Subject(s)
RNA, Transfer/isolation & purification , Amino Acids , Chromatography, High Pressure Liquid/methods , Escherichia coli/chemistry , Fluorenes , Hydrophobic and Hydrophilic Interactions , RNA, Bacterial/isolation & purification , RNA, Transfer/chemistry , RNA, Transfer, Ala/isolation & purification , RNA, Transfer, Amino Acid-Specific/isolation & purificationABSTRACT
The relationship between tRNA structure and function has been widely investigated by site-directed mutagenesis. This method has been a very useful tool to reveal the critical bases in tRNAs that are important for recognition and aminoacylation, but has been limited by the large number of possible base combinations in tRNA molecules. We have devised a new method that uses tRNA knockout cells for selection of functional tRNAs from a mutant tRNA gene library to overcome this limitation. To explore the mechanism of tRNA(Ala) recognition, the bases of the acceptor-stem region were randomized and active mutants were selected in a tRNA(Ala) knockout strain. Mutants of tRNA(Ala) having diverse sequence combinations in the acceptor-stem region and a broad range of functional activity to support knockout cell growth were isolated. The mutant tRNAs selected by the method included molecules containing novel base substitutions as well as extensively altered base combinations that would not be readily generated by rationally designed site-directed mutagenesis. Our results emphasize the importance of the acceptor stem as a structural unit in which some nucleotides may carry more weight than others, but in summation every nucleotide contributes to the interaction with the enzyme.
Subject(s)
Escherichia coli/genetics , RNA, Transfer, Ala/genetics , RNA, Transfer, Ala/isolation & purification , Amino Acyl-tRNA Synthetases/metabolism , Base Sequence , Cloning, Molecular , Escherichia coli/cytology , Escherichia coli/metabolism , Gene Library , Models, Structural , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Oligoribonucleotides/chemical synthesis , Oligoribonucleotides/metabolism , Plasmids/genetics , RNA, Transfer, Ala/metabolism , Substrate SpecificityABSTRACT
The G.U wobble base-pair in the acceptor helix of Escherichia coli tRNAAlais critical for aminoacylation by the alanine synthetase. Previous work by several groups probed the mechanism of enzyme recognition of G.U by a structure-function analysis of mutant tRNAs using either a cell assay (amber suppressor tRNA) or a test tube assay (phage T7 tRNA substrate and purified enzyme). However, the aminoacylation capacity of particular mutant tRNAs was about 10(4)-fold higher in the cell assay. This led us to scrutinize the cell assay to determine if any parameter exaggerates the extent of aminoacylation in mutants forming substantial amounts of alanyl-tRNAAla. In doing so, we have refined and developed experimental designs to analyze tRNA function. We examined the level of aminoacylation of amber suppressor tRNAAlawith respect to the method of isolating aminoacyl-tRNA, the rate of cell growth, the cellular levels of alanine synthetase and elongation factor TU (EF-Tu), the amount of tRNA and the characteristics of EF-Tu binding. Within the precision of our measurements, none of these parameters varied in a way that could significantly amplify cellular alanyl-tRNAAla. A key observation is that the extent of aminoacylation of tRNAAlawas independent of tRNAAlaconcentration over a 75-fold range. Therefore, the cellular assay of tRNAAlareflects the substrate quality of the molecule for formation of alanyl-tRNAAla. These experiments support the authenticity of the cellular assay and imply that a condition or factor present in the cell assay may be absent in the test tube assay.
Subject(s)
RNA, Transfer, Ala/metabolism , RNA, Transfer, Amino Acyl/metabolism , Acylation , Alanine-tRNA Ligase/metabolism , Base Sequence , Blotting, Northern , Escherichia coli/cytology , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/growth & development , Genes, Suppressor/genetics , Guanosine Triphosphate/metabolism , Lysine/analysis , Mutation , Peptide Elongation Factor Tu/metabolism , Protein Binding , RNA, Bacterial/genetics , RNA, Bacterial/isolation & purification , RNA, Bacterial/metabolism , RNA, Transfer, Ala/genetics , RNA, Transfer, Ala/isolation & purification , RNA, Transfer, Amino Acyl/genetics , RNA, Transfer, Amino Acyl/isolation & purification , Reproducibility of Results , Structure-Activity Relationship , Suppression, GeneticABSTRACT
The complete chemical synthesis of an E. coli tRNA(Ala) with its specific minor nucleosides, dihydrouridine, ribothymidine and pseudouridine, is reported. The method makes use of protected 2'-O-tertiobutyldimethylsilyl-ribonucleoside-3'-O-(2-cyanoethyl-N- ethyl-N- methyl)phosphoramidites. The exocyclic amino functions of the bases were protected by the phenoxyacetyl group for purines and acetyl for cytosine. The assembling has been performed on a silica support with coupling yield better than 98% within 2 min of condensation. Triethylamine tris-hydrofluoride allowed a clean and complete deprotection of the tBDMS groups. The synthetic tRNA(Ala) has been transcribed into cDNA by reverse transcriptase and sequenced. With E. coli alanyl-tRNA synthetase the alanyl acceptance activity and kcat/Km were 672 pmol/A260 and 6 x 10(4)M-1s-1, respectively.
