Joining of yeast alanine transfer ribonucleic acid half molecules to form a whole molecule by T4 RNA ligase

Wang, Gui-hai; Zhu, Li; Yuan, Jia-Gui; Liu, Fang; Zhang, Lanfang

doi:10.1016/0005-2787(81)90211-2

Cited by 10 publications

(2 citation statements)

References 5 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Particularly strong evidence indicated a role for the nucleotide base at the wobble position of the anticodon in the discrimination process. T4 RNA ligase has recently been used for the synthesis of normal and variant RNA sequences for a variety of structure-function studies (2)(3)(4)(5)(6)(7)(8)(9)(10)(11). In this paper, we report the synthesis of tRNAfMet molecules containing C,U,A and G in the wobble position of the anticodon using RNA ligase and examine the effect of base substitutions at this site on recognition of the tRNA by its cognate aminoacyl-tRNA synthetase.…”

Section: Introductionmentioning

confidence: 99%

Base substitutions in the wobble position of the anticodon inhibit aminoacylation of E. colitRNAfMet by E. coli Met-tRNA synthetase

Schulman¹,

Pelka²,

Susani³

1983

Nucleic Acids Research

View full text Add to dashboard Cite

Derivatives of E. coli tRNAfMet containing single base substitutions at the wobble position of the anticodon have been enzymatically synthesized in vitro. The procedure involves excision of the normal anticodon, CAU, by limited digestion of intact tRNAfMet with RNase A. RNA ligase is then used to join each of four trinucleotides, NAU, to the 5' half molecule and to subsequently link the 3' and modified 5' fragments to regenerate the anticodon loop. Synthesis of intact tRNAfMet containing the anticodon CAU by this procedure yields a product which is indistinguishable from native tRNAfMet with respect to its ability to be aminoacylated by E. coli methionyl-tRNA synthetase. Substitution of any other nucleotide at the wobble position of tRNAfMet drastically impairs the ability of the synthetase to recognize the tRNA. Measurement of methionine acceptance in the presence of high concentrations of pure enzyme has established that the rate of aminoacylation of the AAU, GAU and UAU anticodon derivatives of tRNAfMet is four to five orders of magnitude slower than that of the native or synthesized tRNA containing C as the wobble base. In addition, the inactive tRNA derivatives fail to inhibit aminoacylation of normal tRNAfMet, indicating that they bind poorly to the enzyme. These results support a model involving direct interaction between Met-tRNA synthetase and the C in the wobble position during aminoacylation of tRNAfMet.

show abstract

Section: Introductionmentioning

confidence: 99%

Base substitutions in the wobble position of the anticodon inhibit aminoacylation of E. colitRNAfMet by E. coli Met-tRNA synthetase

Schulman¹,

Pelka²,

Susani³

1983

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…After autoradiography terminal nucleosides were identified by comparison with unlabelled nucleoside 5' or 3'-monophosphates. The addition of p u p [IS] and the dephosphorylation with T4 polynucleotide kinase [6] were conducted on the halfmolecule after reannealing [16] to the TI fragment, previously blocked by the addition of pCp at the 3' OH end. The S1 fragment with an additional nucleoside (U,,-OH) was isolated by polyacrylamide gel electrophoresis as above.…”

