Search

An eicosanucleotide C-G-C-G-G-G-G-U-G-G-A-G-C-A-G-C-C-U-G-Gp corresponding to the bases 1–20 of the nascent sequence for the Escherichia coli tRNAfMet has been synthesized by the joining of the chemically synthesized oligonucleotides C-G-C-G, G-G-G-U-G-G and A-G-C-A-G-C-C-U-G-Gp using RNA ligase from T4-infected E. coli. The hexanucleotide and decanucleotide were phosphorylated with polynucleotide kinase and [γ-32p] ATP prior to the joining reactions. The decanucleotide and eicosanucleotide were reconstituted respectively with the 3′-three-quarter molecule obtained by limited digestion with RNase T1 of the natural tRNAfMet from E. coli and the activity of the complex as a methionine acceptor was tested using purified methionyl-tRNA synthetase from E. coli. The amino acid acceptor activity of the reconstituted molecules was 11% and 84% with respect to that of the intact tRNAfMet. [ABSTRACT FROM AUTHOR]

Starting from 2-thiodeoxythymidine, the mono- and triphosphate of the nucleoside analog were prepared by enzymatic phosphorylation with thymidine kinase and thymidylate kinase, respectively. 2-Thiodeoxythymidine triphosphate was also obtained by chemical pyrophosphorylation of the monophosphate. The analog nucleoside-triphosphate is a substra@ for Escherichia coli DNA polymerase. A copolymer consisting of alternating deoxyadenosine- and 2-thiodeoxythyunidine nucleotides is synthesized extensively by the enzyme. The polymer was characterized by nearest neighbour analysis, sedimentation and buoyant density in the analytical ultracentrifuge, ultraviolet spectra and stability against thermal and alkaline denaturation. [ABSTRACT FROM AUTHOR]

The almost complete sequence of a region of the 23-S ribosomal RNA from Escherichia coli, 175 nucleotides in length, located between the 550th and the 1000th nucleotide at the 3′ end of 23-S RNA and containing the binding site of protein L1, is given. Possible models of secondary structure are proposed. [ABSTRACT FROM AUTHOR]

Ribonucleoproteins were obtained by T1 ribonuclease digestion of reconstituted complexes of ribosomal protein L1 and 23-S RNA from Escherichia coli. The RNA region of the main ribonucleoprotein 2 was totally digested with Tl ribonuclease. The oligonucleotide products were characterised and they showed that this region comprises 148 nucleotides located between the 550th and 1000th nucleotides from the 3′ end of the 23-S RNA. Of the other two ribonucleoproteins, the largest ribonucleoprotein 1 contained an extra RNA sequence, of at least 15 nucleotides, that was located at the 5′ end of the RNA region. The smallest ribonucleoprotein 3 lacked an RNA section towards the 3′ end of the region. The order of the RNA subfragments and the enzymic cutting positions in the whole RNA region are given for the ribonucleoproteins. It is shown that protein L1 most strongly protects a continuous section of 115 nucleotides at the 5′ end of the main RNA region. Finally, evidence is presented for a methylated base, and for two sequence heterogeneities, in this region of the 23-S RNA. [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]

Signal transduction ATPases with numerous domains ( STAND) are widespread proteins, whose activation involves inducer-dependent conversion of resting ADP-bound monomers into active ATP-bound multimers. This process notably comprises opening of the nucleotide-binding oligomerization domain ( NOD), nucleotide exchange and NOD-mediated multimerization. How inducer binding to the sensor domain, whose structure is not conserved throughout the STAND family, causes protein activation remains unclear. We used MalT, an Escherichia coli transcription factor, as a STAND model system, to address this question by dissecting the signalling pathway in vitro. We have found that inducer binding to the sensor is the first step of the activation pathway. It both triggers opening of the NOD and makes the MalT multimer competent for binding promoter MalT sites via its DNA-binding domains. Based on available data, we proposed that inducer trigger of NOD opening is a conserved STAND feature, irrespective of the sensor structure. As discussed, an additional role for the inducer, as found for MalT, might pertain to other types of STANDs. [ABSTRACT FROM AUTHOR]

