DETERMINATION OF 2'-HYDROXYL AND PHOSPHATE GROUPS IMPORTANT FOR AMINOACYLATION OF ESCHERICHIA-COLI TRNA(ASP) - A NUCLEOTIDE ANALOG INTERFERENCE STUDY

2'-Deoxynucleoside 5'-alpha-thiotriphosphates have been incorporated r andomly, replacing any of the four nucleotides separately and at a low level in Escherichia coli tRNA(Asp) transcripts. After some tRNAs wer e charged with the cognate aminoacyl-tRNA synthetase and biotinylated, charged and uncharged tRNAs were separated by binding to Streptavidin . A comparison of the iodine cleavage pattern of charged and uncharged tRNAs indicated positions of 2'-deoxyphosphorothioate interference wi th charging. To separate the 2'-deoxy from the phosphorothioate effect , the same sequence of reactions was performed with the corresponding NTP alpha S. Several positions were identified with a 2'-deoxy or a ph osphorothioate effect. tRNAs with single deoxy substitutions at the id entified positions were prepared by enzymatic ligation of chemically s ynthesized halves. The kinetics of charging these tRNAs were determine d. The 2'-deoxy effects identified by the interference assay were conf irmed, showing a reduction in charging efficiency of between 2.5-6-fol d, except for the terminal A76 with a 25-fold reduction. Inspection of the X-ray structure of the tRNA-synthetase complex showed consistency of most of these findings. Critical 2'-deoxy groups are localized mai nly on the proposed contact surface with the synthetase or at the inte rface of the two tRNA domains. The same overall picture emerged for cr itical phosphorothioates. With the exception of 2'-deoxy-adenosine-con taining tRNAs, multiple 2'-deoxy-substituted tRNAs, prepared by ligati on of halves, showed a much larger reduction in charging efficiency th an the mono-substituted tRNAs, indicating an additive effect.