Immunochemical analysis of molecular forms of protein synthesis initiation factors in crude cell lysates of Escherichia coli

Three initiation factors (IF1, IF2, and IF3) have been highly purified from Escherichia coli and extensively characterized, but little is known about the molecular forms of these proteins as they occur in vivo. We have analyzed molecular-weight and charge forms in crude cell lysates by polyacrylamide gel electrophoresis followed by immunoblotting with antibodies specific for the initiation factors. Freshly grown bacterial cells were lysed by sonication in buffer containing sodium dodecyl sulfate, and the lysate was fractionated by gel electrophoresis. Proteins from the gel were electrotransferred to a nitrocellulose sheet which was treated with a specific rabbit antiserum followed by radiolabeled Staphylococcus aureus protein A. Autoradiography showed only one major band each for IF1 and IF3, exactly corresponding to the isolated factors. For IF2, two molecular-weight forms were detected which were identical with purified IF2a and IF2b. No evidence for precursor forms was found. Two-dimensional gel analysis showed no charge heterogeneity for IF1, IF2a, and IF3, but multiple forms were seen for IF2b. Analysis of phosphoproteins from cells grown in radioactive phosphate medium ruled out the possibility that phosphorylation occurs on the initiation factors, elongation factors, or ribosomal proteins.     

Further sequence analysis of the phage lambda receptor site. Possible implications for the organization of the lamB protein in Escherichia coli K12

We present the DNA sequence alterations due to seven lamB missense mutations yielding resistance to phages lambda and K10. They reveal five different amino acid positions in the LamB protein. Three positions (245, 247 and 249) define a new region required for phage adsorption. The two other positions (148 and 152) belong to a region where mutations to phage resistance has already been detected. These two regions are hydrophilic and could belong to turns of the protein located at the surface of the cell. All the missense mutational alterations to phage resistance sequenced in the LamB protein correspond to 10 sites located in four different segments of the polypeptide chain. We discuss their location in terms of the notion of phage receptor site and of a working model for the organization of this protein in the outer membrane of Escherichia coli.     

The secondary attachment site for bacteriophage lambda in the proA/B gene of Escherichia coli

We have determined the nucleotide sequence of a secondary λ attachment site in $\text{pro}\text{A}\text{B}$, a site that accounts for 3% of lysogens isolated from Escherichia coli strains deleted for the primary site. Direct sequence analysis of the transducing bacteriophages carrying the left and right att junctions, as well as the recombinant pro⁺ phage reveals that the $\text{pro}\text{A}\text{B}$ site shares an 11-nucleotide interrupted homology with the core sequence of the primary site. We have compared the $\text{pro}\text{A}\text{B}$att site with other secondary attachment sites to gain insights into the structural features important for λ integration.     

Non-random layout of the amino acid loci on the genome of Escherichia coli

A strong correlation exists between the relative frequencies of occurrence of the amino acids in bulk Escherichia coli protein and their genetic map positions when the latter are indexed against the position of the origin of DNA synthesis. The greater the production of the amino acid, the closer its operon is to the origin.     

Nucleotide sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli

The pstA(=phoT), pstB and phoU genes are situated at 84 minutes on the Escherichia coli genetic map. All of them are involved in the negative regulation of the phosphate regulon, and all of them except for phoU are required for the binding-protein-mediated, highly specific phosphate transport. We have determined the DNA sequence of about 4 X 10(3) bases of chromosomal segment containing these genes. Four translational reading frames (TRFs) were detected in the region. We attempted to assign the TRFs to the mutant alleles. Plasmids were constructed so that each contained only one of the TRFs, downstream from the lac promoter, to be used for the complementation tests. By this test, TRF-2, TRF-3 and TRF-4 were identified with the pstA(=phoT), pstB and phoU genes, respectively. Alkaline phosphatase-constitutive mutations of the two strains in our collection were complemented by the plasmid with the TRF-1 region. Therefore, we propose to designate the allele phoW. The order of the genes in this region has been established to be phoS-phoW-pstA(=phoT)-pstB-phoU counterclockwise on the E. coli genetic map.     

