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At least 19 recordsLinked to original sources
PubMed1978

Incomplete aminoacylation of tRNALeu catalyzed in vitro by leucyl-tRNA synthetase from Escherichia coli B.

The extent of esterification of [14C] leucine into Escherichia coli B tRNALeu apparently depends on the concentration of leucyl-tRNA synthetase. The effect is more pronounced at pH 9.0 than at pH 7.4. When reciprocals of leucyl-tRNA concentration at plateau [aa-tRNA]-1 are plotted against reciprocals of initial velocities vo-1 of aminoacylations a straight line is obtained with a slope equal to the rate constant of non-enzymatic deacylation of leucyl-tRNA. Factors which change the stability of leucyl-tRNA, e.g. pH and temperature, also change the shape of the function [aa-tRNA]-1 vs. vo-1. The data are consistent with the idea that the rate constant of spontaneous deacylation of aminoacyl-tRNA is the factor which accounts for the dependence of the level of aminoacylation on initial velocity of aminoacylation.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Initiation of polypeptide synthesis with various NH2-blocked aminoacyl-tRNAs under the direction of alfalfa mosaic virus RNA 4.

Initiation of polypeptide synthesis in a cell-free system of Escherichia coli directed by alfalfa mosaic virus RNA 4 was studied by using either fMet-tRNA or Ac-Phe-tRNA as initiator tRNA. Initiation with fMet-tRNA yielded a product that was identical to the authentic viral coat protein except that the NH2-terminal serine was preceded by fMet instead of being acetylated. When Ac-Phe-tRNA was used as initiator, the biosynthetic product was 10-12 amino acid residues longer, the extra amino acids being located at the NH2-terminus. fMet-tRNA and Ac-Phe-tRNA did not compete for ribosomes during initiation of protein synthesis, as became evident from incorporation studies using both initiator tRNAs simultaneously. It is concluded that E. coli ribosomes recognize two sites on the 5' end of alfalfa mosaic virus RNA 4 that are separated by a region of about 30 nucleotides. The results are in complete agreement with the 5'-terminal nucleotide sequence of this RNA [Koper-Zwarthoff, E. C., Lockhard, R. E., RajBhandary, U. L., Alzner-deWeerd, B. & Bol, J. F. (1977) Proc. Natl. Acad. Sci. USA 74, 5504-5508].

Cell-Free System↗
PubMed1978

"Chemical aminoacylation" of tRNA's.

Incubation of abbreviated tRNA's (tRNA-C-COH's) with (chemically) preaminoacylated P1, P2-di(adenosine 5'-)diphosphates in the presence of purified RNA ligase effected transfer of an aminoacyladenylate moiety to the 3'-terminus of the abbreviated tRNA's in good yield. Aminoacylated (or misacylated) tRNA's may thus be prepared from fractionated or unfractionated tRNA-C-COH's; each of the five aminoacylated dinucleoside diphosphates tested was utilized as a substrate by RNA ligase. That the resulting "chemically aminoacylated" tRNA's were identical with those prepared by enzymatic aminoacylation was judged by comparison of 1) chromatographic properties on benzolated diethylaminoethyl-cellulose, 2) rates of chemical deacylation, and 3) affinities for elongation factor Tu, as well as 4) the ability of misacylated tRNA's so derived to be deacylated chemically and then reactivated enzymatically with their cognate amino acids.

Adenine Nucleotides↗
PubMed1979

Transfer RNA control of the activation of isomeric tRNATrp's.

Previous studies of the homologous aminoacylations of Escherichia coli and yeast tRNATrp's terminating in 2'- and 3'-deoxyadenosine established that E. coli tryptophanyl-tRNA synthetase activates its cognate tRNA preferentially on the 2' position, while the corresponding yeast enzyme utilizes the 3' position on its homologous substrate tRNA. As this seemed to be the only change in positional specificity during evolution, the heterologous activations were investigated in an effort to determine the basis for this change. Remarkably, E. coli tRNATrp terminating in 3'-deoxyadenosine was found to be the preferred substrate for both the E. coli and yeast activating enzymes, while the same tryptophanyl-tRNA synthetase preparations both activated the isomeric yeast tRNATrp's preferentially on the 3' position. Thus, the preferred position of activation was found to be specified by the tRNA rather than the activating enzyme and, additionally, to be due to some process not reflected in initial velocity measurements. The variable utilization of individual modified aminoacyl-tRNA's as substrates in an enzyme-catalyzed deacylation process appears to provide the most likely explanation for the experimental observations.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Energy cost of proofreading to increase fidelity of transfer ribonucleic acid aminoacylation.

The paradox of relatively error free function in biological systems composed of relatively error prone components has recently come under intensive investigation. In the case of tRNA aminoacylation, aminoacyl-tRNA synthetases were discovered to have a separate function that allows misacylated molecules to be hydrolyzed more rapidly than correctly acylated molecules. This additional function of the synthetases provides a proofreading or verification mechanism that is believed to improve significantly the overall accuracy of tRNA aminoacylation. In this paper we provide an explicit relationship between the accuracy achieved by proofreading and the energy cost. Experimental data available in the literature are examined in light of this relationship. The following are the principal conclusions from our study: (1) high-accuracy proofreading of tRNA aminoacylation has a high energy cost, as much as 100 times greater than indications from early experimental work; (2) the minimum net error derived in previous theoretical studies is never actually reached; (3) mechanisms in which misacylation and subsequent proofreading occur on the surface of the same synthetase molecule achieve a much higher accuracy than mechanisms in which these functions occur on the surface of different synthetase molecules.

