dUTP. In the case of the binary system of photoaffinity reagents, a FAB group is activated by energy transfer from sensitizer localized in the dNTP-binding site of DNA polymerase in the triple complex, comprised by reagent, DNA polymerase, and Pyr-6(8)-dUTP. Direct activation of the FAB group under these conditions is negligible. The most efficient photolabeling of DNA polymerases was observed with a primer containing a FAB-4-dUMP group at the 3"-end, and Pyr-6-dUTP as a photosensitizer. Using 10-fold molar excess of photoreagent to DNA polymerase , the labeling efficiency was shown to achieve 60%, which is 2-fold higher than the efficiency of the direct DNA polymerase labeling under harsher conditions (334-365 nm).     

Evaluation of the electrical capacitance in biological membranes at different phospholipid to protein ratios

Photosynthetic chromatophores of Rhodobacter capsulatus were differently enriched in phospholipid content by freezing, thawing and sonicating in the presence of phospholipid vesicles. Closed vesicles, characterized by different phospholipid to protein molar ratios and increasing average radius at increasing phospholipid enrichment, were collected after sucrose density gradient sedimentation. The electrical capacitance of these systems was evaluated from the ratio of reaction center content, photooxidized by single turnover flash in the presence of antimycin, to the corresponding membrane potential difference, measured from the electrochromic red shift of the endogenous carotenoid band. The values obtained, normalized per protein content, increased at increasing phospholipid enrichment, and correlated linearly with the increasing phospholipid to protein molar ratios. The charging capacitance of chromatophores was evaluated to be 3–6×10^-17 F and was found to increase at increasing average radius of the phospholipid enriched vesicles, as predicted by the equation of the spherical shell dielectric. The carotenoid signal, elicited in the dark by imposing diffusion potentials of known extent with K⁺-valinomycin pulses, significantly decreased at high phospholipid enrichment, indicating that in the presence of large phospholipid excess, a partial displacement of the carotenoid molecules sensing the induced electric field is produced. Concomitantly, the energy transfer efficiency from carotenoids to core light harvesting complexes (B-875) was also partially affected, particularly at high phospholipid to protein molar ratio. All together, these results suggest that the reaction center complexes are dispersed within the lipid bilayer upon fusion and that carotenoids sense a delocalized light-induced transmembrane field.Abbreviations BChl bacteriochlorophyll - [BChl]₂ reaction center - PL phospholipid - cyt cytochrome - transmembrane electrical potential difference - TES 2-2-Hydroxy-1,1-bis-(hydroxymethyl)ethyl-amino-ethanosulfonic acid - mg_p mg protein     

Isolation of RNA polymerases from the water mold Achlya

The DNA-dependent RNA polymerases I, II, and III (ribonucleosidetriphosphate: RNA nucleotidyl-transferase, EC 2.7.7.6) from Achlya ambisexualis E87 (male), have been isolated. The highly purified RNA polymerase I was found to be composed of polypeptides with the following molecular weights (·10^-4): 18.5, 14, 11.8, 7.3, 6.1, 4.9, 4.4, 2.8. RNA polymerase II showed a 400-fold higher resistance against -amanitin than mammalian or higher plant RNA polymerase II.     

Complexes prepared from protein A and human serum,IgG, or Fcγ fragments: characterization by immunochemical analysis of ultracentrifugation fractions and studies on their interconversion

