Use of single-stranded DNA oligonucleotides in programming ribosomes for translation.
Single-stranded DNA (ssDNA) oligomers were compared to synthetic RNA oligomers in their ability to program E. coli ribosomes in vitro. AUG and dATG-containing oligomers promoted the non-enzymatic binding of fmet-tRNA to ribosomes, with similar dependence on time and magnesium concentration; only at 10 mM Mg++ or at low oligomer concentration was RNA slightly preferred in complex formation. These initiation complexes were biologically active in that fmet-tRNA, bound in response to ssDNA or RNA, was fully reactive with puromycin. While dAUG could not function as an initiation codon, p-dAUG functioned as well as AUG or dATG. However, dUAA and p-dUAA could not replace UAA in directing release-factor (RF) activity, and dTAA functioned only to a slight extent. Release factors had specificity for termination complexes containing dATGTAA, dATGTAG, or dATGTGA. At Mg++ concentrations of 15 mM or higher, these hexamers directed peptidyl transferase-dependent fmet-tRNA hydrolysis in the absence of RF. We suggest this RF-independent activation of peptidyl transferase as a unique system for studying the mechanism of termination. Overall, these results indicate that ssDNA can be used in place of RNA for certain studies of protein synthesis.
Ricker
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RD
University of Pennsylvania, School of Medicine, Department of Microbiology, Philadelphia 19104-6076.
Kaji
A
A
D003062:Codon; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000378:metabolism; D010442:Peptide Chain Initiation, Translational; D010458:Peptidyl Transferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013728:Terminator Regions, Genetic
D003062:Codon; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; D012358:RNA, Transfer, Met; D012313:RNA; D010458:Peptidyl Transferases; D008274:Magnesium