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H Kersten

Publications and source records attributed to H Kersten.

At least 55 records · Page 3Linked to original sources
PubMed1981

On the role of ribosylthymine in prokaryotic tRNA function.

tRNAPhe and tRNALys were isolated from an Escherichia coli K12 mutant deficient in ribosylthymine (rT) and from the wild-type strain. The sequence G-rT-psi-C which is common to loop IV of practically all tRNAs used in the elongation cycle of protein synthesis reads G-U-psi-C in the tRNAs of the mutant strain. The purified tRNAs were compared in various steps of protein biosynthesis. The poly(U)-dependent poly(Phe) synthesis performed with purified Phe-tRNAPhe and purified elongation factors showed no dependence on the presence or absence of ribosylthymine in the respective tRNAs. In contrast, the corresponding poly(A)-dependent poly(Lys) synthesis was markedly increased when Lys-tRNALys lacking rT was used. The analysis of individual functional steps of the poly(A)-dependent elongation cycle demonstrated that the absence of rT reduced the binding to the A-site and improved the translocation reaction, whereas the formation of the ternary complex EF-Tu . GTP . aa-tRNA as well as both tRNA binding to the P-site and the peptidyltransferase reaction remained unaffected. The presence of U in place of rT in tRNA increases the misincorporation of leucine in an optimized poly(U)/poly(Phe) system from about 3 in 10 000 to 3 in 1000. Our results are in agreement with the view that rT is involved in tRNA binding to the A-site in contrast to the P-site, and suggested that the presence of rT in tRNA improves the fidelity of the decoding process at the A-site of the ribosome.

Bacterial Proteins↗
PubMed1981

Reduced aminoacylation of asparagine-transfer RNA early in the developmental cycle of Dictyostelium discoideum: modification pattern and possible significance of the uncharged isoacceptor tRNAAsn3.

Changes in the patterns of isoacceptors of tRNAAsn and alterations in modification of the guanine residue 34, the first position of the anticodon of tRNAAsn, have been observed in eukaryotes during differentiation. We use Dictyostelium discoideum as a model system to elucidate the possible involvement of tRNAAsn in developmental processes. Vegetative amoebae were induced to undergo developmental transition by nutrient starvation. Since amino acid starvation alone is a specific stimulus initiating development and unacylated tRNAs might be involved in control mechanisms of protein synthesis, the level of aminoacylation of tRNAAsn isoacceptors has been investigated. As early as two minutes after the onset of development, the aminoacylation of tRNAAsn specifically was reduced to about 30%, whereas at the same time 10 other tRNA species were found to be charged normally, i.e. to 70-100%. One of the two major isoacceptors, tRNAAsn3, was completely deacylated, whereas the other one, tRNAAsn2, accounted for the residual aminoacylation. Analyses of the modified nucleosides of highly purified tRNAAsn2 and tRNAAsn3 are respectively, show that both isoacceptors are identical in their modification patterns except for the modification at the first position of the anticodon; tRNAAsn2 comprises queuine (Q), 7-[(4,5-cis-dihydroxy-2-cyclopenten-1-ylamino)methyl]-7-deazaguanine, whereas tRNAAsn3 contains guanine.

Amino Acids↗
PubMed1980

Prevention of alcohol withdrawal symptoms in surgical patients.

Serious withdrawal symptoms in patients with alcoholism who undergo operation can be prevented or effectively treated by providing small amounts of alcohol throughout the withdrawal period. Delirium tremens is a potentially fatal symptom complex which may develop when a prolonged daily drinking pattern is interrupted by illness or injury. It was first described by Thomas Sutton in 1813. The withdrawal period is usually less than 72 hours but may be as long as ten days. It is not necessary to maintain a measurable blood level of alcohol for this therapy to be effective. Ten to 15 milliliters of ethanol given hourly, either orally or intravenously, is usually sufficient to prevent or control serious withdrawal symptoms.

Administration, Oral↗
PubMed1979

Role of ribothymidine in the thermal stability of transfer RNA as monitored by proton magnetic resonance.

In order to elucidate the functional role of the modified uridines at position 54 of tRNA, the 270 MHz high-field proton NMR spectra of methionine tRNAs from E. coli, from a mutant thereof, and from T. thermophilus, containing ribothymidine, uridine and 2-thioribothymidine, respectively, have been measured as a function of temperature. A comparison of the NMR melting profiles of the minor nucleosides from these tRNAs shows that the melting temperature of uridine containing tRNA is 6 degrees C lower than that of the wild type tRNA whereas that of the 2-thioribothymidine tRNA is 7 degrees C higher than that of the wild type tRNA. These results, therefore, demonstrate that these modifications serve for stabilization of the tertiary structure of tRNA.