Control of DNA replication initiation is essential for cell growth. A unifying characteristic of DNA replication initiator proteins is their distinctive AAA+ nucleotide-binding domains. The bacterial initiator DnaA assembles into a right-handed helical oligomer built upon interactions between neighbouring AAA+ domains to form an active initiation complex. Recently we developed a unique cross-linking assay that specifically detects ATP-dependent DnaA helix assembly. Here we have utilized this assay to show that two DnaA regulatory proteins in Bacillus subtilis, YabA and DnaD, inhibit DnaA helix formation. These results, in combination with our previous finding that the regulatory factor Soj/ ParA also targets DnaA filament formation, highlight the critical importance of regulating DnaA helix formation during the initiation reaction. Moreover, these observations lead us to suggest that DnaA oligomerization may be the main regulatory step of the initiator assembly pathway in B. subtilis, in contrast to the prevailing model of bacterial DNA replication based on Escherichia coli DnaA where ATP binding appears to be the targeted activity. [ABSTRACT FROM AUTHOR]

Starvation for DNA precursor dTTP, known as ‘thymineless death’ (TLD), kills bacterial and eukaryotic cells alike. Despite numerous investigations, toxic mechanisms behind TLD remain unknown, although wrong nucleotide incorporation with subsequent excision dominates the explanations. We show that kinetics of TLD in Escherichia coli is not affected by mutations in DNA repair, ruling out excision after massive misincorporation as the cause of TLD. We found that the rate of DNA synthesis in thymine-starved cells decreases exponentially, indicating replication fork stalling. Processing of stalled replication forks by recombinational repair is known to fragment the chromosome, and we detect significant chromosomal fragmentation during TLD. Moreover, we report that, out of major recombinational repair functions, only inactivation of recF and recO relieves TLD, identifying the poisoning mechanism. Inactivation of recJ and rep has slight effect, while the recA, recBC, ruvABC, recG and uvrD mutations all accelerate TLD, identifying the protection mechanisms. Our epistatic analysis argues for two distinct pathways protecting against TLD: RecABCD/Ruv repairs the double-strand breaks, whereas UvrD counteracts RecAFO-catalyzed toxic single-strand gap processing. [ABSTRACT FROM AUTHOR]

Thiamine and its three phosphorylated derivatives (mono-, di- and triphosphate) occur naturally in most cells. Recently, we reported the presence of a fourth thiamine derivative, adenosine thiamine triphosphate, produced in Escherichia coli in response to carbon starvation. Here, we show that the chemical synthesis of adenosine thiamine triphosphate leads to another new compound, adenosine thiamine diphosphate, as a side product. The structure of both compounds was confirmed by MS analysis and 1H-, 13C- and 31P-NMR, and some of their chemical properties were determined. Our results show an upfield shifting of the C-2 proton of the thiazolium ring in adenosine thiamine derivatives compared with conventional thiamine phosphate derivatives. This modification of the electronic environment of the C-2 proton might be explained by a through-space interaction with the adenosine moiety, suggesting U-shaped folding of adenosine thiamine derivatives. Such a structure in which the C-2 proton is embedded in a closed conformation can be located using molecular modeling as an energy minimum. In E. coli, adenosine thiamine triphosphate may account for 15% of the total thiamine under energy stress. It is less abundant in eukaryotic organisms, but is consistently found in mammalian tissues and some cell lines. Using HPLC, we show for the first time that adenosine thiamine diphosphate may also occur in small amounts in E. coli and in vertebrate liver. The discovery of two natural thiamine adenine compounds further highlights the complexity and diversity of thiamine biochemistry, which is not restricted to the cofactor role of thiamine diphosphate. [ABSTRACT FROM AUTHOR]

The filamentous fungus Trichoderma reesei forms two specific, xylan-inducible xylanases encoded by xyn1 and xyn2 to degrade the β-1,4-D-xylan backbone of hemicelluloses. This enzyme system is formed in the presence of xylan, but not glucose. The molecular basis of the absence of xylanase I formation on glucose was the purpose of this study. Northern blotting of the xyn1 transcript as well as the use of the Escherichia coli hygromycin B phosphotransferase-encoding gene (hph) as a reporter consistently showed that the basal expression of xyn1 was affected by glucose, whereas its induction by xylan remained uninfluenced. The repression of basal xyn1 transcription is mediated by the carbon catabolite repressor protein Cre1, which in vivo binds to two of four consensus sites (5'-SYGGRG-3) in the xyn1 promoter, which occurred in the form of an inverted repeat. T. reesei strains, bearing a xyn1::hph reporter construct, in which four nucleotides from the middle of the inverted repeat had been removed, expressed hph on glucose at a level comparable to that observed during growth on a carbon catabolite derepressing carbon source. Northern analysis of xyn1 expression in a T. reesei mutant strain (RUT C-30), which contains a truncated, non-functional cre1 gene, also confirmed basal transcription of xyn1. In this strain, xyn1 transcription was still inducible by xylose or xylan to an even higher degree than in the wild-type strain, suggesting that induction overcomes glucose repression at the level of xyn1 expression. Based on these data, we postulate that basal transcription of xyn1 is repressed by glucose and mediated by an inverted repeat of the consensus motif for Cre1-mediated carbon catabolite repression. [ABSTRACT FROM AUTHOR]