EcoA and EcoE: alternatives to the EcoK family of type I restriction and modification systems of Escherichia coli

The genes (hsd A) encoding EcoA, a restriction and modification system first identified in Escherichia coli 15T-, behave in genetic crosses as alleles of the genes (hsd K) encoding the archetypal type I restriction and modification system of E. coli K12. Nevertheless, molecular experiments have failed to detect relatedness between the A and K systems. We have cloned the hsd A genes and have identified, on the basis of DNA homology, related genes (hsd E) conferring a new specificity to a natural isolate of E. coli. We show that the overall organization of the genes encoding EcoA and EcoE closely parallels that for EcoK. Each enzyme is encoded by three genes, of which only one, hsdS, confers the specificity of DNA interaction. The three genes are in the same order as those encoding EcoK, i.e. hsdR, hsdM and hsdS and, similarly, they include a promoter between hsdR and hsdM from which the M and S genes can be transcribed. The evidence indicates that EcoA and EcoE are type I restriction and modification enzymes, but they appear to identify an alternative family to EcoK. For both families, the hsdR polypeptide is by far the largest, but the sizes of the other two polypeptides are reversed, with the smallest polypeptide of EcoK being the product of hsd S, and the smallest for the EcoA family being the product of hsdM. Physiologically, the A restriction and modification system differs from that of K and its relatives, in that A-specific methylation of unmodified DNA is particularly effective.     

Location and orientation of the chromophore in bacteriorhodopsin: Analysis by fluorescence energy transfer

The spatial location and orientation of the retinal chromophore in bacteriorhodopsin were estimated from a fluorescence energy transfer study. The energy donor used in this study was a fluorescent retinal derivative, which was obtained by partial reduction of the purple membrane with sodium borohydride, and the energy acceptor was the native chromophore remaining in the same membrane. Since bacteriorhodopsin forms a two-dimensional crystal with P₃ symmetry in the purple membrane, and the membrane structure is maintained after the reduction, the rate of energy transfer from a donor to any acceptor existing in the same membrane can be calculated as a function of the location and orientation of the chromophores in the unit cell. Quantitative analyses of the fluorescence decay curve and the quantum yield, with various extents of reduction, enabled us to determine the most probable location and orientation. The result suggested that the chromophore was situated near the centre of the protein in such an orientation that the dipole-dipole interaction with neighbouring chromophores was close to minimum.     

Sequence information within the lamB genes in required for proper routing of the bacteriophage lambda receptor protein to the outer membrane of Escherichia coli K-12

The structure of Satellite tobacco necrosis virus (STNV) has been determined to 3.0 Å resolution by X-ray crystallography. Electron density maps were obtained with phases based on one heavy-atom derivative and several cycles of phase refinement using the 60-fold non-crystallographic symmetry in the particle. A model for one protein subunit was built using a computer graphics display. The subunit is constructed mainly of a β-roll structure forming two β-sheets, each of four antiparallel strands. The N-termini of the subunits form bundles of three α-helices extending into the RNA region of the virus at the 3-fold axis. The topology of the polypeptide chain is the same as, and the conformation clearly similar to, that of the shell domains of the Tomato bushy stunt virus (TBSV) and Southern bean mosaic virus (SBMV) protein subunits. The subunit packing in the T = 1 STNV structure is, however, significantly different from the packing of these T = 3 viruses: parts of some of the structural elements facing the RNA in TBSV and SBMV are utilized for subunit-subunit contacts in STNV. No RNA structure is obvious in the present icosahedrally averaged electron density maps. The protein surface facing the RNA contains mainly hydrophilic residues, especially lysine and arginine.     

Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-lambda PR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening

