Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein.

HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone, which is required for highly specific and efficient reverse transcription. Here, we demonstrate that local structure of acceptor RNA at a potential nucleation site, rather than overall thermodynamic stability, is a critical determinant for the minus-strand transfer step (annealing of acceptor RNA to (-) strong-stop DNA followed by reverse transcriptase (RT)-catalyzed DNA extension). In our system, destabilization of a stem-loop structure at the 5' end of the transactivation response element (TAR) in a 70-nt RNA acceptor (RNA 70) appears to be the major nucleation pathway. Using a mutational approach, we show that when the acceptor has a weak local structure, NC has little or no effect. In this case, the efficiencies of both annealing and strand transfer reactions are similar. However, when NC is required to destabilize local structure in acceptor RNA, the efficiency of annealing is significantly higher than that of strand transfer. Consistent with this result, we find that Mg2+ (required for RT activity) inhibits NC-catalyzed annealing. This suggests that Mg2+ competes with NC for binding to the nucleic acid substrates. Collectively, our findings provide new insights into the mechanism of NC-dependent and -independent minus-strand transfer.

Alteration of nucleic acid structure and stability modulates the efficiency of minus-strand transfer mediated by the HIV-1 nucleocapsid protein.

During human immunodeficiency virus type 1 minus-strand transfer, the nucleocapsid protein (NC) facilitates annealing of the complementary repeat regions at the 3'-ends of acceptor RNA and minus-strand strong-stop DNA ((-) SSDNA). In addition, NC destabilizes the highly structured complementary trans-activation response element (TAR) stem-loop (TAR DNA) at the 3'-end of (-) SSDNA and inhibits TAR-induced self-priming, a dead-end reaction that competes with minus-strand transfer. To investigate the relationship between nucleic acid secondary structure and NC function, a series of truncated (-) SSDNA and acceptor RNA constructs were used to assay minus-strand transfer and self-priming in vitro. The results were correlated with extensive enzymatic probing and mFold analysis. As the length of (-) SSDNA was decreased, self-priming increased and was highest when the DNA contained little more than TAR DNA, even if NC and acceptor were both present; in contrast, truncations within TAR DNA led to a striking reduction or elimination of self-priming. However, destabilization of TAR DNA was not sufficient for successful strand transfer: the stability of acceptor RNA was also crucial, and little or no strand transfer occurred if the RNA was highly stable. Significantly, NC may not be required for in vitro strand transfer if (-) SSDNA and acceptor RNA are small, relatively unstructured molecules with low thermodynamic stabilities. Collectively, these findings demonstrate that for efficient NC-mediated minus-strand transfer, a delicate thermodynamic balance between the RNA and DNA reactants must be maintained.

Effect of transcription on folding of the Tetrahymena ribozyme.

Sequential formation of RNA interactions during transcription can bias the folding pathway and ultimately determine the functional state of a transcript. The kinetics of cotranscriptional folding of the Tetrahymena L-21 ribozyme was compared with refolding of full-length transcripts under the same conditions. Sequential folding after transcription by phage T7 or Escherichia coli polymerase is only twice as fast as refolding, and the yield of native RNA is the same. By contrast, a greater fraction of circularly permuted variants folded correctly at early times during transcription than during refolding. Hybridization of complementary oligonucleotides suggests that cotranscriptional folding enables a permuted RNA beginning at G303 to escape non-native interactions in P3 and P9. We propose that base pairing of upstream sequences during transcription elongation favors branched secondary structures that increase the probability of forming the native ribozyme structure.

Perturbed folding kinetics of circularly permuted RNAs with altered topology.

The folding pathway of the Tetrahymena ribozyme correlates inversely with the sequence distance between native interactions, or contact order. The rapidly folding P4-P6 domain has a low contact order, while the slowly folding P3-P7 region has a high contact order. To examine the role of topology and contact order in RNA folding, we screened for circular permutants of the ribozyme that retain catalytic activity. Permutants beginning in the P4-P6 domain fold 5 to 20 times more slowly than the wild-type ribozyme. By contrast, 50% of a permuted RNA that disjoins a non-native interaction in P3 folds tenfold faster than the wild-type ribozyme. Hence, the probability of rapidly folding to the native state depends on the topology of tertiary domains.