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During human immunodeficiency virus type 1 minusstrand 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 deadend 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.Reverse transcription consists of a complex series of reactions catalyzed by the virion-associated enzyme reverse transcriptase (RT) 1 that lead to conversion of the single-stranded RNA genome into an integration-competent linear doublestranded DNA (1). This process is facilitated by host and viral accessory proteins, one of which is the viral nucleocapsid protein (NC). Human immunodeficiency virus type 1 (HIV-1) NC is a small, highly basic, nucleic acid-binding protein with two zinc fingers, each containing the invariant CCHC metal ion-binding motif (2-5). NC functions as a nucleic acid chaperone in an ATP-independent manner (6) and catalyzes nucleic acid conformational rearrangements that lead to the formation of the most thermodynamically stable structure ). This activity is required for efficient reverse transcription and allows NC to promote intermolecular annealing of nucleic acids with significant stretches of base complementarity (7, 12-26), destabilization of secondary structures in RNA and DNA templates that are responsible for RT pausing (27-30), unwinding of primer tRNA (19, 31-34), primer placement (12,15,19,32,33,(35)(36)(37)(38), and the initiation step (39 -41).NC nucleic acid chaperone activity is also critical for the minus-strand (reviewed in Refs. 8, 10, and 11) and plus-strand (18,20,22,42) transfer events that are required to complete elongation of minus-and plus-strand DNAs and to generate the long te...

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 nonnative 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.

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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.

To study the role of sequence and topology in RNA folding, we determined the kinetic folding pathways of two circularly permuted variants of the Tetrahymena group I ribozyme, using time-resolved hydroxyl radical footprinting. Circular permutation changes the distance between interacting residues in the primary sequence, without changing the native structure of the RNA. In the natural ribozyme, tertiary interactions in the P4-P6 domain form in 1 s, while interactions in the P3-P9 form in 1-3 min at 42 degrees C. Permutation of the 5' end to G111 in the P4 helix allowed the stable P4-P6 domain to fold in 200 ms at 30 degrees C, five times faster than in the wild-type RNA, while the other domains folded five times more slowly (5-8 min). By contrast, circular permutation of the 5' end to G303 in J8/7 decreased the folding rate of the P4-P6 domain. In this permuted RNA, regions joining P2, P3 and P4 were protected in 500 ms, while the P3-P9 domain was 60-80% folded within 30 s. RNase T(1) digestion and FMN photocleavage showed that circular permutation of the RNA sequence alters the initial ensemble of secondary structures, thereby changing the tertiary folding pathways. Our results show that the natural 5'-to-3' order of the structural domains in group I ribozymes optimizes structural communication between tertiary domains and promotes self-assembly of the catalytic center.

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