In this study, the IH-IH, lH31P and13C-3lP 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 y+ and p 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% stacked axis in the tetramer 2. In accordance with this observation, it has been clearly established that y+ and pt 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'+SA3'+5% axis. The NOES observed at 270 MHz between CH6 with GH8, and UH6 with GHB suggest also a spatial proximity between S-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 IH-IH, lH-31P and 13C-31P coupling constants and chemical shifts of analogues of the branched trimer I in this laboratory and elsewhere have shown that the molecular conformation of the branched trimer 1 is governed by A2'4'G stack. The introduction of a Y-terminal widine residue in m*mer 1 to tetramer 2 shifts the molecular conformation from an A2'4'G stack in the trimer 1 to a A3'4'C stack in the tetramer
2. This is a new example of 5'-terminal residue promoted cot$ormational transmission.
In group II splicing of RNA precursors, a lariat RNA is formed at the penultimate step of ligation of exons. In the final step of splicing, this lariat is excised upon the Mg2+ promoted ligation of two exons. The nucleotide sequence at the branch site in the lariat is highly conserved*. In the lariat formed, the branch-point is always an adenosine residue which is linked through its 2'-phosphate to the S-hydroxy group of a guanosine nucleotide, and through its 3'-phosphate to the S-hydroxy group of a pyrimidine nucleotide. In the group II splicing reaction, the lariat formed always carries a uridine residue next to the S-end of the branch point adenosine while in the Nuclear pre-messenger RNA splicing reaction, it is always an adenosine nucleotide. Mutation experiments have shown that replacement of the branch point adenosine by guanosine, uridine or cytidine either stops the splicing reaction, or promotes wrong splicing in the upstream region of the pre-mRNA2.
It has also been
shown that guanosine as the 2'+5' linked nucleotide is necessary for the completion of the second step of the splicing reaction. In last few years, we have been trying to understand the structural significance of formation of branch-RNA in the splicing reaction, and the structural and conformational basis for the choice of adenosine as the branch-point nucleotide and of guanosine as the 2'+5' linked nucleotide.