Summary Protein A of Staphylococcus aureus is an Fc receptor for IgG that has been used as a therapeutic reagent to treat cancer in humans and experimental animals. We used ultracentrifugation combined with analysis of isolated fractions by radioimmunoprecipitation and competitive radioimmunoassay with chicken antibodies that bind free protein A or protein A in complexes but do bind free immunoglobulin reagents to localize and characterize the types of complexes formed with different molar ratios of ¹²⁵I-protein A and human ¹³¹I-IgG alone or in serum, and ¹³¹1-Fc fragments. This approach offers a distinct advantage over direct counting of radioactivity in the fractions because resolution of complexes and free reagents is much improved. With excess ¹³¹I-IgG or ¹³¹1-Fc, all the ¹²⁵I-protein A is present only in complexes that contained 4 molecules of immunoglobulin reagent and 2 molecules of protein A (4:2 complexes), whereas with excess ¹²⁵I-protein A the stoichiometry of the complexes was 1:1. We have also shown the preformed 4:2 and 1:1 complexes will interconvert in the presence of added excess protein A or IgG, respectively, and that fresh IgG will exchange with IgG or Fc in preformed complexes. Because protein A has been found to elute from an immobilized reagent used in serotherapy of human cancer and is present in a large excess of IgG, the 4:2 complexes may play an active role in the tumoricidal or toxic reactions observed.Abbreviations SpA protein A of Staphyloccus aureus - VBS EDTA gel, 0.0055 M veronal buffered saline containing 0.01 M EDTA and 0.1% gelatin, pH 7.4 - PBS 0.01 M phosphate buffered saline, pH 7.4     

Chemical cross-linking of protein to RNA within intact ribosomal subunits from Escherichia coli.

Summary Bifunctional reagents, namely bis-(2-chloroethyl)-amine (nitrogen mustard) and activated esters of 3-(2-bromo-3-oxobutane-1-sulphonyl)-propionic acid (bromo-ketone reagent) are used to cross-link protein to RNA within intact ribosomal subunits. The cross-linked proteins are analysed on two different two-dimensional gel electrophoresis systems, and the existence of a stable cross-linkage is demonstrated by isolating cross-linked protein-oligonucleotide complexes from subunits containing ³²P-labelled RNA. Proteins S3, S4, S5, S9/S11 and S13 from the 30S subunit, and proteins L1 and L2 from the 50S subunit were cross-linked to RNA by the nitrogen mustard, together with a number of other so far unresolved proteins. Correspondingly S3, S4, S7, S9/S11, and L2 were cross-linked by the bromoketone reagent, although in lower yield. The reagents should prove useful for topographical studies on ribosomal subunits, and arguments are presented favouring the use of non-cleavable and relatively non-specific RNA-protein cross-linking reagents for such studies.     

Uptake of homologous single-stranded fragments by superhelical DNA. III. The product and its enzymic conversion to a recombinant molecule

When superhelical DNA (RFI)² of phages φX174 or G4 takes up a homologous single-stranded fragment, RF DNA and fragment are linked by as many as 300 base-pairs, and a corresponding length of one strand of the RFI is displaced, forming a displacement loop (D-loop). The length of the base-paired region was estimated from the fraction of the associated ³²P-labeled fragment that was resistant to digestion by exonuclease VII, as well as by electron microscopy. Dissociation of the fragment by heating was characterized by a sharp melting curve. The displaced strand of the RF DNA was digested by two endonucleases that act on single-stranded DNA, the S₁ nuclease of Aspergillus oryzae and the recBC DNAase of Escherichia coli. Acting on complexes, both enzymes converted the form I [³H]DNA into form II DNA, and left some of the associated ³²P-labeled fragment undigested. The remaining ³²P-labeled fragment could no longer be displaced by branch migration, as expected if the displaced strand of the RF DNA were digested. The action of S₁ nuclease also produced the amount of acid-soluble ³H expected from digestion of the D-loop. Treatment of such digested complexes with polynucleotide ligase covalently linked about 35% of the remaining ³²P-labeled fragment to ³H-labeled strands, which proves that S₁ nuclease digested the D-loop.     