Drug Stability↗
PubMed1979

Synthesis of transfer ribonucleic acids with uridine or 2'-O-methylribothymidine at position 54 in developing Dictyostelium discoideum.

In amoebae of Dictyostelium discoideum the ribothymidine (rT) content of tRNA is 0.9 mol%, but decreases progressively during development into spores. To elucidate which nucleosides replace rT at position 54 in developmental tRNA we have characterized 'vegetative' and 'developmental' tRNAs from the slime mould. Specific tRNAs were separated by two-dimensional gel electrophoresis. During early developmental stages, all tRNA species that could be separated by this method were newly synthesized. A new tRNA with uridine in place of rT and having an electrophoretic mobility similar to 'vegetative' tRNAAsn was detected during the early preaggregation stage. This 'development' tRNA was also extracted from purified polysomes. When development proceeds from preaggregation to postaggregation, tRNAs accumulate with 2'-O-methylribothymidine in place of rT. We suggest that these developmental tRNAs are important for the synthesis of specific developmental proteins.

Dictyostelium↗
PubMed1979

Function of modified nucleosides 7-methylguanosine, ribothymidine, and 2-thiomethyl-N6-(isopentenyl)adenosine in procaryotic transfer ribonucleic acid.

To elucidate subtle functions of transfer ribonucleic acid (tRNA) modifications in protein synthesis, pairs of tRNA's that differ in modifications at specific positions were prepared from Bacillus subtilis. The tRNA's differ in modifications in the anticodon loop, the extra arm, and the TUC loop. The functional properties of these species were compared in aminoacylation, as well as in initiation and peptide bond formation, at programmed ribosomes. These experiments demonstrated the following. (i) In tRNA(f) (Met) the methylation of guanosine 46 in the extra arm to 7-methylguanosine by the 7-methylguanosine-forming enzyme from Escherichia coli changes the aminoacylation kinetics for the B. subtilis methionyl-tRNA synthetase. In repeated experiments the V(max) value is decreased by one-half. (ii) tRNA(f) (Met) species with ribothymidine at position 54 (rT54) or uridine at position 54 (U54) were obtained from untreated or trimethoprim-treated B. subtilis. The formylated fMet-tRNA(f) (Met) species with U54 and rT54, respectively, function equally well in an in vitro initiation system containing AUG, initiation factors, and 70s ribosomes. The unformylated Met-tRNA(t) (Met) species, however, differ from each other: "Met-tRNA(f) (Met) rT" is inactive, whereas the U54 counter-upart effectively forms the initiation complex. (iii) Two isoacceptors, tRNA(1) (Phe) and tRNA(2) (Phe), were obtained from B. subtilis. tRNA(1) (Phe) accumulates only under special growth conditions and is an incompletely modified precursor oftRNA(2) (Phe): in the first position of the anticodon, guanosine replaces Gm, and next to the 3' end of the anticodon (isopentenyl)adenosine replaces 2-thiomethyl-N(6)-(isopentenyl)adenosine. Both tRNA's behave identically in aminoacylation kinetics. In the factor-dependent AUGU(3)-directed formation of fMet-Phe, the undermodified tRNA(1) (Phe) is always less efficient at Mg(2+) concentrations between 5 and 15 mM than its mature counterpart.

Bacillus subtilis↗
PubMed1978

On the biosynthesis of 5-methoxyuridine and uridine-5-oxyacetic acid in specific procaryotic transfer RNAs.

The uridine-5-0-derivatives, 5-methoxyuridine (mo5U) and uridine-5-oxyacetic acid (cmo5U) occupy the first position of anticondons in certain tRNA species of B. subtilis and E. coli, respectively. Here we present experimental evidence showing that both modifications are derived from a common precursor, 5-hydroxyuridine. Incompletely modified tRNASer and tRNAVal from E. coli met- rel-. All five tRNAs accepted methyl groups from S-adenosylmethionine with B. subtilis extracts in vitro and mo5U was formed. In B. subtilis tRNAs the mo5U was proved to be at the specific site; in E. coli tRNAVal the mo5U was demonstrated to be present in the oligonucleotide that comprises the anticodon. In submethylated E. coli tRNAVal,5-hydroxyuridine was detected whereas considerable amounts of cmo5U were lacking.

Alanine↗
PubMed1978

Recognition of individual procaryotic and eucaryotic transfer-ribonucleic acids by B subtilis adenine-1-methyltransferase specific for the dihydrouridine loop.

