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Nucleic Acids Research, 1991, Vol. 19, No. 23 6573-6578
© 1991

MOLECULAR BIOLOGY

Use of Single-stranded DNA oligonucleotides in programming ribosomes for translation

Robert D. Ricker and Akira Kaji*

University of Pennsylvania, School of Medicine, Department of Microbiology Philadelphia, PA 19104–6076, USA

*To whom correspondence shoul be addressed

Received July 17, 1991. Revised November 5, 1991. Accepted November 5, 1991.

Single-stranded DNA (ssDNA) oligomers were compared to synthetic RNA oligomers in their ability to program E. coli ribosomes in vitro. AUG and dATGcontaining 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 transferasedependent 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.


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