Biosynthesis of the Escherichia coli CS31A surface antigen was subject to phase-variation control and repressed by L-alanine. Nucleotide sequence analysis of the clp operon, encoding the biosynthesis of CS31A, reveaied the presence of a regulatory gene, clpB. The amino acid sequence of the regulatory protein ClpB showed similarity to the primary structure of PapB, FaeB and AfaA, involved in the regulation of expression of Pap, K88, and Afa-3 fimbriae, respectively. The clp regulatory region contained two deoxyadenosine methylase sites (GATC-I and GATC-II). The leucine-responsive regulatory protein (Lrp) was required for specific methylation inhibition of the GATC-II site. The activity of the clp promoter was monitored in a clp--lacZYA single-copy fusion. The cloned DNA used in this study did not contain a related papi gene, in these conditions, we showed, as expected, that phase variation did not occur. However, transcription of the clp operon was negatively controlled by ClpB and Lrp, and was maximal in the absence of Dam methylase. in the presence of AfaF, a Papl equivalent, the phase-variation control was restored. We concluded that two regulatory mechanisms were superimposed to control the clp expression. Phase variation, mediated by Lrp and a Papl equivalent, controlled the number of cells producing CS31A in a single colony. The second mechanism, described in this report, was mediated by ClpB and Lrp and controls the level of CS31A produced by a single cell. Furthermore, we showed that L-aianine reduced, by about twofoid, the clp promoter activity independently of a Papl equivalent, ClpB, Lrp or Dam methylase. in addition, the presence of L-aianine prevented the phase-variation control mediated by AfaF. [ABSTRACT FROM AUTHOR]

Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to the translocation of protons across a membrane. The NAD(H)-binding domain of transhydrogenase (domain I protein) from Rhodospirillum rubrum and from Escherchia coli were overexpressed and purified. Nucleoude binding to the domain I proteins was determined by equilibrium dialysis. NADH and its analogue, acetylpyridine adenine dinucleotide (reduced form), bound with relatively high affinity (Kd = 32 μM and 120 μM). respectively, for the R. rubrum protein). The binding affinity was similar at pH 8.0 and pH 9.0 in zwitterionic buffers and at pH 7.5 in sodium phosphate buffer, NAD+hound with lower affinity (Kd = 300 μM). NADPH bound only very weakly (Kd1> 1 mM). Using a centrifugation procedure, Yamaguchi and Hatefi [Yamaguchi, M & Hatefi, Y. (1993) J. Biol. Chem, 268, 1781-17877 ] found that mitochondrial transhydrogenase, and a proteolytically derrived domain I fragment from that exnzyme, bound one NADH per dimer. They suggested that this result implied half-of-the-sites reactivity for the interaction between the nucleotide ligand and the protein. However our studies on both the E. coli and the R. rubrum recombinant transhydrogenase domain I protein using equilibrum dialysis show that the binding stoichiometry for both NADH and the reduced form of acetylpyridine adenine dinucleotide (AcPdADH) is two nucleotides per dimer: no interaction between the monochondrial transhydrogenases are discussed. [ABSTRACT FROM AUTHOR]

The tRNAPhe nucleotides required for recognized by phenylalanyl-tRNA synthetase of Thermus Thermophilus have been determined using Escherichia coli tRNAPhe transcripts with various mutations. The anticodon nucleotides are shown to be the most important recognition elements. The discriminator necleotide, A73, involved in the recognition set of yeast, E. coli and human phenylalanyl-tRNA synthetases contributes only slightly to tRNAPhe recognition by Th. thermophilus phenylalanyl-tRNA synthetase. Nucleotide 20 and some tertiary nucleotides, including the conserved G19 · C56 base pair, are proposed to participate in stabilization of the precise tRNA conformation required for efficient aminoacylation. The 3′-CCA terminus, common to all tRNAs, in the specific interaction of tRNA with phenylalanyl-tRNA synthetase is discussed. [ABSTRACT FROM AUTHOR]

Examines the biochemical properties of the putative new profilm isoform by expressing both profilin 1 and profilin II in Escherichia coli. Comparison of interactions with actin; Isoform showing similar affinity for poly(L-proline) and PtdIns with similar effects on nucleotide exchange from actin.

Investigates the interaction Tyr103 and Tyr264 of the Escherichia coli RecA protein with DNA and nucleotide cofactors. Fluorescence of tryptophan residues of wild-type RecA; Cofactor-binding site in RecA; Antagonistic effects of adenosine triphosphate and adenosine diphosphate.

