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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]

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]

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]

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]

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.