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Considering enzymatic activities found in bacteria and in animal cells, there are two possible mechanisms for repair of N-methylated purines produced by methylating agents such as the mutagen and carcinogen N-methyl-N′-nitro-N-nitrosoguanidine. Both mechanisms involve first an enzymatic removal of the methylated bases by glycosylases. The resulting apurinic sites could then be repaired by (a) direct insertion of the correct bases purine insertases or (b) opening of the polynucleotide chain by apurinic endonuclease followed by repair synthesis. As the methods commonly used to detect lesions induced by methylating agents involve alkali, it was thus far not possible to decide between the above possibilities because apurinic sites are by themselves alkali labile. In this paper I describe two methods which avoid alkali and therefore allow the clarification of some aspects of the repair reaction. One of these methods makes use of 95% formamide at 40 °C in place of alkali to denature DNA with pre-existing single-strand breaks, the other measures the capacity of DNA scissions with free 3′-OH groups to act as primer for Escherichia coli DNA polymerase I. Results obtained with both methods make it unlikely that purine insertases play a major role in the repair of apurinic sites. Kinetics of production and repair of single-strand breaks, produced in 3T6 mouse fibroblasts by incubation with N-methyl-N′-nitro-N-nitrosoguanidine, were also examined using the methods of alkaline elution and alkaline sucrose gradient centrifugation. [ABSTRACT FROM AUTHOR]

Escherichia coli K 12 appears to behave as an enzyme. We show in the present paper that, in fact, phosphofructokinase 2 also presents some regulatory properties in vitro: at high concentrations, ATP is an inhibitor of phosphofructokinase 2 and it provokes the tetramerization of the dimeric native enzyme. The binding of the two substrates to phosphofructokinase 2 is sequential and ordered as for phosphofructokinase 1, but in the former case fructose 6-phosphate is the first substrate to be bound and ADP the first product to be released. Each dimer of phosphofructokinase 2 binds two molecules of fructose 6-phosphate but only one molecule of the product fructose 1,6-bisphosphate. Although both phosphofructokinases of E. coli K 12 present regulatory properties in vitro, the mechanism of regulation of the activity of the two enzymes is strikingly different. It can be asked whether or not these mechanisms operate in vivo. [ABSTRACT FROM AUTHOR]

A highly folded form of the ribosomal protein L25 from Escherichia coli can be obtained from urea-denatured preparations. Proton NMR data show that this form of the molecule must have a compact, globular tertiary structure. Spectroscopically it is indistinguishable from L25 prepared by methods which avoid denaturing solvents. Thus L25 is a protein which can be reversibly denatured. The stability and solubility of the folded form of the protein are discussed and primary assignments made for a number of resonances in its NMR spectrum. The paper shows that this folded form of the protein can be characterised using NMR spectroscopy. High-resolution NMR spectroscopy provides a sensitive and general way for the characterisation of protein folds. [ABSTRACT FROM AUTHOR]

Poly(uridylic acid) [poly(U)] and poly(xanthidylic acid) [poly(X)] strongly stimulate the IF-2-dependent binding of fMet-tRNA to 30-S ribosomal subunits from Escherichia coli [Van der Laken et al. (1979) FEBS Lett. 100, 230–234]. The N-formylmethionine moiety is incorporated into poly(phenylalanine) upon subsequent addition of other components required for protein synthesis when poly(U) is used as template. This paper shows that N-acetylated Phe-tRNAPhe (AcPhe-tRNA), but not Phe-tRNAPhe or tRNAPhe, competes with fMet-tRNA for binding to poly(U)-programmed 30-S ribosomal subunits. The two species of N-blocked aminoacyl-tRNA are bound to poly(U)-programmed and poly(X)-programmed 30-S subunits in a ratio that is linearly dependent on the ratio of the two species added. With poly(U) as template there is no apparent preference for either fMet-tRNA or AcPhe-tRNA, whereas with poly(X) there is a 2–3-fold preference for fMet-tRNA. The initiation factor IF-2, which is strictly required for the binding of N-blocked aminoacyi-tRNAs, has a higher affinity for fMet-tRNA than for AcPhe-tRNA. It is concluded that (a) interaction of the 30-S ribosomal subunit with poly(U) or poly(X) leads to IF-2-dependent binding of N-blocked aminoacyl-tRNA; (b) the initiation factor IF-2 discriminates in favour of fMet-tRNA; (c) the presence of the cognate codon discriminates in favour of the corresponding N-blocked aminoacyl-tRNA. [ABSTRACT FROM AUTHOR]

The DNA-binding protein HU from Escherichia coil is a heterodimer constituted of two poly- peptide chains termed HU-1 and HU-2, of 90 residues each. Their primary structures were established from structural data obtained from tryptic peptides of each monomer in addition to the structural data provided by the automated Edman degradation of the dimer and by peptides derived from cleavage of the dimer with trypsin, chymotrypsin, V8 staphylococcal protease and dilute acid. The results presented in this paper confirm the amino-terminal and carboxy-terminal sequences of the dimer HU reported previously [Laine et al. (1978) FEBS Lett. 89, 116–120]. The amino acid sequences of proteins HU-1 and HU-2 are identical to those of proteins NS-1 and NS-2 respectively, determined independently by Mende et al. [FEBS Lett. (1978) 96, 395–398]. The amino acid sequences of proteins HU-1 and HU-2 are closely related but differ by 28 residues. These proteins are characterized by their high content of hydrophobic residues represented mostly by alanine. In both proteins, half of the basic residues are scattered along the polypeptide chain and the remainder is found within two short sequences located in the carboxy-terminal part of the molecule. No sequence homology could be established between the proteins HU-1 and HU-2 and any one of the five histones from different eukaryotes. [ABSTRACT FROM AUTHOR]

The complete nucleotide sequence of the 16-S RNA from Escherichia coli has been determined using rapid RNA-sequencing gel methods. The experimental data are fully described in this paper. The specificities of the ribonucleases, especially the ribonuclease PhyI are discussed and the consequences of the persistence of stable secondary structure are considered. The proposed sequence contains 1541 nucleotides and agrees completely with the DNA sequence of the rrnB cistron deduced by Brosius, J., Palmer, M. L., Kennedy, P.J., and Noller, H. F. [Proc. Natl Acad. Sci. U.S.A. (1978) 75, 4801–4805]. But there are several cistron heterogeneities of which we described 16 single-base heterogeneities, 7 of the deletion/insertion type and 9 of the transition or transversion type. Our observations suggest the existence, among the 7 ribosome RNA cistrons, of one or two mutated ones. The respective advantages and disadvantages of both RNA and DNA sequencing methods are discussed. [ABSTRACT FROM AUTHOR]