The sequence of the tufA gene from the extreme thermophilic eubacterium Thermus aquaticus EP 00276 was determined. The GC content in third positions of codons is 89.5%, with an unusual predominance of guanosine (60.7%). The derived protein sequence differs from tufA- and tufB- encoded sequences for elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, another member of the genus Thermus, in 10 of the 405 amino acid residues. Three exchanges are located in the additional loop of ten amino acids (182–191 ). The loop, probably involved in nucleotide binding, is absent in EF-Tu of the mesophile Escherichia coli. Since EF-Tu from E. coil is quite unstable, the protein is well-suited for analyzing molecular changes that lead to thermostabilization. Comparison of the EF-Tu domain I from E. coli and Thermus strains revealed clustered amino acid exchanges in the C-terminal part of the first helix and in adjacent residues of the second loop inferred to interact with the ribosome. Most other exchanges in the guanine nucleotide binding domain are located in loops or nearest vicinity of loops suggesting their importance for thermostability. The T. aquaticus EF-Tu was overproduced in E. coil using the tac expression system. Identity of the recombinant T. aquaticus EF-Tu was verified by Western blot analysis, N-terminal sequencing and GDP binding assays. [ABSTRACT FROM AUTHOR]

Escherichia coli RH1 contains a mutation causing complete loss of pyridine nucleotide transhydrogenase activity. A single base change in the chromosomal DNA resulted in the replacement of Gly314 of the β subunit by a Glu residue. The mutant enzyme was partially purified and its trypsin cleavage products examined. The distinct pattern of polypeptides given by proteolysis of the normal transhydrogenase in the presence of NADP(H) was absent when the mutant enzyme was treated with trypsin. However, the β subunit of the mutant enzyme retained its ability to bind to NAD-agarose. Further substitutions were made at Gly314 converting it to Ala, Val or Cys by the use of site-directed mutagenesis. All substitutions for Gly314 abolished the activity completely. The enzyme containing the Gly314→Ala mutation was studied in detail and behaved exactly as the enzyme containing the Gly314→Glu mutation. It is concluded that the mutation in the β subunit abolished the NADP(H)-induced conformational change in the mutant enzyme. This conformational change, caused by NADP(H) binding, is required to cleave the normal β subunit at Arg265 by trypsin. The genes encoding the pyridine nucleotide transhydrogenase were completely resequenced and several corrections have been made to the previously published sequence. [ABSTRACT FROM AUTHOR]

cAMP receptor protein from Escherichia coli has been purified on a large scale. Analogues of cAMP modified on the 6-NH2 group of the adenosine ring, the ribose 2'OH group or the cyclic phosphate are able to displace cAMP from its binding site with dissociation constants of similar magnitude to that of cAMP. More extensive modification produces weaker binding. Ultraviolet/visible difference spectroscopy and fluorescence spectroscopy show that the environment of the bound adenosine moiety is considerably less polar than that in aqueous solvent, while an anthraniloyl group substituted on the 2'OH position remains accessible to solvent. The 2-NH2 group of cGMP appears to be protonated in the bound form, while no change in the charge state of cAMP is apparent. [ABSTRACT FROM AUTHOR]

RNA polymerase was treated in the presence of promoter-containing templates with 16 affinity reagents, derivatives on NMPs, NDPs and NTPs with reactive substituents at the terminal phosphate. This treatment was followed by addition of a pyrimidine [α-32P]NTP. Due to 'catalytic competence' of some of the residues of the affinity reagents bound covalently near the active center at the first stage, active-center-catalyzed synthesis of a phosphodiester bond occurred, and radioactive residues with the general formula -pNpN (where p = radioactive phosphate) appeared covalently attached to the enzyme. Such affinity labelling was super-selective because affinity reagent residues bound outside the active center were not elongated and thus remained non-radioactive. Labelling took place only when the combination of the reagent and [α-32P]NTP corresponded to the sequence of nucleotides of the promoter. With reagents having short 'arms', only the β subunit was labelled; the targets were His and/or Lys residues. With reagents having longer 'arms', the a subunit was also labelled. [ABSTRACT FROM AUTHOR]

