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F Cramer

Publications and source records attributed to F Cramer.

At least 109 records · Page 6Linked to original sources
PubMed1977

Hydrolytic action of aminoacyl-tRNA synthetases from baker's yeast. "Chemical proofreading" of Thr-tRNA Val by valyl-tRNA synthetase studied with modified tRNA Val and amino acid analogues.

The properties of native and of two modified tRNA Val species in the correction of misactivated threonine by valyl-tRNA synthetase have been studied. Whereas Thr-tRNA Val-C-C-A could not be isolated in the valyl-tRNA synthetase catalyzed reaction, Thr-tRNA Val-C-C-3'dA is isolable in up to 50% yield in this system and tRNA Val-C-C-3'NH2A is fully aminoacylated with threonine by the same enzyme. The hydrolysis of preformed Thr-tRNA Val-C-C-A by free valyl-tRNA synthetase is 30 times faster than the corresponding breakdown of Val-tRNA Val-C-C-A. This hydrolytic activity is also observed with Thr-tRNA Val-C-C-3'dA although the rate is reduce to that of the reaction of Val-tRNA Val-C-C-A. Modification of the threonine to O-methylthreonine, which is also a substrate for valyl-tRNA synthetase, leads to stabilization of the O-methylthreonyl-tRNA esters. The AMP/PP independent hydrolysis under aminoacylating conditions, which is a measure of the correction process, indicates that O-MeThr-tRNA Val-C-C-A is only very slowly corrected while the tRNA Val-C-C-3'dA and tRNA Val-C-C-3'NH2A esters are completely stable. Removal of the methoxy group of O-methylthreonine as in alpha-amino-butyric acid increases the rate of the hydrolytic reaction and once again alpha-Abu-tRNA Val-C-C-A and alpha-Abu-tRNA Val-C-C-3'dA are unstable under aminoacylating conditions and not isolable.

Amino Acyl-tRNA Synthetases↗
PubMed1977

[Structure of periodated ribonucleosides and ribonucleotides].

Periodate oxidation converted adenosine, guanosine, cytidine, and uridine into the corresponding dialdehydes. No free dialdehydes are present in solution, but several hydrated species occur (i.r. and n.m.r.). In the solid state, the dialdehydes are completely polymerized. The m.s. molecular peaks corresponding to the free daildehydes were of very low intensity. The c.d spectra of adenosine- and cytidinedialdehyde showed heavily diminished Cotton effects in comparison to the parent nucleosides. For uridine-dialdehyde, the signal intensity of the strongest transition was diminished by about one half, indicating a looser structure allowing free rotation of the base. By contrast, the c.d. signal of guanosine-dialdehyde was increased, indicating self-association of oligomers. The nucleoside-dialdehydes gave, with hydrazides, morpholine derivatives, which posses rigid, stable structures (c.d).

Circular Dichroism↗
PubMed1977

Introduction of antigenic determining 2,4-dinitrophenyl residues into 4-thiouridine, N3-(3-L-amino-3-carboxypropyl) uridine and tRNA-Phe from E. coli.

The introduction of antigenic determining 2,4-dinitrophenyl residues into the rare ribonucleosides 4-thiouridine (1a), and N3-(3-L-amino-3-carboxypropyl) uridine (2) as well as into tRNA-Phe from E. coli has been investigated. Alkylation of 1a with omega-bromo-2,4-dinitroacetophenone (3b) gives S-(2,4-dinitrophenacyl)-4-thiouridine (5A). Applying the reaction to the 5'-monophosphate of 1a, 5b is formed, but this product decomposes at pH 7. However, acylation of 2 with 2,4-dinitrobenzoic acid N-hydroxysuccinimide ester (4b) leads to N3-[3-carboxy-3-L-(2,4-dinitrobenzamido)propyl]uridine (6) which is stable in aqueous solution. The latter reaction was used for the introduction of an antigenic determining 2,4-dinitrophenyl residue into tRNA-Phe from E. coli. The modified tRNA-Phe was isolated and by degradation of the molecule with RNase T2 and alkaline phosphatase the nucleoside derivative 6 was obtained and found to be identical with the synthetic product.

Chemical Phenomena↗
PubMed1977

Properties of phenylalanine transfer ribonucleic acid with modified 3'-terminal end in protein biosynthesis using a rabbit reticulocyte cell-free system: effect of the replacement of cytidine residues from the CpCpA end of tRNA by 5-iodocytidine or 2-thiocytidine.

