Role of hypermodified bases in transfer RNA. Solution properties of dinucleoside monophosphates.

The hypermodified dinucleoside monophosphates, uridylyl(3’-5’)-N-[9-(~-~-ribofuranosyl)purin-6ylcarbamoyl]threonine (Upt6A), adenylyl(3’-5’)-N6-(A2isapenteny1)adenosine (ApihA), adenylyl( 3’-5’)-N6-( A2-isopentenyl)-2-methylthioadenosine (Apms2i6A), and adenylyl(3’-5’)-l ,N6-ethenoadenosine (AptA, a synthetic model for adenylyl(3’-5’)wybutosine, ApyW), which represent the most common sequences found as the third letter of the anticodon triplet and its adjacent 3’ neighbor, have been isolated. Their solution properties have been investigated using ultraviolet absorption, circular dichroism (CD), and high resolution proton magnetic resonance. The properties of these molecules have been compared with those of their unmodified counterparts, uridylyl( 3’-5’)adenosine (UpA) and adenylyl(3’-5’)adenosine (ApA). These properties measured as a function of temperature have been analyzed employing a two-state intramolecular stacking model. All of the properties show that the stacking of Upt6A is stabilized relative to UpA, while Api6A, Apms2i6A, and AptA are slightly destabilized relative to ApA. Thus, Upt6A, Api6A, Apms2i6A, and AptA have comparable stacking equilibria, indicating that the modifications remove the large difference in stacking between UpA and ApA. Furthermore, cytidylyl(3’-5’)adenosine (CpA), which is the most common unmodified sequence in this particular anticodon region, exhibits a stability similar to those of the hypermodified dinucleoside phosphates. Hypermodification therefore seems to keep the flexibility of this crucial part of the t R N A constant. It is proposed that this may result i n a more smoothly regulated translation step. Also, it is proposed that the enhanced stacking of UpthA relative to UpA prevents the incorrect wobble base pairing of this U residue in the tRNA during translation. M/ e have attempted in this investigation to define and understand on a molecular level the functions of hypermodified bases in tRNAs. That these bases perform some function during protein synthesis has been supported by experiments with polymer directed polypeptide synthesis, and binding assays using ribosomes and synthetic polymer messages (Gefter & Russell, 1969; Odom et al., 1974; Kitchingman et al., 1976; Miller et al., 1976). tRNAs modified or deficient in their normal amount of hypermodification were generally found to be less efficient in messenger binding and translation. In most of these studies, the aminoacylations of the tRNAs which were changed in their content of hypermodification were affected little if any by the changes. Binding studies with tRNAs and complementary trimers, or with two tRNAs having complementary anticodon triplets have also shown a stabilization of the binding when hypermodified bases are present (Hogenauer et al., 1972; Grosjean et al., 1976). Thus, with only a few exceptions (Litwack & Peterkofsky, 1971; Kimball & SOH, + From the Department of Chemistry and Laboratory of Chemical Biodynamics, University of California, Berkeley, Berkeley, California 94720. Receiced Norember 18. 1977. This work was supported by National Institutes of Health Grant GM10840 and by the Division of Biomedical and Environmental Research of the United States Department of Energy. Presented in preliminary form a t the 61st Annual Meeting of the Federation of American Societies for Experimental Biology, Chicago, Il l . , April 1977, Abstract No. 2234. *This work is taken from a dissertation submitted to the Graduate Division, University of California at Berkeley, in partial fulfillment of the requirement for the Ph.D. degree, June 1977. Present address: The Proctor and Gamble Co., Miami Valley Laboratories, P. 0. Box 391 75, Cincinnati, Ohio 45247. 1974), the presence of the hypermodified bases has been demonstrated to affect in vitro polypeptide synthesis. This effect, a t least in part, appears to be due to a more efficient binding of the t R N A to the ribosome-mRNA complex. Aside from these effects upon translation, some authors have suggested that the role of t6A’ is to prevent wobble on the 3’ side of the anticodon triplet (Ghosh et al., 1967; Takeishi et al., 1968; Dube et al., 1968; Stewart et al., 1971 ; Jukes, 1973; Elkins & Keller, 1974). Also, on the basis of crystal structures of the hypermodified bases, several workers have proposed that the role of hypermodification is to prevent the formation of a fourth base pair between the t R N A and m R N A (Bugg & Thewalt, 1972; Parthasarathy et al., 1974a,b). Except for the hypothesis of the prevention of a fourth base pair, it is not known how these possible functions are related to the molecular properties of the hypermodified bases. Therefore, we have chosen to approach this problem through the study of the smallest unit of a hypermodified tRNA which still exhibits the characteristics of a polynucleotide-the dinucleoside monophosphate. In this study, the solution spectral properties of Upt6A, Api6A, Apms2ihA, AptA, and their ’ Abbreviations used: t6A, N-[9-(~-~-r ibofuranosylJpurin-6-ylcarbamoyllthreonine; i6A, N6-(A2.isopentenyl)adenosine; ms2i6A, N6-(A*isopentenyl)-2-methyIthioadenosine; EA, l-N6-ethenoadenosine; y W , wybutosine (Kasai et al., 1976); pt6A, 5’-monophosphate of t6A; Upt6A, uridylyl(3’-5’)-N-[9-(~-~-ribofuranosyl)purin-6-ylcarbamoyl]~ threonine; Api6A, adenylyl(3’-5’)-N6-(A2-isopentenyl)adenosine; Apms2i6A, adenylyl(3’-5’)-N6-(~2-isopentenyl)-2-methylthioadenosine; ApcA, adenylyl(3’-5’)-1 ,N6-ethenoadenosine; ApyW, adenylyl(3’-5’)wybutosine. OOO6-2960/78/04I7-2455$01 .OO/O