SOLUTION STRUCTURE OF BRANCHED U3'P5'A3'P5'C(2'P5'G) AND ITS COMPARISON WITH A3'P5'U(2'P5'G) BY 500 MHZ NMR-SPECTROSCOPY

In this study, the H-1-H-1, H-1-P-31 and C-13-P-31 coupling constants of the branched RNA tetramer 2 have been measured at two temperatures to obtain detailed information about its backbone conformation. Evaluation of these coupling constants by Karplus-Altona algorithm shows the decrease of populations of gamma+ and beta-t upon temperature-increase for the branch-point A and 3'-terminal C residues, which have been attributed to a destacking along the U3' --> 5'A3' --> 5'C stacked axis in the tetramer 2. In accordance with this observation, it has been clearly established that gamma+ and beta-t populations of constituent 2' --> 5'-linked guanosine nucleotide is rather insensitive to temperature-change. The NOEs seen at 270 MHz between AH8 with UH6, and AH2 with CH6 also support that the tetramer 2 stacks along the U3' --> 5'A3' --> 5'C axis. The NOEs observed at 270 MHz between CH6 with GH8, and UH6 with GH8 suggest also a spatial proximity between 5'-terminal U and 2'-terminal G, and 3'-terminal C and 2'-terminal G residues. These observations have led us to propose a two-state model for the tetramer 2. On the other hand, detailed temperature-dependent measurements of H-1-H-1, H-1-P-31 and C-13-P-31 coupling constants and chemical shifts of analogues of the branched trimer 1 in this laboratory and elsewhere have shown that the molecular conformation of the branched trimer 1 is governed by A2' --> 5'G stack. The introduction of a 5'-terminal uridine residue in trimer 1 to tetramer 2 shifts the molecular conformation from an A2' --> 5'G stack in the trimer 1 to tetramer 2. This is a new example of 5'-terminal residue promoted conformational transmission.