Virginiamycin S is an inhibitor of protein synthesis in vivo. In this paper we show by equilibrium dialysis that it binds specifically to the 50-S subunit of Escherichia coli ribosomes, with one binding site per subunit. This binding is not altered by the presence of chloramphenicol, tetracycline or puromycin but is competed for by erythromycin. Using the splitting-reconstitution method, it could be demonstrated that protein L16 is absolutely required for the binding of virginiamycin S to the 50-S subunit. [ABSTRACT FROM AUTHOR]

Two lipopolysaccharide preparations were obtained from Escherichia coli 058 by extraction with 45% aqueous phenol and fractional precipitation with cetyltrimethyl ammonium bromide (Cetavlon). Chemical analysis and polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that the two preparations differed only in the extent of the O-specific polysaccharide moiety. The O-specific polysaccharide was characterized with proton magnetic resonance and infrared spectroscopy, optical rotation and paper electrophoresis. Using gas-liquid chromatography and ion-exchange chromatography, it was shown to contain D-mannose, 2-acetamido-2-deoxy-D-glucose, 3-O-(R-1'-carboxyethyl)-L-rhamnose (rhamnolactylic acid), and O-acetyl groups in the molar ratios of 2:1:1:1. The polysaccharide and oligosaccharides obtained from it were subjected to methylation and chromic acid oxidation. The results obtained indicated that the polysaccharide consists of tetrasaccharide repeating units in which the trisaccharide β-GlcNAc1-4αMan-1-4(2/3-O-Ac)-Man is substituted at C-3 of the non-acetylated mannose with rhamnolactylic acid. The repeating units are joined through α-mannosyl-1-3-glucosamine bonds. This structure is identical with that of the cell wall polysaccharide of Shigella dysenteriae type 5. [ABSTRACT FROM AUTHOR]

The DNA-stimulated 75 000-Mr ATPase described in the preceding paper is shown to be a further catalytic DNA unwinding principle (DNA unwinding enzyme II) made in Escherichia coli cells (the first being the 180 000-Mr ATPase of the cells: DNA unwinding enzyme I). Unwinding depends, strictly, on the supply of ATP. It occurs only under conditions permitting ATP dephosphorylation and it proceeds as long as enzyme molecules are permitted to enter the enzyme · DNA complex. The enzyme binds specifically to single-stranded DNA yielding a complex of only limited stability. These results are interpreted in terms of a distributive mode of action of the enzyme. It is argued that chain separation starts near a single-stranded DNA region and that, forced by continued adsorption of enzyme molecules to the DNA, it develops along the duplex. This mechanism is different from that deduced previously for DNA unwinding enzyme I. Complicated results were obtained using ATPase prepared from rep3 mutant cells. [ABSTRACT FROM AUTHOR]

The lytic enzyme from bacteriophage T7 has been purified from T7-infected Escherichia coli B cells and characterized. In the purification procedure advantage was taken of the reversible binding of the enzyme to chitin. Molecular weight determinations of the enzyme by Sephadex G-75 chromatography and sodium dodecyl sulphate gel electrophoresis gave estimates of 21000 and 18000 respectively. Because the activity of the enzyme falls rapidly to 1-3% of that observed immediately after lysis of the cells, various means to stabilize the enzyme have been tried. Of the methods attempted only binding to the substrate analogue chitin was successful. The enzymic activity was found to be markedly dependent on ionic strength and pH, maximum activity was observed using 5 mM phosphate buffer pH 7.5. MgCl2 was found to enhance the lytic activity 2–3-fold, the optimal concentration being about 0.5 mM. The enzyme was not sensitive to penicillin G or spermine, but was specifically inhibited by p-chloromercuribenzoate. Following digestion of E. coli peptidoglycan the cell wall peptides L-Ala-D-Glu(A2pm), L-Ala-D-Glu(A2pm-D-Ala) and the dimer of the latter were formed, together with paper chromatographically immobile glycan strands. The enzyme, therefore, acts as an N-acetylmuramyl-L-alanine amidase by cleaving the bond between N-acetylmuramic acid and L-alanine in the E. coli peptidoglycan. The enzyme has apparently no effect on the peptidoglycan from Micrococcus luteus, and seems to have a preference for high-molecular-weight substrates. Thus the disaccharide-tetrapeptide GlcNAc-MurNAc-L-Ala-D-Glu(A2pm-D-Ala) and its dimer are not substrates for the enzyme. [ABSTRACT FROM AUTHOR]

When Escherichia coli 30-S ribosomal subunits are hydrolysed under mild conditions, two ribonucleoprotein fragments of unequal size are produced. Knowledge of the RNA sequences contained in these hydrolysis products was required for the experiments described in the preceding paper, and the RNA sub-fragments have therefore been examined by oligonucleotide analysis. Two well-defined small fragments of free RNA, produced concomitantly with the ribonucleoprotein fragments, were also analysed. The larger ribonucleoprotein fragment, containing predominantly proteins S4, S5, S8, S15, S16 (17) and S20, contains a complex mixture of RNA sub-fragments varying from about 100 to 800 nucleotides in length. All these fragments arose from the 5′-terminal 900 nucleotides of 16-S RNA, corresponding to the well-known 12-S fragment. No long-range interactions could be detected within this RNA region in these experiments. The RNA from the smaller ribonucleoprotein fragment (containing proteins S7, S9, S10, S14 and S19) has been described in detail previously, and consists of about 450 nucleotides near the 3′ end of the 16-S RNA, but lacking the 3′-terminal 150 nucleotides. The two small free RNA fragments (above) partly account for these missing 150 nucleotides; both fragments arose from section A of the 16-S RNA, but section J (the 3′-terminal 50 nucleotides) was not found. This result suggests that the 3′ region of 16-S RNA is not involved in stable interactions with protein. [ABSTRACT FROM AUTHOR]

By treatment of Bacillus subtilis MO-101-P spoA- met thr- su- with ethyl methanesulfonate, a strong suppressor strain of nonsense mutations, B. subtilis MO-101-P spoA- [met-]+ thr- su+44, was isolated. This strain does not suppress phage Φ29 mutant susB47, selected on a B. subtilis strain containing the su+3 suppressor isolated by Georgopoulos. A revertant from this mutant, susB610, was isolated, being suppressed by both the su+3 and su+44 suppressor strains. The efficiency of suppression by strain su+44 is about 50%. The experiments shown in this paper suggest that strain su+44 contains an amber and strain su+3 an ochre suppressor. [ABSTRACT FROM AUTHOR]