Five transcription units of the Pseudomonas solana-cearum hrp gene cluster are required for the secretion of the HR-inducing PopA1 protein. The nucleotide sequences of two of these, units 1 and 3, have been reported. Here, we present the nucleotide sequence of the three other transcription units, units 2, 4 and 7, which are together predicted to code for 15 hrp genes. This brings the total number of Hrp proteins encoded by these five transcription units to 20, including HrpB, the positive regulatory protein, and HpaP, which is apparently not required for plant interactions. Among the 18 other proteins, eight belong to protein families regrouping proteins involved in type III secretion pathways in animal and plant bacterial pathogens and in flagellum biogenesis, while two are related solely to proteins involved in secretion systems. For the various proteins found to be related to P. solanacearum Hrp proteins, those in plant-pathogenic bacteria include proteins encoded by hrp genes. For Hrp-related proteins of animal pathogens,, those encoded by the spa and mxi genes of Shigella flexneri and of Salmonella typhimurium and by the ysc genes of Yersinia are involved in type III secretion pathways. Proteins involved in flagellum biogenesis, which are related to Hrp proteins of P. solanacearum, include proteins encoded by fli and fih genes of S. typhimurium, Bacillus subtilis and Escherichia coli and by mop genes of Erwinia carotovora. P. solana-cearum Hrp proteins were also found to be related to proteins of Rhizobium fredii involved in nodulation specificity. [ABSTRACT FROM AUTHOR]

The genes determining the biosynthesis of the colonization factor CS5 have been cloned from Escherichia coli 0115:H40:PCF8775 isolated during an outbreak of diarrhoea among aboriginal children in Central Australia. Electron microscopy has shown purified CS5 to be of semi-rigid fimbrial type. NH2-terminal analysis has shown the CS5 determinant to be distinct from other fimbriae, although there is some conservation of certain residues. Expression in minicells of the cloned fimbrial genes encoded on pPM1312 has shown that proteins of 70 and 46.5 kD which co-purify with the 23 kD major fimbrial subunit protein are also co-expressed along with proteins of 45, 31, 17 and 14kD. The major CS5 subunit is synthesized in precursor form (approximately 26 kD). A synthetic oligonucleotide to the NH2-terminal amino acid coding sequence of the purified protein has been used in Southern hybridization analyses to define the region on pPM1312 encoding the structural gene for the major pilin subunit. [ABSTRACT FROM AUTHOR]

Incubation of DNA-dependent RNA polymerase from Micrococcus luteus (gram-positive) with the plasmid pBR322 under transcription conditions in vitro leads to the formation of a rather short-chained RNA. This transcript is initiated at the same site on pBR322 as RNA I, a defined Escherichia coli RNA polymerase product. M. luteus RNA polymerase initiates transcription at the RNA I site much more efficiently than the E. coil enzyme, using either a plasmid preparation of pBR322 or an appropriate linear restriction fragment as template. In the latter case, cleavage of the restriction fragment 24 or 71 nucleotides (but not 91 nucleotides) upstream of the initiation site destroys template activity. By sequence analysis it was determined that the 3' terminus of the M. luteus RNA polymerase transcription product is identical with that known for RNA I. Moreover, in agreement with synthesis of RNA I by E. coil RNA polymerase in vitro, termination by the M. luteus enzyme is also a stutter process characterized by the same dependence on the available UTP concentration. These observations lead to the hypothesis that termination by cubacterial RNA polymerases might not be species-specific. [ABSTRACT FROM AUTHOR]

The replication of M13 single-stranded DNA by the 9S DNA polymerase α from calf thymus has been studied in vitro. Priming conditions, the nature of the replication products and conditions for optimal elongation have been investigated. Oligonucleotides comprising only four nucleotides can serve as primers. Both ribo and deoxy oligonucleotides can be elongated. Priming by the short oligonucleotides occurs at multiple sites on the M13 genome. If replication is primed at single sites with a specific pentadecamer or with RNA in the origin of replication, specific pausing sites are observed. These pausing sites can partly be correlated with secondary structures in the template DNA. Addition of Escherichia coli single- stranded DNA binding protein leads to a weakening of pausing sites and to the synthesis of longer products. The 9S enzyme is able to proceed through most of the pausing sites resulting in the synthesis of product molecules as long as 6600 nucleotides. The 9S DNA polymerase α contains a potent DNA primase activity which enables it to initiate replication on a single-stranded template in the presence of the four NTPs . However, priming is also possible in the presence of ATP alone. The priming sites are not randomly distributed over the M13 DNA. [ABSTRACT FROM AUTHOR]

We studied the inhibition of tryptic digestion of the subassembly α2β of Escherichia coli DNA-dependent RNA polymerase to investigate its interaction with RNA and rifampicin. Both agents decreased distinctly the cleavage of subunit β in the subassembly as well as the degradation of the transiently formed polypeptides (Mr > 80000). Short RNAs with a chain length of approximately 35 nucleotides were most protective at a concentration of 1 mg/ml while long RNAs were less effective at the same concentration. DNA did not exert any observable protective effects. The association of RNA with α2β was shown by chromatography on phosphocellulose. which separates α2β bound to RNA from free α2β. The association of α2β with RNA was inhibited by rifampicin. [ABSTRACT FROM AUTHOR]

