SOLUTION STRUCTURES OF NONAMERIC AND DECAMERIC BRANCHED-RNA MODELING THE LARIAT OF GROUP-II AND NUCLEAR PRE-MESSENGER-RNA INTRONS (SPLICING) BY 500 MHZ NMR-SPECTROSCOPY

Conformational analysis of nonameric and decameric branched-RNAs by 500 MHz NMR spectroscopy (HOHAHA, DQFCOSY, NOESY, ROESY and temperature-dependent chemical shifts and coupling constants) in conjunction with our previous studies ox trimers, tetramers, pentamer and heptamer confirms some general trends on the conformational properties of branched RNA : (1) The conformation of the sugar ring of the adenosine branch-point (A4) is determined by the presence or absence of a 5'-terminal nucleotide: The sugar of the A4 is in the S-type conformation when no nucleotide is linked to the 5-hydroxyl of A4 (as in trimer 1 and pentamer 3) while it is in the N-type conformation when at least ow nucleotide is linked to its 5'-hydroxyl (as in tetramer 2, heptamer 4, nonamer 5 and decamer 6). (2) The 2'-linked G8 is anti in the trimer and pentamer. It is syn in the tetramer. heptamer, nonamer and decamer. (3) The conformation of the branched trimer and pentamer is dominated by a strong A4(2'-->5')G8 base-base stacking. The A4(2'-->5')G8 base stacking is weaker in the tetramer, heptamer, nonamer and decamer. (4) A comparison of the tetramer, heptamer, nonamer and decamer shows that the sugar conformation of the nucleotides in the 5'-chain (U3 in hepta, nona and decamer; U3 and C2 in decamer) are not influenced by the introduction of additional pyrimidine nucleotides. (5) The enlargment of the RNA branch system from the 2'- and 3-termini leads however to some conformational differences amongst the nucleotides at the 2'- and 3'-termini in the branched-RNAs possessing at least one 5-terminal nucleotide (as in tetramer 2, heptamer 4, nonamer 5, decamer 6): (a) The introduction of a 2- and 3'-terminal A7 and G10 purine nucleotide shifts the conformation of the U9 and C6 sugars from the N-type in the pentamer and heptamer to the S-type in the nonamer and decamer. (b) All the nucleotides of the 2- and 3' chain have a S-type sugar. (c) The branch-point A4 which was in the C3'-endo, anti conformation in the tetramer and heptamer is in the C3'-endo, syn conformation in the nonamer and decamer. Thus, three distinctly different types of conformational features have been identified from our studies on branched-RNA systems as models for lariat intron: The first group (trimer 1 and pentamer 3) is characterized by A4 in a C2'-endo, syn conformation and a overall conformation dominated by a strong A4(2'-->5')G8 stacking. The second group (tetramer 2 and heptamer 4) is characterized by A4 in a C3'-endo, anti conformation and a weaker A4(2'-->5')G8 stacking. The third group (nonamer 5 and decamer 6) is characterized by C3'-endo, syn conformation for the branch-point A4 residue, weaker A4(2'-->5')G8 stacking and the nucleotides of the 2- and 3'-chains are all in the C2-endo conformation which indicates that the 2'- and 3'-chains in branched nonamer 5 and decamer 6 do not adopt an A-RNA type helix.