From Escherichia coli 0124 two lipopolysaccharide preparations were obtained with phenol/water extraction and cetavlon precipitation. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and chemical analysis showed that the two preparations differed only in the extent of the O-specific polysaccharide moiety. In passive haemagglutination and gel precipitation the two lipopolysaccharide preparations from E. coli 0124 and the corresponding preparations from Shigella dysenteriae type 3 reacted alike. The O-specific polysaccharide moiety was characterized with proton magnetic resonance spectroscopy, optical rotation and paper electrophoresis. The constituents were determined by gas chromatography and ion-exchange chromatography. The polysaccharide contained glucose (Glc), gatactose (Gal), galactosamine (GalN) and 4-O-(1′-carboxyethyl)-D-glucopyranose (glucolactilic acid, GIcLA) in the molar ratios of 1 : 2 : 1 : 1. Glucolactilic acid, which has a structure similar to muramic acid, was first found in Sh. dysenteriae. The polysaccharide from E. coli 0124 and oligosaccharides obtained from it by partial acid hydrolysis were subjected to methylation analysis using the method of combined gas chromatography—mass spectrometry. The results indicated that the pentasaccharide repeating unit of the polysaccharide is[This symbol cannot be presented in ASCII format] In the polysaccharide the repeating units are joined through galactofuranosidic linkages. This structure is identical with that of the somatic polysaccharide of Sh. dysenterae type 3. [ABSTRACT FROM AUTHOR]

An enzyme capable of hydrolyzing the substrate L-alanine p-nitroanilide has been found in the various Escherichia coli strains tested. This enzyme has been called aminoendopeptidase since it shows both activities (see accompanying paper). It is released from the cells by osmotic shock and by lysozyme-EDTA spheroplasting treatment, and 50% of the total activity is directly detectable with suspensions of intact cells. However, the release by osmotic shock or spheroplasting is not as efficient as it is for alkaline phosphatase. This periplasmic aminoendopeptidase is constitutively produced but the differential rate of synthesis is increased 4-fold when the cell growth is limited by Pi. The occurrence of this 'derepression' is simultaneous with that of alkaline phosphatase. Increasing the concentration of inorganic phosphate in the medium has no effect on the constitutive aminoendopeptidase synthesis. The effect of phosphate starvation is specific since starvation for neither nitrogen nor carbon and energy source are effective in derepressing aminoendopeptidase. [ABSTRACT FROM AUTHOR]

Three different phenylalanyl-tRNA synthetases have been purified to near homogeneity, one from a wild-type strain of Escherichia coli and the others from two independently isolated p-fluorophenylalanine-resistant strains. The mutant enzymes were not able to use p-fluorophenylalanine as a substrate for activation and attachment to tRNA. They proved to be indistinguishable from the wild-type enzyme by several electrophoretic and immunological criteria. The α and β subunits of all three enzymes have been prepared by a method described in this paper. The isolated subunits per se did not reveal any significant enzyme activity, but combined they were able to form active phenylalanyl-tRNA synthetase after a defined reconstitution process. Mixed reconstitution experiments between wild-type and mutant subunits indicate that the mutant a subunit is responsible for p-fluorophenylalanine resistance and therefore seems to carry the phenylalanine-binding site or to participate in its formation. [ABSTRACT FROM AUTHOR]

The results presented in this paper establish relationships between structural morphological and functional properties of cytoplasmic membrane vesicles isolated from an Escherichia coli unsaturated fatty acid auxotroph. The membranes were isolated from cells grown in the presence of either oleic, linoleic, linolenic or elaidic acids. High-angle X-ray diffraction studies show that the order-disorder transitions induced by temperature variations and associated with the paraffin chains of the lipids are a function of the fatty acid composition of the membranes. In some cases "cocrystallization" of various lipid species takes place within a single type of ordered domains. In other cases there is segregation of various lipid species into more than one distinct type of ordered domain. The various order-disorder transitions observed induce morphological changes in the hydrophobic core of the membranes which can be detected by freeze-etch electron microscopy. A random distribution of particles on the fracture faces is observed when the paraffin chains of the lipids are disordered. Upon ordering of the paraffin chains, particles are excluded from the ordered domains and as a consequence, smooth areas and areas with densely packed particles are observed. The ratio of the smooth surface to particulated surface is proportional to the amount of ordered paraffin chains present. Moreover, the size of the smooth domains is a function of the fatty acid composition of the membranes. Discontinuities in the rate of D-lactate-dependent proline uptake as a function of temperature correlate with the order-disorder transitions observed. The high energies of activation at low temperatures are attributed to decreased mobility of the carrier proteins upon aggregation. In contrast, phosphoenolpyruvate-dependent vectorial phosphorylation does not respond to the ordering of the paraffin chains. [ABSTRACT FROM AUTHOR]

This paper describes the isolation and the properties of DNA-dependent RNA polymerase from Lactobacillus curvatus. The enzyme is highly labile and shows a tendency to dissociate into fragments. Therefore. a special purification procedure also suitable for the isolation of labile RNA polymerases from other pro-karyotes was developed. Three enzyme species, E (core enzyme). Eσ (full enzyme) and Ey were obtained. The subunit composition corresponds to that of other prokaryote RNA polymerases. In contrast to Echerichia coli enzyme. the Β subunit (Mr = 145000) has a lower molecular weight than the Β subunit (Mr = 1.51000). The Β subunit was identified by its capacity to bind [³H]rifamycin. σ is extremely small (Mr = 44000). y is a peptide chain (Mr = 84000) present once in the Ey monomer. By incomplete dissociation, Β α2, Β'σ and Β'y complexes were obtained. y and 5 have never been found together in the same enzyme particle. In contrast to RNA polymerase from E. coli. Eσ from L. curvatus exhibits optimal activity in the presence of Mn2+ as bivalent metal ion. Co2+ and Mg2+ also activate though with considerably lower efficiency. All subunits except y were isolated in pure state. σ was catalytically active. y could only be obtained as a complex with Β'. On single-stranded DNA. Eσ and E are equally active. For the transcription of double-stranded DNA. (σ is absolutely required. Even the transcription of poly[d(A-T)] · [d(A-T)] is strongly stimulated by σ. σ from L. curvatus is able to replace σ from E. coli on E. coli core enzyme even for the formation of the stable preinitiation complex. This effect requires Mg2+, that is conditions optimal for the E. coli system. Thus, the core and not g appears to determine the requirement for the bivalent metal ion. Different double-stranded templates are transcribed with highly different efficiency. The ionic strength optimum is different for different templates. Ey in contrast to E exhibits a low, though significant background activity on double-stranded DNA. It is stimulated by σ to twice the specific activity of Eσ. Thus, σ and y act synergistically though they appear to exclude each other on the core. [ABSTRACT FROM AUTHOR]