Bulk tRNA from yeast and Rat liver can be methylated in vitro with -adenosylmethionine and B, subtilis extracts. The sole product formed is 1-methyladenosine (m1A). This tRNA (adenine-1) methyltransferase converts quantitatively the 3'-terminal adenosine-residue in the dihydrouridine-loop of tRNAThr and tRNATyr from yeast into m1A. Out of 16 eucaryotic tRNAs with known sequences 6 accepted methyl groups, all at a molar ratio of 1. These tRNAs have in common an unpaired adenosine-residue at the specific site in the sequence Py-A-A+-G-G-C-m2G. Out of 12 tRNAs from E. coli 6 served as specific substrates. These E. coli tRNAs also have an unpaired adenosine-residue at the 3'-end of the D-loop. Besides restrictions in primary structure intact secondary and tertiary structure is important for recognition of the specific tRNAs by the enzyme.

Bacillus subtilis↗
PubMed1977

Methylation of an adenosine in the D-loop of specific transfer RNAs from yeast by a procaryotic tRNA (adenine-1) methyltransferase.

tRNA (adenine-1) methyltransferase occurs in Bacillus subtilis. Eucaryotic tRNAThr and tRNATyr from yeast in which 1-methyladenosine (m1A) is already present in the TpsiC loop, can be methylated in vitro with S-adenosylmethionine and B. subtilis extracts. Each of the specific tRNAs accepts 1 mol of methyl groups per mol tRNA. The enzyme transforms into m1A the 3'-terminal adenylic acid residue of the dihydrouridine loop, a new position for a modified adenosine residue in tRNA. Both tRNAs have the sequence Py-A-A-G-G-C-m2(2)G in the D-loop and D-stem region. Other tRNAs with the same sequence in this region also serve as substrates for the tRNA (adenine-1) methyltransferase.

Adenine Nucleotides↗
PubMed1977

Tetrahydrofolate-dependent biosynthesis of ribothymidine in transfer ribonucleic acids of Gram-positive bacteria.

Trimethoprim, an inhibitor that prevents tetrahydrofolate-dependent transmethylation reactions inbacteria, was used in a comparative study to discriminate between two possible biosynthetic pathways, either the S-adenosylmethionine or the tetrahydrofolate-dependent formation of ribothymidine (rT) in transfer ribonucleic acids (tRNA's) of several strains of gram-positive and gram-negative microorganisms. rT-deficient tRNA's accumulate in trimethoprim-treated gram-positive Streptococcus faecium, Staphylococcus aureus, Corynebacterium bovis, Arthrobacter albidus, and all examined Bacillaceae, except Bacillus stearothermophilus. The rT-deficient rT-deficient tRNA's accept the methyl moiety from S-adenosylmethionine in vitro, with extracts from Escherichia coli (wild type) as a source of methylating enzymes; 90% of the incorporated methyl groups are present in rT. Trimethoprim does not inhibit the biosynthesis of rT in tRNA of gram-negative Enterobacteriaceae, Rhizobium lupini, and Pseudomonadaceae, suggesting that the rT-specific tRNA methyltransferases of these gram-negative strains use S-adenosylmethionine as coenzyme.

Arthrobacter↗
PubMed1976

Purification and characterization of tRNAMet-f, tRNAPhe and tRNATyr2 from Baccillus subtilis.

Three tRNAs specific for methionine, phenylalanine and tyrosine were isolated from the total tRNA of Bacillus subtilis by chromatographic procedures using BD-cellulose and reversed-phase (5) chromatography. The acceptor activities of the purified tRNAs are 1160, 1260 and 1320 pmoles per A260nm unit for tRNAMetf, tRNAPhe and tRNATyr2 respectively. In tRNAMetf and tRNAPhe ribothymidine, pseudouridine and dihydrouridine are present, in addition, in tRNAPhe 7-methyguanosine and a 2'-O-methylated nucleoside were found. The modified nucleosides of tRNATyr2 are ribothymidine, pseudouridine, dihydrouridine, 4-thiouridine and 1-methyladenosine. The results suggest the presence of 2-methylthio-N6(delta 2-isopentenyl)adenosine in tRNAPhe and tRNATyr2. The thermal denaturation profiles of the three tRAN species are presented.

Bacillus subtilis↗
PubMed1976

7-Methylguanine specific tRNA-methyltransferase from Escherichia coli.

A 7-methylguanine (m7G) specific tRNA methyltransferase from E. coli MRE 600 was purified about 1000 fold by affinity chromatography on Sepharose bound with normal E. coli tRNA. The purified enzyme catalyzes exclusively the formation of m7G in submethylated bulk tRNA of E. coli K12 met- rel-. The purified enzyme transfers the methyl group from S-adenosyl-methionine to initiator tRNA of B. subtilis and 0.8 moles m7G residues are formed per mole tRNA. It is suggested that the enzyme specifically recognizes the extra arm unpaired guanylate residue.

Electrophoresis, Disc↗