The diploid genome of all retroviruses is made of two homologous copies of RNA intimately associated near their 5' end, in a region called the dimer linkage structure. Dimerizatlon of genomic RNA is thought to be important for crucial functions of the retroviral life cycle (reverse transcription, translation, encapsidation). Previous in vitro studies mapped the dimer linkage structure of human immunodeficiency virus type 1 (HIV-1) in a region downstream of the splice donor site, containing conserved purine tracts that were postulated to mediate dimerization, through purine quartets. However, we recently showed that dimerization of HIV-1 RNA also involves sequences upstream of the splice donor site. Here, we used chemical modification interference to identify nucleotides that are required in unmodified form for dimerization of a RNA fragment containing nucleotides A ubiquitous property of retroviruses is that their genonme consists of two homologous copies of single-stranded RNA (1). Electron microscopy showed that these two RNAs are joined together in an apparent parallel orientation by a structure called the dimer linkage structure (DLS) located near their 5' end (2-5). Dimerization of genomic RNA is considered to control several essential steps of the retroviral life cycle. First, it was proposed to act as a positive signal for encapsidation (6,7). Second, it was suggested to downregulate the translation ofthe gag gene in Rous sarcoma virus (RSV) and human immunodeficiency virus type 1 (HIV-1) (8, 9). Third, the dimeric nature of the retroviral genome is thought to be of importance in the process of reverse transcription and recombination since it may account for the first strand transfer and template switching during proviral DNA synthesis (10-14). Therefore, dimerization of genomic RNA most likely represents a potential target for the design of antiviral drugs against HIV and other retroviruses.However, the process of dimerization is still poorly understood. Dimerization of synthetic fragments containing the (29). Again, probing data indicate that the same structure is found in synthetic RNA fragments (9,30) and in genomic RNA extracted from infected cells (29).In HIV-1, several reports showed that a RNA region of -100 nucleotides located downstream of the splice donor (SD) site is able to dimerize in vitro (6,(21)(22)(23)(24). This region that includes important components of the packaging signal (29,31,32) was assumed to contain the putative DLS. However, the mechanism of dimerization is still a subject of controversy. In an initial study, we proposed that polypurine tracts, the only common motifs found in the putative dimerization-encapsidation region of most retroviral RNAs, may be involved in the dimerization process through the formation of quartets involving both guanines and adenines (21). This concept has been disputed by recent reports suggesting that dimers formed with short RNA restricted to the postulated DLS are stabilized by quartets containing only guanines (23,24). However, the di...

The majority of structural efforts addressing RNA's catalytic function have focused on natural ribozymes, which catalyze phosphodiester transfer reactions. By contrast, little is known about how RNA catalyzes other types of chemical reactions. We report here the crystal structures of a ribozyme that catalyzes enantioselective carbon-carbon bond formation by the Diels-Alder reaction in the unbound state and in complex with a reaction product. The RNA adopts a λ-shaped nested pseudoknot architecture whose preformed hydrophobic pocket is precisely complementary in shape to the reaction product. RNA folding and product binding are dictated by extensive stacking and hydrogen bonding, whereas stereoselection is governed by the shape of the catalytic pocket. Catalysis is apparently achieved by a combination of proximity, complementarity and electronic effects. We observe structural parallels in the independently evolved catalytic pocket architectures for ribozyme-and antibody-catalyzed Diels-Alder carbon-carbon bond-forming reactions.The discovery of the catalytic activity of RNA 1,2 and the hypothesis of a prebiotic 'RNA world' 3 have expanded the scope of enzymology to include other biopolymers than proteins. The currently known natural ribozymes catalyze only a narrow range of chemical reactions, namely the hydrolysis and transesterification of internucleotide bonds 4,5 , and probably peptide bond formation 6 . However, in vitro selection and evolution have demonstrated that ribozymes are capable of accelerating a much broader reaction spectrum 7 . This finding and Correspondence should be addressed to A.J. (jaeschke@uni-hd.de) or D.J.P. (pateld@mskcc.org). COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.Note: Supplementary information is available on the Nature Structural & Molecular Biology website. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript recent discoveries of metabolite-controlled RNA switches and ribozymes 8,9 suggest that RNA might have performed an even broader range of activities in the preprotein realm, and that in vitro-selected ribozymes could be analogs of the missing links in the transition from an RNA world to modern protein-dominated life 10 . Whereas high-resolution structures and biochemical investigations of several natural ribozymes provide a basic understanding of how RNA carries out phosphodiester chemistry 5,11 , little is known about how RNA catalyzes other reactions. To obtain a comprehensive picture of the catalytic abilities and limitations of ribozymes, it is thus important to expand structural and mechanistic investigations to in vitro-selected ribozymes [12][13][14] . Such structural information can be especially valuable in the determination of the minimal RNA folds required for catalysis and, therefore, could be helpful in the investigation of the origin and evolution of natural ribozymes 15 .Two examples describe the in vitro selection of ribozymes that accelerate the formation of ...

RNA-RNA interactions govern a number of biological processes. Several RNAs, including natural sense and antisense RNAs, interact by means of a two-step mechanism: recognition is mediated by a loop-loop complex, which is then stabilized by formation of an extended intermolecular duplex. It was proposed that the same mechanism holds for dimerization of the genomic RNA of human immunodeficiency virus type 1 (HIV-1), an event thought to control crucial steps of HIV-1 replication. However, whereas interaction between the partially self-complementary loop of the dimerization initiation site (DIS) of each monomer is well established, formation of the extended duplex remained speculative. Here we first show that in vitro dimerization of HIV-1 RNA is a specific process, not resulting from simple annealing of denatured molecules. Next we used mutants of the DIS to test the formation of the extended duplex. Four pairs of transcomplementary mutants were designed in such a way that all pairs can form the loop-loop "kissing" complex, but only two of them can potentially form the extended duplex. All pairs of mutants form heterodimers whose thermal stability, dissociation constant, and dynamics were analyzed. Taken together, our results indicate that, in contrast with the interactions between natural sense and antisense RNAs, no extended duplex is formed during dimerization of HIV-1 RNA. We also showed that 55-mer sense RNAs containing the DIS are able to interfere with the preformed HIV-1 RNA dimer.

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