Exogenously supplied thymine is only poorly utilized by wild-type cells of Escherichia coli for the synthesis of their DNA. It appears that the lack of incorporation of exogenous thymine is due to a lack of endogenous deoxyribosyl groups, which are required for the synthesis of thymidine. Data to be presented in this paper indicate that the ability of different thymine auxotrophs to grow on progressively lower thymine concentrations is a function of their capacity to increase the internal pool of deoxyribose 1-phosphate and/or the level of thymidine phosphorylase. Thymine incorporation in wild-type cells could be observed if the cells possessed an increased deoxyribose 1-phosphate pool. The kinetics of thymine uptake in wild-type cells, in a mutant lacking thymidine phosphorylase and in thymine auxotrophs show that the initial rates of thymine uptake are almost identical and proportional to the external thymine concentration. The experiments in vivo led us to conclude that the incorporation of exogenous thymine occurs via thymidine, which is synthesized from thymine and deoxyribose 1-phosphate, catalyzed by thymidine phosphorylase. In accordance with this studies in vitro with purified thymidine phosphorylase showed that th3Tnidine is synthesized from deoxyribose 1-phosphate and thymine rather than through a deoxyribose transfer from a deoxynucleoside. [ABSTRACT FROM AUTHOR]

Exogenously supplied thymine is only poorly utilized by wild-type cells of Escherichia coli for the synthesis of their DNA. It appears that the lack of incorporation of exogenous thymine is due to a lack of endogenous deoxyribosyl groups, which are required for the synthesis of thymidine. Data to be presented in this paper indicate that the ability of different thymine auxotrophs to grow on progressively lower thymine concentrations is a function of their capacity to increase the internal pool of deoxyribose 1-phosphate and/or the level of thymidine phosphorylase. Thymine incorporation in wild-type cells could be observed if the cells possessed an increased deoxyribose 1-phosphate pool. The kinetics of thymine uptake in wild-type cells, in a mutant lacking thymidine phosphorylase and in thymine auxotrophs show that the initial rates of thymine uptake are almost identical and proportional to the external thymine concentration. The experiments in viva led us to conclude that the incorporation of exogenous thymine occurs via thymidine, which is synthesized from thymine and deoxyribose 1-phosphate, catalyzed by thymidine phosphorylase. In accordance with this studies in vitro with purified thymidine phosphorylase showed that thymidine is synthesized from deoxyribose 1-phosphate and thymine rather than through a deoxyribose transfer from a deoxynucleoside. [ABSTRACT FROM AUTHOR]

The two types of 30-S · 50-S couples described in the preceding paper, differ markedly in their sensitivity to hydrostatic pressure evoked during centrifugation in sucrose gradients. The sedimentation coefficient of about 60 S recorded with couples formed from native subunits is only apparent, the real a value being 70 S. The latter couples can be stabilized against pressure-induced dissociation by bound tRNA. On the other hand this binding renders couples formed from derived subunits more sensitive to this dissociation. As the factor determining this intriguing difference in pressure sensitivity lies in the 50-S subunit, a comparative analysis of a number of ribosomal constituents has been performed for native and derived 50-S particles. No qualitative differences in the proteins L1–L34 have been detected. Quantitative differences cannot yet be excluded. It is considered unlikely that differences in rRNAs or low-molecular-weight components arc responsible for the distinct association behaviour. Partial reconstitution of ribosomal γ-cores and split proteins detached by CsCl reveals that the determinant factor is located in the core particles. This suggests that native and derived 50-S subunits do not differ in conformation only. The possibility may be envisaged therefore, that the two types of 50-S differ in a ribosomal protein. [ABSTRACT FROM AUTHOR]

1. 1. Acapsular (K-) forms were isolated from Escherichia colt strain E56b (serotype O8 :K27(A): H-). A number of different R strains were obtained from the K- forms by selection of spontaneous mutants and by phage selection. 2. The lipopolysaccharides of these R strains (consisting of core and lipid A) were subjected to mild acid degradation and, after removal of insoluble lipid A, the mixtures were fractionated by gel chromatography. Results of chemical analyses showed that the core oligosaccharides of different R mutants differed with respect to their sugar constituents as well as to the molar ratios of these constituents. A group of R mutants did not contain phosphate in their core oligosaccharides (P- mutants). Enzyme determinations indicated that one R mutant had a block involving UDP glucose pyrophosphorylase, whereas the enzymes of activation and interconversion of component sugars were intact in the other R mutants. 4. By allelic exchange (mating with S-form Hfr strains)it could be demonstrated that with one exception the chromosomal site of the S→ R mutation of all mutants maps close to mtl (mannitol utilization). In Salmonella the rfa locus maps fun the same region. All R mutants had an unimpaired (his-linked) rfb locus. 5. The groups of mutants which differed in the sugar composition of their core oligosaccharides could also be differentiated serologically and by their sensitivity to phages. There was serological cross reactivity between the lipopolysaccharides of some R mutants with R mutants of Salmonella minnesota which also have a very incomplete core. It is concluded that the lipopolysaccharides of the R mutants described in this paper have incomplete core structures which result from blocks at successive stages in the synthesis of the coli R1 core. in analogy to Salmonella these E. coli R mutants shoed, with one exception, be termed rfa type mutants. [ABSTRACT FROM AUTHOR]

The experiments reported in this paper lead to the following conclusions: 1. An endonucleolytic activity is associated with the "replication site" in Escherichia coli cells; this endonucleolytic activity attacks the complementary strand of the ψx replicative form DNA only. 2. the viral strand of the ψx-replicative form DNA remains circularly closed during the replication cycle; 3. In the immediate replication product both composing strands are linear; in circularization no strand is preferred; 4. A replication product with both strands closed is transiently found in a structural state which can be, under our experimental conditions, only partially denatured by alkali. Subsequently it is transformed to a typical supercoiled RFI structure. [ABSTRACT FROM AUTHOR]

It is well know that protein synthesis stopped either by chloromycetin or by starvation of an essential amino acid for a RC relstrain, ribosomal RNA synthesis continues, but products do not appear in 50 S and 30 ribosomes. It has also been shown that 5 S RNA synthesis is altered by the addition of chloromycetin but in an unknown manner. In this paper we report that: (a) during amino-acid starvation of a starvation of RCrel strain of Escherichia coli, 5 S RNA is transcribed as 5 S relax RNA located outside the ribosomes, in the supernatant fraction; (b) this 5 S RNA which is slightly longer than the normal product is convertible into a true 5 S RNA when protein synthesis is restored after blockage of RNA synthesis by actinomycin ;(c) part of the 23 S RNA from a 25 S particle appears after resumption of protein synthesis in a 40 S parcel which contains normal 5S RNA; (d) a pool of 5 S precursor RNA exists outside the 50 S ribosome. This 5 S precursor RNA is isolated; (e) this precursor which is isolated from the 105000xg supernatant fraction after pulse labeling, migrates on gel electrophoresis between 5 S relax RNA and true 5 S RNA. [ABSTRACT FROM AUTHOR]