The kinetics of formation and of dissociation of open complexes (RPo) between Escherichia coli RNA polymerase (R) and the lambda PR promoter (P) have been studied as a function of temperature in the physiological range using the nitrocellulose filter binding assay. The kinetic data provide further evidence for the mechanism R + P in equilibrium I1 in equilibrium I2 in equilibrium RPo, where I1 and I2 are kinetically distinguishable intermediate complexes at this promoter which do not accumulate under the reaction conditions investigated. The overall second-order association rate constant (ka) increases dramatically with increasing temperature, yielding a temperature-dependent activation energy in the range 20 kcal (near 37 degrees C) to 40 kcal (near 13 degrees C) (1 kcal = 4.184 kJ). Both isomerization steps (I1----I2 and I2----RPo) appear to be highly temperature dependent. Except at low temperatures (less than 13 degrees C) the step I1----I2, which we attribute to a conformational change in the polymerase with a large negative delta Cp degrees value, is rate-limiting at the reactant concentrations investigated and hence makes the dominant contribution to the apparent activation energy of the pseudo first-order association reaction. The subsequent step I2----RPo, which we attribute to DNA melting, has a higher activation energy (in excess of 100 kcal) but only becomes rate-limiting at low temperature (less than 13 degrees C). The initial binding step R + P in equilibrium I1 appears to be in equilibrium on the time-scale of the isomerization reactions under all conditions investigated; the equilibrium constant for this step is not a strong function of temperature and is approximately 10(7) M-1 under the standard ionic conditions of the assay (40 mM-Tris . HCl (pH 8.0), 10 mM-MgCl2, 0.12 M-KC1). The activation energy of the dissociation reaction becomes increasingly negative at low temperatures, ranging from approximately -9 kcal near 37 degrees C to -30 kcal near 13 degrees C. Thermodynamic (van't Hoff) enthalpies delta H degrees of open complex formation consequently are large and temperature-dependent, increasing from approximately 29 to 70 kcal as the temperature is reduced from 37 to 13 degrees C. The corresponding delta Cp degrees value is approximately -2.4 kcal/deg. We propose that this large negative delta Cp degrees value arises primarily from the burial of hydrophobic surface in the conformational change (I1 in equilibrium I2) in RNA polymerase in the key second step of the mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding of terbium to apoferritin: a fluorescence study

Luminescence measurements show that apoferritin binds three Tb(III) atoms per subunit in accordance with crystallographic evidence. Fe(II) competes with Tb(III) for at least some of the binding sites. This competition may be the molecular basis for the inhibition of iron incorporation into apoferritin brought about by Tb(III). Ca(II), which is generally replaced by Tb(III) in Ca(II) binding proteins, does not compete with the lanthanide for binding to apoferritin.     

Catalase synthesis in Escherichia coli is not controlled by catabolite repression

Catabolite repression is not involved in the regulation of catalase gene expression. The presence of glucose in minimal salts media and LB medium did not affect the basal levels of catalase but did enhance catalase synthesis following induction with either hydrogen peroxide or ascorbate. The cofactor for catabolite gene activator protein, cAMP, did not affect either the basal levels or the rate or extent of catalase synthesis. Catalase synthesis occurred normally in an adenylate cyclase mutant where β-galactosidase, a catabolite-sensitive enzyme was not synthesized.     

Structure of the central spacer region of extrachromosomal ribosomal DNA in Physarum polycephalum

We have analyzed the sequence organization of the central spacer region of the extrachromosomal ribosomal DNA from two strains of the acellular slime mold Physarum polycephalum. It had been inferred previously from electron microscopy that this region, which comprises about one third of the 60 kb³ palindromic rDNA, contains a complex series of inverted repetitious sequences. By partial digestion of end-labeled fragments isolated from purified rDNA and from rDNA fragments cloned in Escherichia coli, we have constructed a detailed restriction map of this region. The 11 kb of spacer DNA of each half molecule of rDNA contains the following elements: (a) two separate regions, one of 1.1 kb and one of 2.1 kb, composed of many direct repeats of the same 30 base-pair unit; (b) a region of 4.4 kb composed of a complex series of inverted repeats of a 310 base-pair unit; (c) another region of 1.6 kb composed of inverted repeats of the same 310 base-pair unit located directly adjacent to the center of the rDNA; (d) two copies of a unique sequence of 0.85 kb, which probably contains a replication origin. Some of the CpG sequences in the spacer resist cleavage by certain restriction endonucleases and thus appear to be methylated. The lack of perfect symmetry about the central axis and the arrangement of inverted repeated sequences explain the complex pattern of branches and forks of the fold-back molecules previously observed by electron microscopy. Comparison of the rDNA restriction maps from the two strains of Physarum suggests that the repeat units in the spacer are undergoing concerted evolution. We propose a model to explain the evolutionary origin of the several palindromic axes in the Physarum rDNA spacer.     

Structure of the filamentous bacteriophage,Pf3, by X-ray fiber diffraction

Fiber diffraction data have been obtained for the filamentous bacteriophage Pf3. The virus crystallizes on a hexagonal net with lattice constants a = b = 5.61 nm and c = 7.50 nm at 0% relative humidity and a = 6 = 6.20 nm and c = 7.99 nm at 98% relative humidity. The X-ray diffraction pattern resembles those of two other bacteriophages, Pf 1 and Xf, and therefore belongs to class II. The data are consistent with a 27₅ helix symmetry with an axial rise of 0.277 (dry) to 0.296 (wet) nm.     