One of the major problems in the study of DNA replication involves the presence of numerous short, nonnascent DNA chains in the cell. Such chains often contaminate nascent DNA preparations, making accurate analysis of the replicative DNA difficult. This complication can be avoided by the use of hydroxyapatite chromatography. Previous results from this laboratory had shown that short nascent DNA chains could be eluted from hydroxyapatite at low phosphate concentrations because of their non-covalent association with protein. Additional data now indicate that this isolation procedure yields a DNA preparation which contains only nascent DNA and is essentially free of non-nascent chains. 5′-Terminal labeling patterns and molecular weight distributions show that only nascent DNA chains form the DNA-protein complex and can therefore be separated from non-nascent chains if the isolation is performed under non-denaturing conditions. The protein involved in the complex appears to be rather specific for DNA chains in the replication fork since short chains resulting from the excision of dUMP immediately after replication also form the DNA-protein complex. Using this isolation technique, the 5′-terminal nucleotides of nascent DNA chains were determined in an effort to learn something about the chain initiation process. DNA was pulse-labeled in several ways, purified by hydroxyapatite chromatography, and labeled at the 5′ end with [32P]phosphate. Two-dimensional thin-layer chromatography of the 32P-labeled nucleotides, obtained after enzymatic hydrolysis, revealed that all four deoxyribonucleotides are present at the 5′ end in approximately equal amounts. In most cases, this 5′-terminal nucleotide distribution does not vary significantly, even when the conditions of pulse-labeling are changed. Only if the cells are allowed to run out of thymine is a variation in the relative amounts of 5′-end nucleotides detected. In this case, the amount of dTMP at the 5′ end decreases significantly, reflecting the low thymine levels and indicating that the cell can compensate for this deficiency by utilizing other available nucleotides. From these results, it appears that DNA chain initiation is essentially random with respect to the nucleotide used in the initiation process. [ABSTRACT FROM AUTHOR]

Formylmethionyl-tRNA has been found to bind to Escherichia coli ribosomal protein SI. Com- plexes of S1 and methionine-labeled fMet-tRNAfMet were detected by binding to cellulose nitrate membranes. Complex formation was stimulated by incubation at low ionic strength, at 37°C and in the absence of Mg2+. Protein S1 also bound [3H]poly(rC) under the same conditions or in buffers of higher ionic strength, 10 mM in Mg2+, and during incubation at 0°C. Unlabeled poly(rC) was an effective inhibitor of the binding of labeled fMet-tRNA when both nucleotides were added simultaneously to the reaction mixtures; however, approximately 80% of previously bound f[35S]Met-tRNA remained bound after the subsequent addition of unlabeled poly(rC). Binding of f[3sS]Met- tRNA, but not the binding of [3H]poly(rC), was inhibited by polyamines or aminoglycoside antibiotics. Protein S1 that had been reacted with N-ethylmaleimide failed to bind f[3sS]Met-tRNA but was still capable of binding [3H]poly(rC). The data support the concept of two distinct RNA-binding sites on the S1 molecule. Initiator tRNA appears to occupy a site concerned with the RNA unwinding property of S1, whereas poly(rC) binds primarily at a separate site. During initiation on the ribosome, the poly(rC) site may interact with the 3′ end of 16-S ribosomal RNA, while the other site may interact with either mRNA or fMet-tRNA. [ABSTRACT FROM AUTHOR]

32-P-labelled 50-S subunits from Escherichia coli ribosomes were hydrolysed under conditions known to give rise to two specific ribonucleoprotein fragments, containing proteins L1, L9, and L5, L18 and L25 respectively. RNA corresponding to these ribonucleoproteins was isolated and purified, and the various RNA fragments obtained were subjected to oligonucleotide analysis. The results showed that the RNA associated with proteins L1 and L9 was very similar to the RNA found with protein L1 after controlled digestion of 23-S-RNA · L1 complexes (described elsewhere); this RNA lies within a region 550–1000 nucleotides from the 3′ terminus of 23-S RNA. The RNA associated with proteins L5, L18, and L25 consisted predominantly of two species of similar size. One was 5-S RNA, and the other a fragment of 23-S RNA, lying within the region 450–1000 nucleotides from the 3′ terminus. [ABSTRACT FROM AUTHOR]