Section: Construction Of Trnacys (Anticodon Uca)mentioning

confidence: 99%

Construction of a UGA suppressor tRNA by modification in vitro of yeast tRNA^Cys

Vacher

Grosjean

Henau

et al. 1984

European Journal of Biochemistry

View full text Add to dashboard Cite

In this paper we describe the construction of a yeast tRNACys UGA suppressor. After specific hydrolysis of the parent molecule, the first base of the anticodon GCA was replaced by a uracil. The resulting molecule, harboring a UCA anticodon, was injected into Xenopus laevis oocytes in order to test its biological activities. The level of aminoacylation was similar to that of the parent molecule. Readthrough of the UGA termination codon in /?-globin mRNA, coinjected with the tRNA, indicated suppressor activity; however, tRNACys (anticodon UCA) was a much less efficient suppressor than others tested under the same conditions. We see no post-transcriptional modification of the uracil in the anticodon wobble position after injection into oocytes. This may be related to the low suppressor activity; however, it is also possible that other features of tRNACyS structure may be unadapted to efficient UCA anticodon function.Several lines of evidence now suggest that the overall functional efficiency of a tRNA molecule in protein synthesis is dependent on the structure of the whole of the molecule and its relation to the anticodon. Thus Gorini, observing the varying efficiencies of different nonsense suppressors remarked [I] "it appears that a 'correct' anticodon obtained by mutation can be out of context in the resulting tRNA suppressor molecule". In addition to the occurrence of invariant nucleotides in the tRNA molecule, it is known that base usage in other positions is often far from random [2]. One of the most versatile ways of investigating the importance of particular bases or regions of the molecule is to construct tRNAs with modified primary structure. This may be done either by modification of the DNA or the RNA sequence. Temple et al. have recently reported the construction of an active suppressor tRNALy" by site-directed mutagenesis of the gene [3]. By modification of the RNA sequence, Bruce et al. have succeeded in synthesising a biologically active amber suppressor by modification at the RNA level of yeast tRNAPhe [4]. Here we describe the application of the RNA mutagenic technology to the construction of a potential UGA suppressor with anticodon UCA, by base substitution in yeast tRNACyS. This methodology, dependent largely on the properties of RNA ligase and polynucleotide kinase from phage T4 [5, 61, was initially developed by the group of Uhlenbeck, and applied successfully to the modification of several yeast tRNAs: tRNAPhe [4], tRNAASp [8] and tRNAkg [9].Essentially three reasons led us to select yeast tRNACy" as an interesting species for modification. Among the anticodon changes that may be easily introduced, one should lead to a UGA suppressor species, the activity of which may be readily assayed in vivo as well as in vitro, The normal tRNACYs anticodon, GCA, is conveniently split by ribonuclease TI. Finally, the availability of high specific activity [35S]cysteine should facilitate characterisation of polypeptides synthesized in vivo or in vitro.Enzymes. RNAse TI (EC 3.1.27.3); nuclease S1 (EC 3.1.30...

show abstract

Effect of the anticodon loop size of yeast alanyl tRNA on its biological activity

Jin

Qiu

et al. 1987

Analytical Biochemistry

View full text Add to dashboard Cite

Joining of yeast alanine transfer ribonucleic acid half molecules to form a whole molecule by T4 RNA ligase

Cited by 10 publications

References 5 publications

Base substitutions in the wobble position of the anticodon inhibit aminoacylation of E. colitRNAfMet by E. coli Met-tRNA synthetase

Base substitutions in the wobble position of the anticodon inhibit aminoacylation of E. colitRNAfMet by E. coli Met-tRNA synthetase

Construction of a UGA suppressor tRNA by modification in vitro of yeast tRNA^Cys

Effect of the anticodon loop size of yeast alanyl tRNA on its biological activity

Contact Info

Product

Resources

About

Joining of yeast alanine transfer ribonucleic acid half molecules to form a whole molecule by T4 RNA ligase

Cited by 10 publications

References 5 publications

Base substitutions in the wobble position of the anticodon inhibit aminoacylation of E. colitRNAfMet by E. coli Met-tRNA synthetase

Base substitutions in the wobble position of the anticodon inhibit aminoacylation of E. colitRNAfMet by E. coli Met-tRNA synthetase

Construction of a UGA suppressor tRNA by modification in vitro of yeast tRNACys

Effect of the anticodon loop size of yeast alanyl tRNA on its biological activity

Contact Info

Product

Resources

About

Construction of a UGA suppressor tRNA by modification in vitro of yeast tRNA^Cys