Multiple species of Escherichia coli transfer RNA for valine and leucine were separated from each other by countercurrent distribution. Each transfer RNA species was charged with the corresponding radioactive amino acid and then allowed to transfer its label into hemoglobin peptides in the ribosomal system from rabbit reticulocytes. Earlier experiments had shown that the minor tRNAIIbLeu would only label one position, no. 48 of the α-hemoglobin chain under these conditions. In this paper we report the coding properties of the other separated leucine tRNAs and of multiple valine tRNAs with the hemoglobin messenger. The leucine codons in the α-chain messenger can be divided into three classes with respect to their tRNA response : (a) position 48 is recognized only by tRNAIIbLeu. (b) The majority of the leucine codons can be recognized by both the two major leucine tRNAs. (c) Some leucine codons can only be recognized by one of the major leucine tRNAs. Similarly, two classes of valine codons can be distinguished in the α-chain messenger. [ABSTRACT FROM AUTHOR]

The behavior of N-acetylphenylalanyl-tRNA as an initiator of phenylalanine polymerisation in an Escherichia coli cell-free system shows some analogy with the behavior of formylmethionyl- tRNA during the synthesis of natural proteins. At low Mg++ concentrations, polyphenylalanine chain initiation depends on N-acetylphenylalanyl-tRNA and the same initiation factors which are required for protein chain initiation with formylmethionyl-tRNA. However in a rabbit reticulocyte cell-free system, N-acetylphenylalanine is not incorporated into polyphénylalanine at any Mg++ concentration. In this paper, we showed that : a) N-acetylphenylalanyl-tRNA did bind to reticulocyte ribosomes in the presence of poly U. This binding was GTP dependent and needed a factor which was extracted by washing ribosomes with salt (NH4CI 1 M); b) the bound N-acetylphenylalanyl-tRNA was partially released by puromycin or by addition of phenylalanyl-tRNA. In this last case, incorporation of N-acetylphenylalanine into polyphenyl- alanine was noticed (ratio N-acetylphenylalanine/phenylalanine = 1/20); c) the ribosomal wash fluid also greatly enhanced phenylalanyl-tRNA binding to salt- treated ribosomes, in the presence of poly U and GTP. The maximum binding was obtained with the same concentrations of Mg++ and K+ as for N-acetylphenylalanyl-tRNA binding. It was not possible to separate on a DEAE-cellulose column the factors that were required for phenyl- alanyl-tRNA and acetylphenylalanyl-tRNA binding. On the other hand, a preparation of the binding enzyme from reticulocyte supernatant enhanced both phenylalanyl-tRNA and acetyl- phenylalanyl-tRNA binding to ribosomes; d) the optimum Mg+÷ concentration for phenylalanine polymerisation was higher with salt- washed ribosomes than with sucrose-washed ribosomes. Adding the wash fluid shifted the Mg++ optimum back to the lower value. N-acetylphenylalanyl-tRNA was not required for this shift as noticed in Escherichia coli. One likely explanation of our results with reticulocytes is the possibility that the ‘binding enzyme’ of Schweet et al., which was present in our ribosome wash fluid, could work with both N-acetyl as well as with phenylalanyl-tRNA. Thus, the inability to incorporate N-acetylphenyl- alanine might be due to a competition between these two compounds for the same enzyme and the same ribosomal site. If this is indeed the case, it is in marked contrast to results obtained with Escherichia coti and could reflect a basic difference in the mechanism of protein chain initiation. [ABSTRACT FROM AUTHOR]

Alkaline phosphatase (Escherichia coli) was found to catalyze the synthesis of monophosphate esters of serine, ethanolamine, propanolamine, butanol, glycerol, L-glucose, and tris(hydroxymethyl)aminomethane by phosphoryl transfer from cysteamine S-phosphate (donor) to the respective alcohols (acceptors). Maximum enzymic synthesis of the above esters occurred at pH 7.8. The newly-formed phosphate esters were measured quantitatively after separation of the products by the aid of paper high voltage electrophoresis techniques or by an amino acid analyzer. The percentage of the enzyme-catalyzed synthesis of the new phosphate esters varied from 15-39 0/0 using different acceptors under otherwise identical experimental conditions. It is pertinent to note that the same compounds were phosphorylated to essentially the same extent when the donor (substrate) compound was serine O-phosphate, aminoethanol O-phosphate or p-nitrophenyl O-phosphate. The rates of enzymic consumption of cysteamine S-phosphate (vh + va) were measured at different pH values and at different concentrations of acceptor (Tris or aminoethanol) in the presence of 1.5 M NaCl using 3 mM substrate (cysteamine S-phosphate). The presence of either Tris or aminoethanol gave the same pH profile for the rate of consumption (vh + va) of cysteamine S-phosphate with a maximum value around pH 7.8. This value differs from that found for the same reaction in barbital buffer (pH 9.0). The rate of enzymic consumption of cysteamine S-phosphate was also measured at various substrate and Tris concentrations at pH 7.8 under constant ionic strength (Γ/2 = 1.0). The same Km value (0.24 mM) was obtained at Tris concentrations varying from 0.02 M to 0.5 M. The Vmax values derived from these experiments were linearly related to the Tris concentrations up to 0.5 M Tris. The ph profile of the rate of consumption of p-nitrophenyl phosphate as well as the Km value (0.26 mM) were similar to the corresponding values obtained for cysteamine S-phosphate. The rate of hydrolysis (vh) could not be measured when cysteamine S-phosphate served as the donor compound due to the instability of cysteamine S-phosphate under the conditions required for inorganic phosphate assay. Therefore, the rate of transfer (va = vRSH - vP1) could not be estimated. The pH dependence of the rate of transfer, va, was calculated from experimental data obtained when p-nitrophenyl phosphate served as the donor compound and Tris served as the acceptor. The pH profile for the rate of transfer, va, obtained from these measurements was very similar to that obtained by direct measurement of Tris O-phosphate formation using cysteamine as the donor. It is suggested that phosphoryl transfer occurs through a phosphorylated enzyme intermediate, and therefore, the types of compounds that could serve as acceptors are independent of the donor compounds and depend only on the bond energy of the particular phosphorylated enzyme intermediate. [ABSTRACT FROM AUTHOR]