RNA polymerase sigma-related proteins in Escherichia coli: detection by antibodies against a synthetic peptide

Summary Antibodies were raised against a synthetic tetradecameric peptide with an amino acid sequence, DLIQEGNIGLMKAV, which corresponds to the most highly conserved region of bacterial RNA polymerase factors. In a Western-blot analysis of total Escherichia coli proteins, the antiserum reacted specifically with at least three proteins with apparent molecular weights of 75 kDa, 27 kDa and 23 kDa, in addition to the known factors (⁷⁰ and ³²). The majorities of ⁷⁰ and ³² were recovered as associated forms with the RNA polymerase on glycerol gradient centrifugation, while the other cross-reacting proteins were not. Unambiguous evidence was obtained which indicated that the intracellular level of ³² increased rapidly upon heatshock, at least in the strain containing high copy numbers of the rpoH gene.     

Variations in the rate of synthesis of beta and beta'' RNA polymerase polypeptides under the influence of certain factors.

Summary In merodiploid cells containing a double dose of structural genes of RNA polymerase subunits-rpoBand rpoC-the rate of and subunits synthesis is 2 times higher than in haploidcells. Missence mutation rpoC1 (tsX) alters polypeptide and inducesthe and subunits synthesis at increased rate, particularly at a nonpermissive temperature. When rpoBCoperon carrying mutation rpoC1 is duplicated no dosage effect is observed. In the rpoC ⁺/rpoC1 heterodiploid the rpoC1 mutation does not significantly accelerate RNA polymerase subunits synthesis i.e. is recessive with respect to rpoC ⁺ Rifampicin causes 6-fold stimulation of RNA polymerase subunits synthesis in a sensitive wild-type strain. The rpoC1 mutation itself accelerates the synthesis of these subunits 3-fold. In the presence of rifampicin the mutant strain produces 13–22-fold faster as compared to wild-type strain without the drug. Thus, the effects of rifampicin and the mutation are multiplied suggesting that these factors act independently. Similar data have also been obtained with rifampicin-treated cells of rpoB22 (ts22) amber-mutant.After UV-irradiation of cells and synthesis is depressed much stronger than the total protein synthesis. Infection with a transducing phage rif ^d-47 which carries rpoB gene provokes a higher rate of synthesis. When pre-irradiated cells (500 erg/mm²) are infected with this phage, the rate of synthesis grows 20-fold compared to irradiated, non-infected cells and 6.5-fold compared to intact cells.The data are discussed in terms of the possible regulatory mechanisms of RNA polymerase subunit synthesis.     

Isolation and characterization of the fission yeast gene rpa42 +, which encodes a subunit shared by RNA polymerases I and III

Eukaryotic RNA polymerases I and III share two distinct α-related subunits that show limited homology to the α subunit of Escherichia coli RNA polymerase, which forms a homodimer to nucleate the assembly of prokaryotic RNA polymerase. To gain insight into the functions of α-related subunits in eukaryotes, we have previously identified the α-related small subunit RPA17 of RNA polymerase I (and III) in Schizosaccharomyces pombe, and have shown that it is a functional homolog of Saccharomyces cerevisiae AC19. In an extension of that study, we have now isolated and characterized rpa42 ⁺, which encodes the α-related large subunit RPA42 of S. pombe RNA polymerase I, by virtue of the fact that its product interacts with RPA17 in the yeast two-hybrid system. We have found that rpa42 ⁺ encodes a polypeptide with an apparent molecular mass of 42?kDa, which shows 58% identity to the AC40 subunit shared by RNA polymerases I and III in S. cerevisiae. Furthermore, we have shown that rpa42 ⁺ complements a temperature-sensitive mutation in RPC40 the gene that encodes AC40 in S. cerevisiae and which is essential for cell growth. Finally, we have shown that neither RPA42 nor RPA17 can self-associate. These results provide evidence that the two distinct α-related subunits, RPA42 and RPA17, of RNA polymerases I and III are functionally conserved between S. pombe and S. cerevisiae, and suggest that heterodimer formation between them is essential for the assembly of RNA polymerases I and III in eukaryotes.     