Ribonucleoproteins were prepared by ribonuclease digestion of a reconstituted complex of ribosomal protein L1 and 23-S RNA from Escherichia coli. Three main ribonucleoproteins were identified. The largest was only obtained in an impure state at low ribonuclease concentrations, whereas the two smaller ones, which were difficult to separate from one another electrophoretically, were stable over a range of enzyme concentrations. The two smaller ribonucleoproteins yielded a total of 13 RNA subfragments that were judged to be homogeneous electrophoretically. The latter were characterized for molecular weight and the subfragment composition of each of these ribonucleoproteins was established. Furthermore, the subfragments were shown to be maintained together in each ribonucleoprotein by RNA-RNA interactions. The primary and specific binding site of protein L1 was localized on one continuous RNA subfragment of about 110 nucleotides in length by two newly developed binding methods. [ABSTRACT FROM AUTHOR]

Quinolinic acid (pyridine 2,3-dicarboxylic acid) which is an immediate precursor of the pyridine nucleotides, is synthesised from L­aspartate and dihydroxyacetone phosphate in Escherichia coli. Extracts from certain nadB mutants complement the extracts prepared from all nadA mutants for the enzymic synthesis of quinolinate. Using the complementation assay, the quinolinate synthetase B protein has been purified more than 300-fold. The quinolinate synthetase B protein exists in all nadA and nadC mutants examined. The quinolinate synthetase A protein was present in all nadC mutants and most (but not all) nadB mutants. The facile separation of the wild-type quinolinate synthetase A and B proteins out of a nadC mutant suggests that quinolinate synthetase does not exist as a tightly bound complex. The partially purified quinolinate synthetase is inhibited by physiological concentrations of NAD and NADH but not by NADP or NADPH. [ABSTRACT FROM AUTHOR]

Limited digestion of the Escherichia coli 50-S subunit permits the isolation of two large fragments of the 23-S RNA. One arises from the 5' end of the 23-S RNA and contains about 1200 nucleotides. The other arises from the 3' end of the 23-S RNA and contains about 2000 nucleotides. Each of the ten 50-S proteins known to bind specifically and independently to the 23-S RNA were tested for their ability to interact with these two RNA fragments. It was determined that the 5' terminal segment contains the specific binding sites for proteins L4, L20 and L24, whereas the 3' terminal segment contains the specific binding sites for proteins LI, L2, L3, L6, L13, L16 and L23. [ABSTRACT FROM AUTHOR]

Infection of Escherichia coli with bacteriophage T5 caused a more than 20-fold increase in ribonucleotide reductase activity in crude cell extracts. The phage induced the formation of a new ribonucleotide reductase and a new thioredoxin. The T5-specific ribonucleotide reductase was defined by its ability to accept the T5-induced thioredoxin as hydrogen donor. However, the enzyme could also accept E. coli thioredoxin as hydrogen donor and it was inhibited by antisera against subunit B2 of E. coli ribonucleotide reductase. The crude phage enzyme preparation studied preferred CTP to CDP and CMP as substrate and required magnesium and ATP for activity. Its behaviour towards allosteric effectors was different from that of the host enzyme. T5-induced thioredoxin chromatographed together with E. coli thioredoxin on DEAEcellulose but could be separated from the latter by immunoadsorbent chromatography. T5 thioredoxin was reduced by E. coli thioredoxin reductase but could not serve as hydrogen donor of E. coli ribonucleotide reductase. Bacteriophage T6 induced a new ribonucleotide reductase and a new thioredoxin upon infection. The T6-induced proteins cross-reacted functionally and immunologically with the corresponding T4-induced proteins. Infection of E. coli B with phage T7 or induction of the lysogens E. coli K-39 (λ+) and E. coli W-3350 (∼c I857) caused no increase in reductase activity and chromatography of cell extracts gave no evidence for the presence of new thioredoxins. [ABSTRACT FROM AUTHOR]