Phe-tRNA Phe from yeast containing 2-thiocytidine or 5-iodocytidine in position 75 of the polynucleotide chain or Phe-tRNA Phe in which both positions 74 and 75 were substituted by 5-iodocytidine were investigated in the poly U-dependent polyphenylalanine synthesis on ribosomes from rabbit reticulocytes. Phe-tRNA Phe-Cps2CpA was nearly as active as the native Phe-tRNA Phe-CpCpA in the overall process. Phe-tRNA Phe-Cpi 5CpA as well as Phe-tRNA Phe-i5Cpi 5CpA were considerably less active than the native species. Investigation of individual steps of protein biosynthesis with these modified substrates revealed that the donor activity of peptidyl-tRNAs which contain 5-iodocytidine in their 3'-terminus is strongly imparied suggesting exacting structural requirements for the interaction of the CpCpA end of tRNA with the ribosomal P-site.

Animals↗
PubMed1977

Enzymatic incorporation of ATP and CTP analogues into the 3' end of tRNA.

Structural analogues of adenosine 5'-triphosphate and cytidine 5'-triphosphate were investigated as substrates for ATP(CTP):tRNA nucleotidyl transferase. Eight out of 26 ATP analogues and six out of nine CTP analogues were incorporated into the 3' terminus of tRNA. In general, for the recognition of the substrates the modification of the cytidine is less critical than is the modification of adenosine. An isosteric substitution on the ribose residue is possible in both CTP and ATP. The free hydroxyls of these triphosphates can be replaced by an amino group or hydrogen atom without loss of substrate properties. Modifications of positions 1, 2, 6, and 8 on the adenine ring of ATP are not allowed whereas modification on positions 2, 4 and 5 on the cytosine ring of CTP are tolerated by the enzyme. No differences can be observed in the substrate properties of ATP(CTP):tRNA nucleotidyl transferase isolated from different sources. Methods for preparation of tRNA species, which are shortened at their 3' end by one or more nucleotides, and analytical procedures for characterisation of these modified tRNAs are described.

Adenosine Triphosphate↗
PubMed1976

Calorimetric investigations on thermal stability of tRNAIle (yeast) and tRNASer (yeast).

Variation with temperature of the partial heat capacities of tRNAIle (yeast) and tRNASer (yeast) has been determined in two buffers at various salt conditions by scanning microcalorimetry. The overall molar transition enthalpy, deltaHt = 320 +/- 20 kcal mol-1 (1339 +/- 84 kJ mol-1) is identical for the two tRNA species within the limits of experimental error. deltaHt does not show any dependence on the nature of the buffer, nor does it vary on addition of 1 mM MgCl2 or 150 mM NaCl. Thermal unfolding of the native structure to the random coil cannot adequately be described by a two-state, concerted transition under the experimental conditions applied in this study, but exhibits a multistep mechanism characterized by sequential unfolding of separable cooperative domains.

Calorimetry↗
PubMed1976

Spermine stabilizes the conformation of tRNAPhe in crystals.

Crystals from yeast tRNAPhe were dissolved and compared with tRNAPhe that had not been srystallized. A number of differences were found regarding the interaction with ethidium bromide, the melting point and the circular dichroic signal. These differences were assigned to the presence of spermine in the dissolved crystals indicating a transient stabilization of the conformation of tRNAPhe, probably as a tRNAPhe-spermine complex, after dissolving.

Binding Sites↗
PubMed1976

Hydrolytic action of aminoacyl-tRNA synthetases from baker's yeast: "chemical proofreading" preventing acylation of tRNA(I1e) with misactivated valine.

