RNA secondary structure: Prediction, three-dimensional modeling, and the significance of alternate folds

The secondary structure of RNA is crucial in determining its three-dimensional conformation. A secondary structure can be predicted by comparative sequence or energy minimization algorithms. The feasibility of a secondary structure can be tested by building it in three dimensions. Predicted secondary structures can be used to develop hypotheses that can be experimentally tested. Finally, prediction algorithms can be improved by including newly identified RNA motifs.; A secondary structure model for the human immunodeficiency virus (HIV-1) RNA/tRNALys3 initiation complex has previously been proposed that includes additional base-pairing between tRNA and HIV-1 RNA beyond the primer binding site. In our efforts to build a three-dimensional model, we have found several problems. The structure is topologically knotted, posing a problem for folding and transcription of the initiation complex. We have also been unable to build all-atom models of this structure based on known RNA conformations.; We propose a new model for the initiation complex that does not include some of these extended HIV/tRNA interactions. Human tRNALys3 is predicted to have an alternate conformation that is almost isoenergetic to the cloverleaf structure and we call this conformation the slingshot. We propose that HIV-1 exploits the slingshot in primer selection and formation of the initiation complex. This prediction is supported by two experimental tests. First, an in vitro transcribed tRNA engineered to form the slingshot promotes initiation complex formation. Second, we found that human tRNAGlu is also predicted to fold into a slingshot conformation with favorable energy. We designed a virus complementary to tRNA Glu in both the PBS and A-loop regions, and our results show that this virus stably uses tRNAGlu as a primer.; Finally, there are a large number of helices in rRNA followed by either a GA or an AA mismatch. To test the hypothesis that these sequences pair and stack on the end of helices, we examined the structural database to determine if they are defined by a distinct base-pairing pattern. We have found that these sequences pair, extend helices, and allow coaxial stacking with adjacent helices. Their three-dimensional conformation is also dependent on their orientation with respect to the helix.