The aminoacylation of 15 fragments from yeast tRNAPhe and of various mixtures of these fragments was studied in detail. The aminoacylation conditions were systematically varied in order to obtain maximal incorporation of phenylalanine. No fragment by itself accepted phenylalanine. Fragment combinations in which parts of the anticodon loop or the dihydrouridine loop were missing, accepted phenylalanine to an extent of 20–75% compared to intact tRNAPhe. Modifications in both regions together inactivated the molecule. With the anticodon stem and/or the manilooop missing, low but significant activity was found. Splits in the dihydrouridine loop and in the T-ψ-C loop together did not abolish the phenylalanine acceptance. Combinations in which parts of the dihydrouridine stem or of the T-ψ-C stem were missing could not be aminoacylated. Complete association of complementary fragments was observed in all cases. The results of charging and heating experiments, however, indicate the coexistence of different flagment combinations in a fragment mixture. The incomplete aminoaeylation of fragment mixtures is explained by the presence of fragment combinations with an unsuitable three-dimensional structure. The rate of aminoacylation was lower for fragment combinations than for tRNAPhe. The Michaelis constants of 3 fragment combinations were identical to the one of t RNAPne. Two fragment mixtures inhibited the rate of charging of tRNAPhe. Phenylalanine was the only amino acid which was accepted by the fragment mixtures with synthetases from yeast or Escherichia coli. No miseharging was observed. The results together with those of related publications are discussed with respect to recognition sites of tRNAPhe for the cognate synthetase. [ABSTRACT FROM AUTHOR]

Cells made permeable to nucleotides by treatment with ether synthesize DNA from exogenous deoxynucleoside triphosphates in a semi-conservative fashion, The reaction proceeds from sites of previous synthesis on the chromosome, and not detectably from new sites. Short chains appear as precursors of long units and have a mean sedimentation coefficient of about 10 S in alkali, like Okazaki-pieces when synthesized in the presence of 10 to 20 μM deoxynucleoside tri- phosphates, and of only 5 S when synthesized in the presence of 1μM dNTP, Under the former conditions short chains contain not more than half of the newly synthesized DNA; under the latter conditions they can contain more than half, the remainder of the newly synthesized DNA being in long units, Nascent short chains labeled after several minutes of synthesis in the presence of 5-bromodeoxyuridine 5'-triphosphate at high deoxynucleoside triphosphate concentrations show bimodal density distribution in an alkaline Cs2SO4 equilibrium gradient with peaks of material corresponding to the density of heavy single strands and almost light single strands, respectively. The implications of these findings are discussed. Judged from controls, repair synthesis was not a source of error in these experiments. In contrast with results reported for intact cells, ether-treated polA1- (Kornberg-polymerase deficient) cells join nascent short chains as efficiently as do polA+ cells. [ABSTRACT FROM AUTHOR]

Glutamine synthetase adenylyltransferase was purified 300-fold from Escherichia coli B grown on minimal medium. The purified enzyme was found to be homogeneous in the ultracentrifuge, but by electrophoresis on polyacrylamide gel two enzymatically active protein components were separated, that differed from batch to batch in their relative proportions. The apparent Km values of the various substrates and activators of the adenylylation of glutamine synthetase were determined. The adenylylating enzyme requires Mg2+ or Mn2+ and ATP for activity. Several other divalent cations and nucleotides were tested and found to be ineffective. In the presence of glutamine the rate of the reaction decreases at ATP concentrations greater than 1 mM. Addition of ADP, CTP, UTP and ITP to the standard assay mixture, containing ATP results likewise in a decrease of the rate of the reaction. Various intermediates of glycolytic pathway and Krebs cycle inhibit the adenylylation reaction. The optimum pH for adenylylation is about 7.6. The activation energy for the adenylylation was estimated from rate measurements. It is 13 kcal/mole in the temperature range from 5° to 32°. Gel filtration on Sephadex G-150 indicates an average molecular weight of 145000 daltons. On storage of the enzyme in solution inactive aggregates of higher molecular weight are formed. Bovine serum albumin prevents inactivation and aggregation. [ABSTRACT FROM AUTHOR]

The effects of nucleotide analogues on DNA synthesis ere studied in nucleotide permeable Escherichia coli cells. 2′,3′,Dideoxyribosylthymine 5′-triphosphate inhibited replicative DNA synthesis far more strongly than endonuclease-induced DNA repair synthesis. In ϕX174-infected cells this analogue caused the formation of short pieces (7 S) of ϕX replicative form DNA in which newly synthesized DNA was found covalently linked to preformed primer. Deoxyuridine triphosphate led to even shorter pieces (3 S) of ϕX replicative form DNA. Nicotinamide mononucleotide, an inhibitor of E. coli DNA ligase, promoted accumulation of short (10 S) E. coil DNA pieces. Newly synthesized 5′-triphosphate ends were not detected in these nucleotide-permeable cells during the early stages of ϕX replicative form replication, nor was evidence obtained that nuoleoside monophosphates are immediate precursors of DNA. [ABSTRACT FROM AUTHOR]

Reports on the availability of cyclic adenylic acid (cAMP) in Germany. Potent stimulator of gene transcription in Escherichia coli; Importance of cyclic nucleotide analogue for the variety of research fields from plants over bacteria to mammalian systems; Method to modulate the cAMP pathway.