Phe-tRNAPhe-C-C-A, Val-tRNAVal-C-C-A, and Ile-tRNAIle-C-C-A, which accept their amino acid on the 2'-OH of the 3'-terminal adenosine, are hydrolyzed readily by their aminoacyl-tRNA synthetase. If the 3'terminal adenosine in these tRNAs is replaced by either 3'-deoxyadenosine or formycin, little if any hydrolysis can be observed. Correspondingly Ser-tRNASer-C-C-A which accepts serine on the 3'-OH of the 3'-terminal adenosine is hydrolyzed by seryl-tRNA synthetase, whereas Ser-tRNASer-C-C-2'dA and Ser-tRNASer-C-C-F are not. Tyr-tRNATyr-C-C-A and all modified Tyr-tRNATyr-C-C-N, which can accept tyrosine on either the 2'OH or the 3'-OH of the 3'terminal adenosine, are not hydrolyzed by tyrosyl-tRNA synthetase. The data can be rationalized assuming that hydrolysis takes place only if the amino acid is bound to the nonaccepting OH and hence is not positioned at the amino acid binding site upon formation of the complex between aminoacyl-tRNA and aminoacyl-tRNA synthetase. In the formycin-carrying tRNA, the amino acid bound to the nonaccepting OH seems to be inaccessible to the enzymatic groups responsible for hydrolysis. Val-tRNAIle-C-C-3'dA and Ile-tRNAIle-C-C-3'DA cannot be hydrolyzed by isoleucyl-tRNA synthetase. Val-tRNAIle-C-C-A is hydrolyzed by the enzyme five times more rapidly than Ile-tRNAIle-C-C-A. Whereas Ile-tRNAIle-C-C-F is absolutely stable, Val-tRNAIle-C-C-F si hydrolyzed immediately. As shown by the earlier finding that valine misactivated by isoleucyl-tRNA synthetase cannot be permanently transferred to tRNAIle-C-C-A but to tRNAIle-C-C-3'dA, the 3'-OH is essential for preventing transfer of misactivated valine. It thus appears that valine is hydrolyzed off Val-tRNAIle-C-C-N if it is bound to the accepting 2'-OH in the binding site for isoleucine. A hypothesis is offered attempting to explain the experimental observations in mechanistic terms. We consider the hydrolytic action of the aminoacyl-tRNA synthetases as a general mechanism of "chemical proofreading" in the protein biosynthesis.

Amino Acyl-tRNA Synthetases↗
PubMed1976

Properties of tRNA species modified in the 3'-terminal ribose moiety in an eukaryotic ribosomal system.

Phe-tRNAPhe species modified on the 3'-terminal ribose residue were investigated for their ability to participate in individual steps of the elongation cycle using eukaryotic ribosomes from reticulocytes. None of the Phe-tRNAs used, namely Phe-tRNAPhe-C-C-3'dA, Phe-tRNAPhe-C-C-3'-NH2A, and Phe-tRNAPhe-C-C-Aoxi-red, can function in the overall process. All modified Phe-tRNAPhe species can be bound nonenzymatically to ribosomes. Phe-tRNAPhe-C-C-3'NH2A exhibits exceptionally high binding at low Mg2+ concentration compared with Phe-tRNAPhe-C-C-A binding. Ac-Phe-tRNAPhe species prepared from the three modified tRNAs, when bound to the donor site, were devoid of donor activity. The enzymatic binding of both Phe-tRNAPhe-C-C-3'dA and Phe-tRNAPhe-C-C-3'NH2A is less efficient than that of Phe-tRNAPhe-C-C-A but these Phe-tRNAPhe species have acceptor activity. Phe-tRNAPhe-C-C--Aoxi-red is not a substrate for the EF-I promoted binding reaction and has no acceptor activity. The nonaminoacylated species, tRNAPhe-C-C-A, tRNAPhe-C-C-3'dA, and tRNAPhe-C-C-3'NH2A, bind to the ribosome to a larger extent than the corresponding aminoacylated tRNAs, both in the presence and in the absence of poly(U). Peptidyl-tRNAPhe-C-C-3'dA bound to the donor site cannot activate the acceptor site for EF-I promoted binding of Phe-tRNAPhe as does peptidyl-tRNAPhe-C-C-A. Further, it was observed that a correct codon-anticodon interaction influences the recognition of the 3' terminus of tRNA. Specificity of eukaryotic ribosomes for the 2'- and/or 3'-aminoacylated tRNA species is discussed and compared with the properties of Escherichia coli system.

Binding Sites↗
PubMed1976

Affinity labelling of tRNA nucleotidyltransferase from baker's yeast with tRNAPhe modified on the 3'-terminus.

2'-Deoxy-2'-amino-cytidylic acid can be incorporated into position 75 of tRNAPhe from yeast by tRNA nucleotidyltransferase yielding tRNAPhe-C-C(2'NH2). tRNAPhe-C-C(2'NH2) can be reacted with the N-hydroxysuccinimide esters of bromoacetic acid of mercuriacetic acid to yield the derivatives tRNAPhe-C-C(2'NHCOCH2Br) and tRNAPhe-C-C(2'NHCOCH2Hg+OH-). Each of these reactive tRNAs inactivates tRNA nucleotidyltransferase from yeast with similar kinetics. The enzyme can be protected against inhibition by its substrates tRNAPhe-C and tRNAPhe-C-C as well as ATP and CTP. A covalent, isolatable 1:1 complex between tRNAPhe-C-C(2'NHCOCH2Br) and the enzyme was formed, but could not be found when the enzyme had previously been inactivated with p-hydroxymercuribenzoate.