Kinetics of beta-glucosidase production by Saccharomyces cerevisiae recombinants harboring heterologous bgl genes

The maximum productivity of -glucosidase by Saccharomyces cerevisiaerecombinants under the control of GALI promoter was 100 IU l^–1 h^–1. The highest productivity of -glucosidase by a S. cerevisiae recombinant was 16-fold more than that supported by Cellulomonas biazotea. The recombinants also co-produced ethanol from cellobiose: maximum product yield and productivity were 0.5 and 1.1 g ethanol g^–1 cellobiose and g ethanol l^–1 h^–1, respectively.     

Alu-family repeat binding protein from HeLa cells which interacts with regulatory region of SV40 virus genome

Using a gel retardation assay the protein which binds selectively to the Alu-family repeat (AFR) has been identified and partially purified from HeLa cell nuclear extract. The protein (AFR-binding protein, ABP) forms multiple discrete complexes with AFR even in the presence of 200 to 2000-fold excess of non-specific (E. coli) DNA. The most stable complex has a relative mobility in 4% polyacrylamide gel (as compared to the free Alu-fragment) of 0.54. Heterogeneity of protein-DNA bands seen in the polyacrylamide gel suggests that ABP is able to form multimeric complexes with AFR. Competition experiments show that ABP do not interact with the RNA polymerase III promoter and with the TGGCA-sequence, but a high affinity binding site for ABP was found within a 660 bp restriction fragment containing the SV40 virus promoter and replication origin.     

Utilization of promoter regions of coliphage lambda in Bacillus subtilis

Summary In vivo studies with galactokinase monitoring system demonstrated that the coliphage lambda P_RP_L promoter regions could be utilized in B. subtilis. These promoter regions were preferentially utilized during the stationary growth phase of B. subtilis. However, these promoter regions were not controlled by the cI₈₅₇ gene at permissive or non-permissive temperature in B. subtilis, although the P _RM promoter was utilized in B. subtilis. S1-nuclease mapping suggests that B. subtilis RNA polymerase recognizes specific sequences in P _R promoter region that is not utilized in E. coli.     

The permeability to water of the eggs of certain marine teleosts

Summary Measurements have been made of the rate of turnover of tritiated water in the eggs of the plaice,Pleuronectes platessa and of the permeability of the vitelline membrane. The rate constant of water exchange in body cavity eggs is 1.0 h^–1, corresponding to a permeability of 0.086 ³/²/sec. When shed into sea water the rate constant of exchange declines to 0.2 h^–1 after 1 hour and to 0.02 h^–1 after one day corresponding to a permeability of 0.0017 [³/²/sec. As the embryo develops the rate of turnover increases again. The buoyancy of the egg is dependent on its low salt content which counteracts the weight of the protein. During development the water loss by osmosis reduces the egg almost to neutral buoyancy.     

Temperature dependent release of beta-beta'' subunits of DNA dependent RNA polymerase from the folded chromosome of a dnaAts mutant of Escherichia coli.

Summary DNA-dependent RNA polymerase has been found to be preferentially released at 43° C from the folded nucleoids of an E. coli dnaA ^ts mutant when compared with the same nucleoids at 30° C or with nucleoids of a dnaA ⁺ strain at either 30° or 43° C. The polypeptides released are identical in molecular weight with those of the and constituent polypeptides of the core enzyme of a known E. coli RNA polymerase. In addition, these polypeptides are precipitated by specific anti-RNA polymerase rabbit IgG. The implications of the interactions of RNA polymerase with the dnaA gene product are discussed.     

Modified sequences in yeast ribosomal RNA

26S and 17S yeast ribosomal RNA were digested with T ₁ plus pancreatic ribonuclease and the products were fractionated by two-dimensional electrophoresis. Besides the expected standard products (type (Ap) _n Np, where N is C, U or G) several non-standard products were found to be present in the digests. The latter products include methylated oligonucleotides and pseudouridylic acid (p)-containing fragments. The primary structure and molar frequency of these modified products were determined. They appeared to be present in approximately integral molar amounts. Several of these oligonucleotides contain more than one modification. The total number of p-residues in 26S and 17S yeast rRNA was estimated to be about 32 and 14, respectively.