Search PubMed⌕ Search

Biomedical subjects

F Cramer

Publications and source records attributed to F Cramer.

At least 91 records · Page 5Linked to original sources
PubMed1978

Reaction of tRNAPhe from yeast with 1-fluoro-2,4-dinitrobenzene. Attachment sites of the potential antigenic-determining 2,4-dinitrophenyl residues.

The reaction of 1-fluoro-2,4-dinitrobenzene with tRNAPhe from yeast, for the introduction of antigenic-determining 2,4-dinitrophenyl residues into tRNA, took place only at adenosine residues in tRNAPhe. After reaction at pH 8.0 and 50 degrees C two kinds of products were detected: one was ribose-modified adenosine which was derived from the 3' terminus of tRNA, and the other was base-modified adenosine. The sites and extent of the modification of each particular adenosine residue of tRNAPhe were determined as follows: 5 (6% modified), 31 (2%), 35 (36%), 67 (5%), and 76 (51%). Thus mainly the terminal adenosine and one adenosine in the anticodon loop bear the 2,4-dinitrophenyl residue.

Base Sequence↗
PubMed1978

Aminoacyl-tRNA synthetases from yeast: generality of chemical proofreading in the prevention of misaminoacylation of tRNA.

The specificity of valyl-, phenylalanyl-, and tyrosyl-tRNA synthetases from yeast has been examined by a series of stringent tests designed to eliminate the possibility of artefactual interference. Valyl-tRNA synthetase, as well as activating a number of amino acid analogues, will accept alanine, cysteine, isoleucine, and serine in addition to threonine as substrates for both ATP-PPi exchange and transfer to some tRNAVal species. The transfer is not observed if atempts are made to isolate the appropriate aminoacyl-tRNAVal-C-C-A but its role in the overall aminoacylation can be suspected from both the formation of a stable aminoacyl-tRNAVal-C-C-A(3'NH2) compound and from the stoichiometry of ATP hydrolysis during the aminoacylation of the native tRNA. Similar tests with phenylalanyl-tRNA synthetase indicate that this enzyme will also activate and transfer other naturally occurring amino acids, namely, leucine, methionine, and tyrosine. The tyrosine enzyme, which lacks the hydrolytic capacity of the other two enzymes (von der Haar, F., & Cramer, F (1976) Biochemistry 15, 4131--4138) is probably absolutely specific for tyrosine. It is concluded that chemical proofreading, in terms of an enzymatic hydrolysis of a misacylated tRNA, plays an important part in maintaining the specificity in the overall reaction and that this activity may be more widespread than has so far been suspected.

Amino Acids↗
PubMed1978

Threonyl-tRNA synthetase from yeast: aminoacylation of tRNA on its non-accepting 3'-terminal hydroxyl group and its behaviour in enzyme-catalyzed deacylation.

Methods have been developed by which tRNA Thr may be aminoacylated at the normally non-accepting 3'-terminal ribose OH. Two of the methods utilize the mischarging ability of the synthetases under special conditions of low salt concentration and presence of organic solvents. The third method demonstrates for the first time that for some synthetases the 2',3' specificity may be manipulated by use of similar special conditions. In the case of threonyl-tRNA synthetase, Thr-tRNAThr-C-C-A(3'd) has been synthesised by this method. The behaviour of threonyl esters of tRNAThr-C-C-A, tRNAThr-C-C-A(2'd) and tRNA Thr-C-C-A-(3'd) in the free enzyme-catalyzed deacylation has been studied and the results indicate that the cis diol functional group is necessary for this hydrolysis. The position on the terminal ribose from which the amino acid is removed in this reaction remains to be identified.

Adenosine Monophosphate↗
PubMed1978

Threonyl-tRNA, lysyl-tRNA and arginyl-tRNA synthetases from Baker's yeast. Substrate specificity with regard to ATP analogues.

Sixteen analogues of ATP have been tested in the aminoacylation reaction of threonyl-tRNA, lysyl-tRNA, and arginyl-tRNA synthetases from baker's yeast. Two compounds are substrates for threonyl-tRNA and for lysyl-tRNA synthetases and five compounds for arginyl-tRNA synthetase. There are six inhibitors for threonyl-tRNA, nine for lysyl-tRNA, and six for arginyl-tRNA synthetase. Their Km and Ki values have been determined. Thus positions 2, 6, 7, 8 and 9 of the purine moiety and 2' and 3' of the sugar moiety of the ATP molecule are important for catalytic action of these aminoacyl-tRNA synthetases. Remarkably arginyl-tRNA synthetase is the first aminoacyl-tRNA synthetase which tolerates bulky substituents at the sugar moiety of ATP. These data fit with the idea that synthetases of subunit structure need magnesium-ion-ATP complexes with an anti conformation as substrates whereas single-chain enzymes accept this substrate in the syn conformation.

Adenosine Triphosphate↗
PubMed1978

Photolabile and paramagnetic derivatives of the nucleoside X and of Escherichia coli tRNAPhe.