It is known from previous work that the regulation of Glutaminesynthetase in coli is effected by two mechanisms. First, the synthesis of GS is repressed by NH4+; and secondly, the activity of GS is regulated by inactivation and reactivation of the enzyme molecule. In the present paper we demonstrate and characterize the reactivation of inactivated GS in intact cells of E. coli. GS is inactivated after addition of NH4+ to the culture medium. Experiments with a cell flee extract from E. coli have shown, that the inactivation is mediated by a specific "inactivating enzyme", ATP, Mg2+, and Glutamine which is probably formed from the added NH4+, The reactivation is observed, when the inactivated ceils are centrifuged from the NH4+ containing medium and then resuspended in an NH4+ free medium. In about 5–7 minutes the activity increases 5–30-fold. Inhibitors of protein synthesis such as chloramphenicol and p-fluoro-phenylalanine do not affect the reactivation process. Only a slight retardation of the reactivation is observed. A carbon source is required for reactivation. When inactivated cells are incubated in a medium containing only salts and buffer no reactivation is observed. Probably the carbon sources is metabolized to form intermediates and energy, which are needed for glutamine catabolizing reactions or for prevention of glutamine synthesis. Reactivation does not take place m the presence of NH4+ i.e. under inactivating conditions. However if inactivation is inhabited by fluoride, reactivation can be observed in the presence of NH4+. When inactivated cells are incubated with fluoride only, without a carbon source in the reactivation medium, a reactivation is found. Inactivation as well as reactivation arc inhibited by thiol-group reagents. From these results we conclude that: (a) Reactivation is independent of protein synthesis; (b) Reactivation does not require energy; (c) Probably thiol-groups are involuted in the reactivation process. [ABSTRACT FROM AUTHOR]

We have studied the ribonucleosidediphosphate reductase of a deoxyuridine-requiring mutant LD195 of Escherichia coli K. This enzyme was purified 50 fold from the mutant and a control strain. In all steps of the purification the mutant strain yielded normal activity of the Bi subunit of the enzyme but approximately 10 % of the wild-type activity of the B2 subunit The activity is similarly reduced for all three substrates tested CDP, UDP, and GDP. The mutation appears to affect the physical structure of the B2 subunit, since addition of high concentrations of sodium acetate can restore enzymatic activity to the partially purified B2 subunit from the mutant strain. In contrast, the same concentrations strongly inhibit the wild- type B2 subunit. Although the mutant has a requirement of deoxyuridine at 42 °C but not at 30 °C (as shown in an accompanying paper), mutant protein B2 is not more thermo-labile than wild-type protein B2 This requirement therefore probably reflects an increased demand of deoxyribonucleotides at higher temperatures. Hydroxyurea inhibits mutant and wild-type ribonucleosidediphosphate reductase to the same extent in vitro. In spite of this, hydroxyurea inhibits growth of the mutant strain at a 20 fold lower concentration than that required for comparable inhibition of growth of the parent. This is explained by the greatly decreased protein B2 activity of the mutant. [ABSTRACT FROM AUTHOR]

A procedure to isolate Escherichia coli mutants defective in ribonucleotide reduction is described. Using a parental strain defective in dCTP deaminase (paxA) and thus unable to synthesize dUMP from deoxycytidine nucleotides, we selected for cells unable to reduce UDP at a rate sufficient to satisfy the cell's need for thymidine nucleotides. This was accomplished by selecting for cells resistant to 2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine (trimethoprim) in the presence of thymidine and screening these cells for a thymidine requirement that could be satisfied by deoxyuridine. Genetic analysis as well as enzyme assays of four such mutants chosen for study indicated that three had a defective thymidylate synthetase. One, LD195, which had normal thymidylate synthetase activity, was found to have a conditional deoxyuridine requirement that is only expressed under favourable growth conditions. Since restoration of a functional dCTP deaminase to derivatives of LD195 eliminated the deoxyuridine requirement, it was concluded that a combination of two mutations is responsible for the expression of this phenotype. Analysis of nucleotide pools of LD195 grown exponentially under permissive conditions revealed that both the dTMP and dTTP pools were 6-fold lower than in the parent indicating a deficiency in the production of a thymidine nucleotide precursor. Enzyme assays of crude extracts of LD195 indicated that this deficiency was due to a defective ribonucleosidediphosphate reductase. This conclusion is proven by an extensive enzymatic analysis presented in an accompanying paper. [ABSTRACT FROM AUTHOR]

Initiation of protein synthesis in Escherichia coli is a catalytic process, provided that conditions for hydrolysis of GTP are present. This means that the initiation factors IF-1, IF-2 and IF-3 are released at a certain stage of initiation complex formation, whereafter they recycle and are reutilized in another round of initiation. Experiments presented in this paper provide evidence for the mechanism of recycling, in which the hydrolysis of GTP and IF-1 both play a predominant role. We demonstrate that binding of fMet-tRNA, which is dependent on IF-2, is a stoichiometric process in a system devoid of IF-1. Addition of IF-1 converts it into a catalytic process, however, only if GTP can be hydrolysed. No such conversion takes place with 5′-guanylylmethylenediphosphonate. The same observation is valid for the uncoupled IF-2 dependent GTPase activity. In the process of initiation IF-1 acts as a recycling promoting factor, which displays analogous properties to elongation factor EF-Ts. We postulate that a ribosomal-bound IF-2 · GDP complex is converted into an IF-2 · GTP complex, which is released and is capable of starting a new cycle of initiation. Furthermore, kinetic evidence is presented that IF-3 recycles even at the level of 30-S initiation complex formation, irrespective of whether conditions for hydrolysis of GTP are provided. [ABSTRACT FROM AUTHOR]