Affinity Labels↗
PubMed1976

Valyl-tRNA, isoleucyl-tRNA and tyrosyl-tRNA synthetase from baker's yeast. Substrate specificity with regard to ATP analogs and mechanism of the aminoacylation reaction.

Nineteen analogs of ATP have been tested in the aminoacylation of valyl-tRNA, isoleucyl tRNA and tyrosyl-tRNA synthetases from baker's yeast. Four compounds are substrates for valyl tRNA and two for isoleucyl-tRNA synthetase, but there is no modified substrate for the tyrosyl tRNA synthetase. There is one inhibitor for valyl-tRNA synthetase, eight compounds inhibit isoleucyl-tRNA synthetase and two compounds inhibit tyrosyl-tRNA synthetase. Their Km and Ki and V values have been determined. The substrate specificity shows that positions 2, 6, 7, 8, 9, 2', and 3' of ATP are important for catalytic action of these aminoacyl-tRNA synthetases.

Adenosine Triphosphate↗
PubMed1976

Phenylalanyl-tRNA and seryl-tRNA synthetases from baker's yeast. Substrate specificity with regard to ATP analogs and mechanism of the aminoacylation reaction.

Eighteen analogs of ATP have been tested in the aminoacylation reaction of phenylalanyl-tRNA and seryl-tRNA synthetases from baker's yeast. Four compounds are substrates for phenylalanyl-tRNA synthetase, five for seryl-tRNA synthetase, one compound is an inhibitor for both enzymes; their Km and Ki and V values have been determined. The substrate specificity shows that for the catalytic action of both enzymes with these substrates positions 6, 7, 8 and 9 of the purine moiety and positions 2' and 3' of the ribose moiety are important.

Adenosine Triphosphate↗
PubMed1976

The kinetics of binding of U-U-C-A to a dodecanucleotide anticodon fragment from yeast tRNA-Phe.

The kinetics of U-U-C-A binding to the dodecanucleotide (A-Cm-U-Gm-A-A-Y-A-psi-m5C-U-Gp) isolated from the anticodon region of yeast tRNA-Phe are similar to the kinetics of binding of U-U-C-A to intact tRNA-Phe. A large enhancement in binding constant over that predicted for U-U-C-A-U-G-A-A is observed for both the complexes of dodecanucleotide and tRNA-Phe with U-U-C-A. This strongly suggests that both the anticodon loop in tRNA-Phe and the dodecanucleotide can form four base pairs with U-U-C-A. Furthermore, the enhanced stability cannot be attributed to a special conformation of the anticodon loop, but instead the anticodon loop is probably flexible. A likely explanation for the increased binding is the effect of non-base-paired ends. This increased thermodynamic stability comes from a larger entropy gain rather than a larger enthalpy decrease.

Adenine Nucleotides↗
PubMed1975

Proton magnetic resonance studies on the conformation of the hexanucleotide, GmpApApYpApsiP, and Related fragments from the anticodong loop of baker's yeast phenylalanine transfer ribonucleic acid.

A hexanucleotide, GmpApApYpAppsiP, and 11 related compounds from the anticodon loop of Baker's yeast tRNA-Phe were studied by proton magnetic resonance from 100 to 250 MHz. Totally 19 resonance lines from all the base protons (H8, H6, and H2), H1', and methyl proton resonances of the hexamer have been assigned by a systematic "incremental procedure" in comparing all the related shorter fragments. Emphasis is given to the Y base and its stacking conformation with respect to its nearest neighboring bases. The results reveal a strong tendency of the purine bases to have a maximal extent of base-base overlap with their neighbors in the sequence. This tendency is manifested in a zigzag (or balcony-like) mode of base-stacking pattern of the -ApYpA-sequence in the hexamer in which the -pA-residue tends to stack toward the adduct ring (C10, C11, and N12) of Y. This tendency is also shown in the formation of a stack of GmAAA closing the gap left behind by the excision of Y in the hexamer GmpApAp--pApp. The implication of these findings to the structure and function of tRNA is discussed.

Codon↗