SOLUTION STRUCTURE OF PENTAMERIC AND HEPTAMERIC BRANCHED-RNA MODELING THE LARIAT STRUCTURE OF GROUP-II OR NUCLEAR MESSENGER-RNA INTRONS STUDIED BY ONE-DIMENSIONAL AND 2-DIMENSIONAL NMR-SPECTROSCOPY AT 500 MHZ

A detailed 500 MHz H-1 NMR study of the branched RNA systems 3 (pentamer) and 4 (heptamer) is reported. The conformational properties of 3 and 4 were compared with those of the previously studied smaller branched RNAs 1 (trimer) and 2 (tetramer). Compounds 1 - 3 are structural constituents of 4, which corresponds to the sequence at the branch site of Group II intro b11 from yeast mitochondria. The present data provide further substantiation to our previous preliminary conclusion that trimer 1 and pentamer 3 show very similar conformational features, which are remarkably distinct from tetramer 2 and heptamer 4. It is found that the absence of a nucleotide unit at the 5' -site of the branch point (A) leads to a conformation at the branch point which is characterized by C2'-endo (S) ribose, syn orientation of the A-base, and A2' --> 5'G base stacking. This conformation is encountered for trimer 1, and -to a larger extent- also for pentamer 3. The introduction of a mono- or dinucleotide unit at the 5'-site of the branch point introduces a marked conformational transition at the branch point, in such a way that the conformation becomes C3'-endo (N) ribose, anti orientation of the base, and disruption of A2' --> 5'G base stacking. This conformation is found for tetramer 2 and heptamer 4. The structural differences between 1,3 and 2,4 are highly reminiscent of the 5'-conformational transmission effect for a series of linear trimer and tetramer RNA molecules. We therefore propose that the 5'-conformational model is also of great interest with respect to our understanding of the structural preferences of branched RNAs. However, our chemical shift vs. temperature profiles and NOE data on 3 and 4 show several additional conformational features. We propose a model for the secondary structure of pentamer 3, which entails peculiar backfolding of the 3'-linked chain in such a way that stacking is achieved between the remote bases C + 2 (in the 3'-linked chain), and branch point A. Also, our NOE data on heptamer 4 led to the suggestion that a stack exists between the bases U + 2 (in the 2'-linked chain), and U-1 (upstream of A). All our experimental observations on 3 and 4 were integrated into preliminary structural models, shown in Figure 10. It was impossible, however, to obtain strong support for these models from NOE data, since virtually no interresidual NOE crosspeaks appeared in our 2D NOESY spectra. Only in the case of 4, we could observe four weak interresidual NOEs at mixing times of 300, 500 and 900 ms in the aromatic spectral region, provided that a sample temperature of 292 K was used; elevation of the sample temperature to 303 K rendered these NOEs invisible.