ABSTRACT
A combination of hydrophobic chromatography on phenyl-Sepharose and reversed phase HPLC was used to purify individual tRNAs with high specific activity. The efficiency of chromatographic separation was enhanced by biochemical manipulations of the tRNA molecule, such as aminoacylation, formylation of the aminoacyl moiety and enzymatic deacylation. Optimal combinations are presented for three different cases. (i) tRNA(Phe) from Escherichia coli. This species was isolated by a combination of low pressure phenyl-Sepharose hydrophobic chromatography with RP-HPLC. (ii) tRNA(Ile) from E. coli: Aminoacylation increases the retention time for this tRNA in RP-HPLC. The recovered acylated intermediate is deacylated by reversion of the aminoacylation reaction and submitted to a second RP-HPLC run, in which deacylated tRNA(Ile) is recovered with high specific activity. (iii) tRNA(i)(Met) from Saccharomyces cerevisiae. The aminoacylated form of this tRNA is unstable. To increase stability, the aminoacylated form was formylated using E.coli: enzymes and, after one RP-HPLC step, the formylated derivative was deacylated using peptidyl-tRNA hydrolase from E.COLI: The tRNA(i)(Met) recovered after a second RP-HPLC run exhibited electrophoretic homogeneity and high specific activity upon aminoacylation. These combinations of chromatographic separation and biochemical modification can be readily adapted to the large-scale isolation of any particular tRNA.
Subject(s)
Chromatography, High Pressure Liquid/methods , Chromatography/methods , RNA, Transfer/isolation & purification , Acylation , Carboxylic Ester Hydrolases/isolation & purification , Carboxylic Ester Hydrolases/metabolism , Chromatography, Agarose , Escherichia coli/genetics , RNA, Bacterial/isolation & purification , RNA, Fungal/isolation & purification , RNA, Transfer/chemistry , RNA, Transfer, Ile/isolation & purification , RNA, Transfer, Met/isolation & purification , RNA, Transfer, Phe/isolation & purification , Saccharomyces cerevisiae/genetics , Sensitivity and Specificity , Time FactorsABSTRACT
A yeast 50-kDa mRNA-binding protein (50mRNP) is found selectively associated with the 48S and 80S initiation complexes. This protein is structurally related to the translational elongation factor EF-1alpha. The protein reacts with antibodies directed against EF-1alpha and, similarly, EF-1alpha recognizes antibodies against the 50mRNP protein. This is evidence that they share at least one epitope which allows a similar antigenic behavior. In addition, both proteins show similar cleavage patterns upon treatment with the endoproteinase Lys-C. A murine antibody raised against 50mRNP inhibits both 48S and 80S initiation complex formation. The inhibitory effect is relieved by preincubating anti-50mRNP with EF-1alpha. Antibody to EF-1alpha manifests a similar inhibitory pattern for the formation of 48S and 80S complexes. These data strongly suggest that 50mRNP is an EF-1alpha-like polypeptide essential for the formation of the above complexes.