The synthesis of N3-[3-L-(5-azido-2-nitrobenzamido)-3-carboxypropyl]uridine (4b) and N3-[3-carboxy-3-L-(2,2,5,5-tetramethyl-3-pyrroline-3-carbonylamino)propyl]uridine Npyr-oxyl (4c) starting from the nucleoside X (4a) and the appropriate N-hydroxysuccinimide ester 1 or 2 is described. After acylation of tRNAPhe from E. coli (5a) with 1 or 2, the photolabile tRNAPhe derivative 5b and the paramagnetic tRNAPhe derivative 5c could be isolated. The position of modification in the polynucleotide chain was elucidated by comparison of the ribonuclease II/alkaline phosphatase digestion products of the substituted and unsubstituted tRNAPhe samples, and was identified as being exclusively the amino group of the nucleoside X in position 47 of E. coli tRNAPhe.

Aminobutyrates↗
PubMed1977

Specificity of elongation factor Tu from Escherichia coli with respect to attachment to the amino acid to the 2' or 3'-hydroxyl group of the terminal adenosine of tRNA.

Modified Tyr-tRNATyr and Phe-tRNAPhe species from yeast having the aminoacyl residue bound specifically to the 2' and 3' position of the terminal adenosine, respectively, were investigated for their ability to form ternary complexes with Escherichia coli elongation factor Tu and GTP. Both Tyr-tRNATyr-CpCpA (2'd) and Tyr-tRNATyr-CpCpA(3' d) derivatives which are esterified with the amino acid on the 3' and 2' position respectively and which lack the vicinal hydroxyl were able to form ternary complexes. The stability of these ternary complexes was lower than in the case of native Tyr-tRNATyr-CpCpA. Tyr-tRNATyr-CpCpA(3' d) having the amino acid attached to the 2' position interacted considerably more strongly with EF-Tu - GTP than Tyr-tRNATyr-CpCpA(2' d). Ternary complex formation was observed with neither Phe-tRNAPhe-CpCpA(2'NH2) nor Phe-tRNAPhe-CpCpA(3'NH2). It is concluded that 2' as well as 3' isomers of native aminoacyl-tRNA can be utilized for ternary complex formation but in a following step a uniform 2'-aminoacyl-tRNA - EF-Tu - GTP complex is formed. Although the free vicinal hydroxyl group of the terminal adenosine is not absolutely required, replacement of the ester linkage through with the amino acid is attached to tRNA by an amide linkage leads to loss of ability to interact with elongation factor Tu.

Adenosine↗
PubMed1977

On the stereochemistry of activation of phenylalanine by phenylalanyl-tRNA synthetase from baker's yeast.

Because of its chiralic alpha-phosphorus atom adenosine 5'-O-(1-thiotriphosphate) (ATPalphaS) exists in two diastereomeric forms, arbitrarily named (A) and (B). For phenylalanyl-tRNA synthetase ATPalphaS (A) is a substrate whereas ATPalphaS (B) is neither a substrate nor an inhibitor. During the ATPalphaS (A)/PPi exchange reaction with phenylalanyl-tRNA synthetase the configuration at the alpha-phosphorus is retained. The mechanistic implications of these findings are discussed. Preliminary investigations with several other aminoacyl-tRNA synthetases show that the stereochemical requirement with respect to the alpha-phosphorus of ATP is not identical for all aminoacyl-tRNA synthetases.

Adenosine Triphosphate↗
PubMed1977

Tyroslyl-tRNA synthetase from baker's yeast. Rapid isolation by affinity elution, molecular weight of the enzyme, and determination of essential sulfhydryl groups.

Tyrosyl-tRNA synthetase (EC 6.1.1.1) has been isolated from baker's yeast with an overall purification factor of more than 5000. After opening the cells, pH 4.8 precipitation, ammonium sulfate fractionation, removal of the nucleic acids with DEAE-cellulose and chromatography on CM-Sephadex, the critical purification step is the elution of the cation-exchanger-bound tyrosyl-tRNA synthetase with tRNATyr. The homogeneous enzyme exhibits a molecular weight of 40 000 as estimated by sedimentation equilibrium centrifugation and dodecylsulfate-gel electrophoresis under reducing and non-reducing conditions. Gel filtration experiments show a molecular weight of about 100 000 indicating the existence of an active dimeric form. The possibility of proteolytic cleavage of the enzyme is excluded. The reaction of tyrosyl-tRNA synthetase with p-chloromercuribenzoate and N-ethylmaleimide reveals two repidly reacting sulfhydryl groups per subunit of molecular weight 40 000, as demonstrated by the inhibition of aminoacylation and the isolation of enzyme-inhibitor complexes. In addition an efficient purification method is described for isolating tRNATyr from soluble ribonucleic acid from baker's yeast in three chromatographic steps in a yield of 28%.

Amino Acyl-tRNA Synthetases↗