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

Studies on transfer RNA from mycobacteria.

Active preparations of tRNA and aminoacyl-tRNA synthetases have been isolated from exponentially growing cells of Mycobacterium smegmatis and Mycobacterium tuberculosis H37Rv. Though the aminoacyl-tRNA synthetases of older cells retain their activity, the tRNAs seem to undergo modification and show poorer activity. The mycobacterial enzyme preparations catalyse homologous and heterologous aminoacylation between tRNA from the two species (M. smegmatis and M. tuberculosis H37Rv) or from Escherichia coli, with equal efficiency; tRNA samples from eukaryotic cells (yeast and rat liver) do not serve as substrates for the mycobacterial synthetases. The analytical separation of the different amino acid specific tRNAs from M. smegmatis resembles the pattern found in other bacteria. Purification of valine- (three species) and methionine-specific tRNA (two species) to 70-80% purity has been accomplished by using column-chromatographic techniques. Of the two species of tRNAMet, one can be formylated in the presence of formyl tetrahydrofolate and the transformylase from mycobacteria.

Amino Acyl-tRNA Synthetases↗
PubMed1978

The mechanism of the aminoacylation of transfer ribonucleic acid. The kinetics and stoichiometry of the lysis of aminoacyl-tRNA.

It is often stated that the aminoacylation of transfer RNA proceeds in discrete steps: (formula: see article). If this is a complete description of the reaction, the reverse overall formation of ATP should not be more rapid than the formation of Enz . (AA approximately AMP). We show for four different amino acid:tRNA ligases that lysis of AA-tRNA (with PPi and AMP) to ATP is faster than lysis of AA-tRNA (with AMP only) to Enz . (AA approximately AMP). This requires that the transition state proceeds from a quaternary complex of PPi, AMP, AA-tRNA and Enz. From the law of microscopic reversibility, this requires that in the forward reaction the AA-tRNA bond be formed before PPi leaves the enzyme complex. Therefore, the forward reaction passes through the quaternary complex Enz . ATP . AA . tRNA. (In view of recent evidence of the specific requirement of two cations, the complex is accurately described as senary).

Adenosine Monophosphate↗
PubMed1978

Aminoacylation of undermethylated mammalian transfer RNA.

To study the role of 5-methylcytidine in the aminoacylation of mammalian tRNA, bulk tRNA specifically deficient in 5-methylcytidine was isolated from the livers of mice treated with 5-azacytidine (18 mg/kg) for 4 days. For comparison, more extensively altered tRNA was isolated from the livers of mice treated with DL-ethionine (100 mg/kg) plus adenine (48 mg/kg) for 3 days. The amino acid acceptor capacity of these tRNAs was determined by measuring the incorporation of one of eight different 14C-labeled amino acids or a mixture of 14C-labeled amino acids in homologous assays using a crude synthetase preparation isolated from untreated mice. The 5-methylcytidine-deficient tRNA incorporated each amino acid to the same extent as fully methylated tRNA. The tRNA from DL-ethionine-treated livers showed an overall decreased amino-acylation capacity for all amino acids tested. The 5-methylcytidine-deficient tRNA from DL-ethionine-treated mice were further characterized as substrates in homologous rate assays designed to determine the Km and V of the aminoacylation reaction using four individual 14C-labeled amino acids and a mixture of 14C-labeled amino acids. The Km and V of the reactions for all amino acids tested using 5-methylcytidine-deficient tRNA as substrate were essentially the same as for fully methylated tRNA. However, the Km and V were increased when liver tRNA from mice treated with DL-ethionine plus adenine was used as substrate in the rate reaction with [14C]lysine as label. Our results suggest that although extensively altered tRNA is a poorer substrate than control tRNA in both extent and rate of aminoacylation, 5-methylcytidine in mammalian tRNA is not involved in the recognition of the tRNA by the synthetase as measured by aminoacylation activity.

Amino Acids↗
PubMed1979

Transfer RNATyr of melanoma tissues and cells: relevance to melanin synthesis?

The tRNA present in swine melanoma tumor tissue and normal gray skin tissue were compared by aminoacylation of the unfractionated tRNA preparations. Of the seventeen amino acids studied, seven showed differences in rate of acceptance to tRNAs from normal and tumor tissues; the tRNAs of two amino acids, tyrosine and glycine, showed dramatic three fold increases in melanoma tumor. As melanin biosynthesis proceeds from tyrosine oxidation the investigations focused on the increase in tyrosine tRNA. Kinetic analysis of tyrosine aminoacylation to normal and melanoma tRNAs revealed no differences. Analysis of the isoaccepting species of tRNATyr from normal skin and melanoma tumor tissues identified three isoacceptors; tRNATyr, represented the predominant species in normal gray skin, while tRNA2Tyr predominated in melanoma tumor tissue. The tyrosine acceptances by tRNAs from three human melanoma cell lines were analyzed and found to be variable, but isoaccepting species analysis of the tRNATyr of these three cell lines still showed a correlation between the preponderance of tRNA2Tyr and extent of tyrosine acceptance. Additionally the enzymatic activity for the oxidation of tyrosine was found to be related to tyrosine acceptance and tRNA2Tyr predominance..

Amino Acyl-tRNA Synthetases↗