After the recent investigation on the biochemical properties of the 2-keto-3-deoxy-gluconokinase and 2-keto-3-deoxy-6-phospho-gluconate aldolase in Escherichia coli K12, we report in this paper the induction mechanism of these two enzymes and the genetic control of a new enzymatic activity: the 2-keto-3-deoxy-gluconate transport system. The D-glucuronate and D-galacturonate raise the high basal levels of the kinase and aldolase on glycerol by a factor of 5 and 3-4, respectively, the kinase and aldolase being enzymes of the common degradative pathway of D-glucuronate and D-galacturonate. The differential rates of synthesis of these two enzymes are constant from the addition of the galacturonate. Under a variety of conditions (different inducers and substrates of growth) kinase and aldolase have been found to be synthesized non coordinately. The strongest decoordination is carried out by gluconate: this compound which is a good inducer of the aldolase, not only is unable to induce the kinase but also represses it. Moreover, the aldolase seems to be less sensitive to the catabolic repression than the kinase. By means of appropriate negative mutants we have established that the two hexuronates and two keto-uronates do not induce directly kinase and aldolase, but by sequential conversion into 2-keto-3-deoxy-gluconate. Furthermore, the fact that the hexuronates induce the kinase in aldolase-negative mutants (kdg A) and besides, "over" induce the aldolase in kinase negative mutants (kdg K) strongly suggests that, 2-keto-3-deoxy-gluconate is a true inducer for both enzymes. In addition, the gluconate which still induces the aldolase in an edd strain suggests that, this compound and/or the 6-phospho-gluconate are/is also inducer(s) of this enzyme. Moreover, we have isolated spontaneous mutants able to utilize the 2-keto-3-deoxy-gluconate as a unique carbon source. Two classes have been identified. The mutants of these classes which arise at high frequency (10-5 to 10-6) are either simultaneously constitutive for a specific 2-keto3-deoxy-gluconate transport system, kinase and aldolase (class kdg Rc) or only constitutive for the transport system (class kdg Pc). The 2-leto-3-deoxy-gluconate as exogenous substrate, which cannot induce its transport system in the wild type, behaves like a non-inducer substrate. So the strains kdg Rc and kdg Pc are respectively i- and oc lac-like mutants. These findings and the fact that we have found thermosensitive mutants mapping in the kdg R locus (34.5 min) and which are simultaneously derepressed for the permease, kinase and aldolase at 42 °C but not at 28 °C, strongly suggest that the synthesis of the three sequential enzymes degrading the 2-keto-3-deoxy-gluconate is negatively controlled by a common regulator gene product. The decoordinated syntheses of these three enzymes are in agreement with the scattering of the corresponding operons on the chromosome: transport system operon (76.5 min), kinase operon (69 min), aldolase operon (34 min). [ABSTRACT FROM AUTHOR]

D-Mannonate : NAD oxidoreductase of Escherichia coli K12 has been purified. The purification consists of four steps: precipitation of nucleic acids with protamine sulfate; ammonium sulfate precipitation; ion-exchange chromatography on DEAE-cellulose and Sephadex G-200 gel filtration. The overall procedure results in a 53-fold purification with a yield of 70%. Mannonate oxidoreductase is an inducible enzyme: glucuronate and fructuronate are good inducers, galacturonate is not. Optimal activity is obtained at pH 6.3 fro fructuronate reduction, and at pH 8.4 for mannonate oxidation. The apparent Michaelis constants for the purified enzyme have been determined: for fructuronate, the Km is 0.5 mM; for NADH 0.03 mM; for mannonate, 1.8 mM and for NAD+, 0.2 mM. Mannonate oxidoreductase and altronate oxidoreductase have been separated on DEAE-Sephadex in order to study mannonate-oxidoreductase specificity. After separation, mannonate oxidoreductase is found to be active with both fructuronate and tagaturonate as substrates. Kinetic studies, differential inhibition of activities by p-chloromercuribenzoate and thermal inactivation studies confirm this double specificity. Other carbohydrates tested are unsuitable as substrates. Mannonate oxidoreductase is active with NADPH as coenzyme and the apparent Km value for NADPH is 0.7 mM. Kinetic inhibition studies of the enzyme by direct and reverse reactional products suggest a random mechanism for mannonate oxidoreductase. Enzyme inhibition specificity appears to be restricted. The value of the energy of activation is 34 kJ/mol (8 kcal/mol); the Q10 is 1.5. The results of thermal inactivation of mannonate oxidoreductase (53 °C) indicate homogeneous-enzyme activity fructuronate in the crude extract. In the absence of inducer, basal activity is dependent on mannonate oxidoreductase only. Thermal inactivations also indicate that the same enzyme, mannonate oxidoreductase, catalyzes the direct reactions with NADH or NADPH, and the reverse reaction. Enzyme substrates, such as NADH, NADPH, fructuronate, tagaturonate, NAD+, mannonate and altronate protect enzymatic activity from degradation by heat. NADH in particular, is effective as a stabilizer; this ability is proportional to its concentration. p-Chloromercuribenzoate is an active inhibitor: at 5 μM, activity is 100% inhibited. Inhibition is non-competitive, with an apparent Ki of 0.1 μM. The end product of the reaction of mannonate oxidoreductase with fructuronate has been identified by means of paper chromatography as D-mannonate. [ABSTRACT FROM AUTHOR]

D-Altronate: NAD-oxidoreductase of Escherichia coli K12 has been purified. The purification consists of four steps: nucleic acids precipitation with protamine sulfate; ammonium sulfate precipitation; ion exchange chromatography on DEAE-cellulose and Sephadex G-150 gel filtration. The overall procedure results in a 53-fold purification with a yield of 60%. Altronate oxidoreductase is an inducible enzyme: galacturonate, tagaturonate, glucuronate and fructuronate are good inducers. The activity remains stable at 4 °C or at — 20 °C for several months. Optimal activity is obtained at pH 6.3 for tagaturonate reduction, and at pH 8.9 for altronate oxidation. Altronate oxidoreductase does not require any cofactor, except NADH. The apparent Michaelis constants have been determined: for tagaturonate, the Km is 350 μM; for NADH, 60 μM; for altronate, 90 μM, and for NAD+, 110 μM. Moreover, altronate oxidoreductase is active with NADPH as coenzyme and the apparent Km value for NADPH is 400 μM. Other carbohydrates tested are unsuitable as substrates. Many other carbohydrates tested do not prove to be inhibitors. The value for the energy of activation is 38 kJ/mol (9 kcal/mol); the Q10 is 1.5. The results of heat inactivation of altronate oxidoreductase (58.5 °C) indicate homogeneous enzyme activity in crude extract. Thermal inactivations also indicate that the same enzyme, altronate oxidoreductase, is induced by galacturonate and glucuronate, and catalyzes the direct reactions with NADH or NADPH. Finally, it has been confirmed by heat inactivation that there is no interference between specific assays of altronate and mannonate oxidoreduetases. Compounds such as NADH, NADPH, altronate and mannonate protect enzymatic activity from degradation by heat, whereas NAD+, tagaturonate and fructuronate do not. NADH, in particular, is effective as a stabilizer; this ability is proportional to its concentration. Enzyme inhibition with para-chloromercuribenzoate is non-competitive and the apparent Ki is 1 mM. para-Chloromercuribenzoate also inactivates altronate oxidoreductase indicating homogeneous activity and specificity of the assay method in crude extracts. Chromatographic properties of the enzyme are identical whether the inducer is galacturonate or glucuronate. Using paper chromatography, the reaction product of enzyme action on D-tagaturonate is identified as D-altronate. [ABSTRACT FROM AUTHOR]