Cloning and deletion mapping of the recFdnaN region of the Escherichia coli chromosome

By cloning a 3.6-kb EcoRI fragment of the Escherichia coli chromosome with pBR322 we located more precisely recF relative to dnaN. By deletion mapping we localized functional recF to a 1.65-kb region of the cloned fragment and allowed rough mapping of the C terminus of dnaN. Cloned recF⁺, separated from functional flanking genes dnaN and gyrB, complemented chromosomal recF mutations presumably by coding for a cytodiffusible product. The protein encoded by dnaN was observed as a band on a polyacrylamide gel from minicells. Identification of a recF protein was not made.     

A local antigenic determinant distribution in a continuous antigenic region at residues 38-54 of hen egg white lysozyme

The precise location of the antigenic determinants in a continuous antigenic region at residues 38–54 of hen egg white lysozyme (lysozyme) was investigated using the inhibition test of binding of N^α-[¹⁴C]acetyl fragment 38–54 with goat (three individuals) and sheep (four individuals) anti-lysozyme antisera by various synthetic and proteolytic fragments of lysozyme. From these inhibition studies, we found that in this region there were three independent antigenic determinants, consisting of residues 38–45, 40–48, and 44–54, respectively. The existence and the specificity of the antibodies directed to these determinants were further examined with isolating the specific antibodies by affinity chromatography on columns to which the fragment 38–45, 44–48, and 46–54 were bound. The results indicated that these determinants partially overlapped one another in amino acid sequence, but the antibodies directed to them could recognize only each corresponding determinant. These antibodies were also shown to be reactive with native lysozyme as well as a reduced and S-carboxymethylated derivative of lysozyme, and to be found in goat and sheep anti-lysozyme antibodies. The amounts of these antibodies calculated from the binding capacities were in the range from 0 to 48 μg/ml of antisera. These values corresponded to a small fraction of the total precipitable anti-lysozyme antibodies and were as high as 0.8% of the total. The ratios of the amounts of these antibodies differ in individuals or in different species of animals. The binding affinities of N^α-[¹⁴C]acetyl fragment 38–54 with these antibodies were in the range from 1.3 × 10⁷ to 2.6 × 10⁸m^?1. The double-reciprocal plots of the antigen binding with these antibodies drew almost a straight line compared with those of a mixture of several antibody populations, that is, whole antisera.     

gamma-Glutamyl transpeptidase and glutathione in aging IMR-90 fibroblasts and in differentiating 3T3 L1 preadipocytes

γ-Glutamyl transpeptidase, glutathionase, and phosphate-independent glutaminase activities, and glutathione contents were measured in IMR-90 cells, human embryonic lung diploid fibroblasts, “aging” in culture, and in cultures of 3T3 L1 mouse preadipocytes differentiating to adipocytes. γ-Glutamyl transpeptidase, glutathionase, and phosphateindependent glutaminase activities in IMR-90 cells were about 8-, 8-, and 7-fold higher in “old” cells as compared to the activities in “young” cells. Total and reduced glutathione contents, expressed in relation to either cell number or DNA content, of IMR-90 cells were 3- and 12-fold higher in “young” cells (population doubling level 22) as compared to “old” cells (population doubling level 49). The ratio of reduced to oxidized glutathione was relatively constant in “young” cells over a range of population doubling levels 18–25; however, relative to the “young” cells, the ratio was decreased in “old” cells over a range of population doubling levels 49–55. γ-Glutamyl transpeptidase, glutathionase, and phosphate-independent glutaminase activities in 3T3 L1 cells were about 4-, 5-, and 5-fold higher in differentiating cells as compared to the activities in undifferentiating cells. Total glutathione contents in 3T3 L1 cells differentiating to adipocytes were similar to glutathione contents in undifferentiating cells. As the culture differentiated to the extent of 50%, almost all of the glutathione was present as oxidized glutathione. These results suggest that the several enzymatic activities and glutathione contents may be used as markers for aging or differentiation in cells. In the course of these studies, a sensitive fluorometric assay was developed, using l-γ-glutamyl-4-methoxy-2-naphthylamide as substrate.