In this paper we report on the properties of thc purified isoleucyl-tRNA synthetase from Bacillus stearothermophilus. The enzyme which was purified approximately 500-fold, has a molecular weight of about 110 000. The effect of temperature on the activation and transfer activities has been studied. The thermostable enzyme shows an optimal activity at 65 °C for the isoleucine-dependent ATP-PPi exchange reaction and at 45 °C for the formation of isoleucyl-tRNA. Attempts to heat inactivate the amino-acid-transfer activity selectively, i.e. without loss of the activation reaction, were unsuccessful. The protection of the enzyme against heat inactivation in presence of tRNA makes it possible to study the binding of this molecule to the protein. An association constant of 0.11 nM-1 was determined at 70.5 °C. Formation of this tRNA · enzyme complex was also demonstrated by binding onto nitrocellulose filters. By this technique one tRNA site per enzyme molecule was detected and an affinity constant of 0.4 nM-1 measured. The same results were obtained either with tRNA from Escherichia coli or B. stearothermophilus, showing close resemblance of the two nucleic acids. Periodate-oxidation of the 3'-terminal adenosine of the tRNA does not perceptibly affect the binding or the degree of protection of the enzyme against heat inactivation. These effects are still strictly dependent on magnesium ions and a clearcut requirement of these ions for the formation and/or stabilization of the tRNA · enzyme complex has been demonstrated. The intermediate isoleucine · AMP · enzyme complex with a molar ratio of 1:1:1 was isolated by molecular sieving and by chromatography on DEAE-cellulose. Mg2+ ions are required for the formation of the complex but are not necessary to stabilize the isoleucyl-adenylate once this is bound to the enzyme. The transfer of activated isoleucine to tRNA again requires magnesium ions and must probably be related to the formation of a tRNA enzyme complex. Part only of the activated isoleucine is transferred to tRNA; this results essentially from the breakdown of a fraction of the isoleucine · AMP · enzyme complex, induced by tRNA and Mg2+ ions, acting together. Periodate-oxidized tRNA of B. stearothermophilus or of E. coli in presence of Mg2+ ions completely releases isoleucine from the complex. [ABSTRACT FROM AUTHOR]

The 09 antigen from Escherichia coli 09:K30 (A):H12 contains mannose (90%) and glucose (10%). Incorporation of [14C]mannose from guanosine diphosphate [14C]mannose (GDP-[14C]Man) into acetic-acid precipitates has been studied with a preparation from cells ground with alumina. The time-course of mannose incorporation was biphasic (rapid reaction for 2-5m in followed by a slower, linear reaction for 5-20 rain) and the incorporation was stimulated by uridine diphosphate glucose (UDP-Glc) during the early part of the reaction. The reactions (mannose incorporation after 2 rain and 15 min, ± UDP-Glc) were nevertheless proportional to protein concentration. [14C]Glucose was incorporated into acetic-acid precipitates from UDP[14C]Glc and this reaction was slightly stimulated by GDP-Man. Radioactivity from GDP[14C]Man was incorporated into butanol extracta and the early part of the reaction (2-5 min) was markedly stimulated by UDP-Glc. Bacitracin did not inhibit the incorporation of mannose into acetic-acid precipitates in the early part of the reaction, but did inhibit incorporation about 20% between 5 and 30 min. Larger-scale incubation mixtures were extracted with 45% aqueous phenol. The product was of high molecular weight as indicated by Sephadex G-100 chromatography and yielded a single radioactive peak corresponding to mannose after total hydrolysis and paper chromatography. This high-molecular-weight product precipitated with anti-09 antiserum in an antigen-binding assay, indicating that it was serologically active. Thus, it is likely that the 09 antigen of E. coli 09:K30(A):H12 is synthesized from GDP-Man and UDP-Glc through a butanol-extractable (lipid-linked) intermediate. Reactions appear to be analogous to those described for lipopolysaccharide biosynthesis in Salmonella. [ABSTRACT FROM AUTHOR]

The binding of purified Gly3-Phe-tRNA to Escherichia coli ribosomes was compared to that of crude Gly3-Phe-tRNA (containing bulk tRNA). Gly3-phe-tRNA from the purified preparation binds preferentially to the P site (peptidyl site) at both a high (30 mM) and a low (10 mM) magnesium acetate concentration, while peptidyl-tRNA from the crude preparation bound more to A site (aminoacyl site) than to the P site at 30 mM magnesium acetate under the same experimental conditions. The addition of tRNAPhe to the purified Gly3-Phe-tRNA preparation strongly inhibited the binding at 10 mM. At 30 mM Mg2+ the addition of the same amount of tRNAPhe caused the Gly3-Phe-tRNA to bind partially to the A site, but did not inhibit the total amount of Gly3-Phe-tRNA which binds to the ribosomes. The binding of purified Phe-tRNA in the presence of T factor and GTP is not inhibited by tetracycline. However, tetracycline strongly inhibited the enzymatic binding of Phe-tRNA when tRNAPhe was added. From the results presented in this paper it is proposed that in the presence of poly(U), Phe-tRNA or peptidyl-tRNAPhe can bind to A sites only of those ribosomes which have their P site occupied by tRNA or one of its derivatives (aminoacyl-tRNA or peptidyl-tRNA). [ABSTRACT FROM AUTHOR]

We have previously reported that the catalytic RNA subunit of RNase P of Escherichia coli (M1 RNA) cleaves Drosophila initiator methionine tRNA (tRNA[sup Met][sub i]) within the mature tRNA sequence to produce specific fragments. This cleavage was dependent on the occurrence of an altered conformation of the tRNA substrate. We call this further cleavage hyperprocessing. In the present paper, to search for another tRNA that can be hyperprocessed in vitro, we used total mature tRNAs from Drosophila as substrates for the in vitro M1 RNA reaction. We found that some tRNAs can be hyperprocessed by M1 RNA and that two such tRNAs are an alanine tRNA and a histidine tRNA. Using mutant substrates of these tRNAs, we also show that the hyperprocessing by M1 RNA is dependent on the occurrence of altered conformations of these tRNAs. The altered conformations were very similar to that of tRNA[sup Met][sub i]. We show here that M1 RNA can be used as a powerful tool to detect the alternative conformation of tRNAs. The relationship between these hyperprocessing reactions and stability of the tRNA structure will also be discussed. [ABSTRACT FROM AUTHOR]