No abstract

The pseudoknot is an important RNA structural element that provides an excellent model system for studying the contributions of tertiary interactions to RNA stability and to folding kinetics. RNA pseudoknots are also of interest because of their key role in the control of ribosomal frameshifting by viral RNAs. Their mechanical properties are directly relevant to their unfolding by ribosomes during translation. We have used optical tweezers to study the kinetics and thermodynamics of mechanical unfolding and refolding of single RNA molecules. Here we describe the unfolding of the frameshifting pseudoknot from infectious bronchitis virus (IBV), three constituent hairpins, and three mutants of the IBV pseudoknot. All four pseudoknots cause -1 programmed ribosomal frameshifting. We have measured the free energies and rates of mechanical unfolding and refolding of the four frameshifting pseudoknots. Our results show that the IBV pseudoknot requires a higher force than its corresponding hairpins to unfold. Furthermore, its rate of unfolding changes little with increasing force, in contrast with the rate of hairpin unfolding. The presence of Mg(2+) significantly increases the kinetic barriers to unfolding the IBV pseudoknot, but has only a minor effect on the hairpin unfolding. The greater mechanical stability of pseudoknots compared to hairpins, and their kinetic insensitivity to force supports the hypothesis that -1 frameshifting depends on the difficulty of unfolding the mRNA.

The dimerization of viral RNA through noncovalent interactions at their 5 ends is a key step in the life cycle of retroviruses. In Moloney murine leukemia virus, three stem-loops are important in this process. One is a self-complementary tetraloop (H1), but the other two stem-loops (H2, H3) contain highly conserved GACG tetraloops that are not self-complementary sequences. Using twodimensional NMR, we determined the structure of the H3 stemloop. Surprisingly, it forms a stable, homodimeric kissing complex through only two intermolecular G⅐C base pairs. Cross-strand interactions of the adenines adjacent to the intermolecular G⅐C base pairs, plus unusual strong electrostatic interactions around the base pairs, contribute to the unexpected stability. This structure shows how even stem-loops without self-complementary sequences can facilitate the intermolecular recognition between two identical RNAs, and thus initiate dimerization and encapsidation of retroviral RNAs. RNA dimerization is a key step in the life cycle of retroviruses (1-4) and is closely tied to the RNA encapsidation process (5-7). In Moloney murine leukemia virus, a dimer linkage structure overlaps the encapsidation domain (8, 9), where there are three conserved stem-loop structures (10) (Fig. 1A, H1, H2, and H3). Based on in vitro dimerization studies (11,12), the H1 stem-loop was postulated to trigger RNA dimerization through base pairing of its self-complementary tetraloop. Two other stem-loop structures containing loop sequences that are not self-complementary tetraloops (H2, H3) also participate in this process (13). Because the H2 and H3 are essential for RNA packaging during encapsidation (9, 14), these two stem-loops may be key structural elements in connecting the dimerization and encapsidation processes. Both H2 and H3 hairpins contain GACG tetraloops that are highly conserved among murine type-C retroviruses (15) and that are thought to facilitate the recognition between the two genomic RNAs (13). However, it is not known how the H2 and H3 use their tetraloops in these processes. In this study, by using high-resolution NMR, the solution structure of the kissing complex of an 18-mer RNA oligonucleotide mimicking the H3 GACG tetraloop motif (H3-18; Fig. 1B) was determined. We found that GACG tetraloops form stable loop-loop kissing complexes. MethodsRNA Sample Preparation and Characterization. H3-18 RNA was transcribed from DNA templates in vitro by using T7 RNA polymerase (16). The preparation and purification of the RNA (17) and of the 13 C, 15 N-labeled NTPs (18) has been described. Native gel electrophoresis and RNA melting studies were done as described (17). The purified RNAs were heated at 95°C for 1 min and cooled to room temperature. For NMR Spectroscopy. NMR spectra were recorded on Bruker (Billerica, MA) DRX 500-MHz and Bruker AMX 600-MHz spectrometers. Spectra were processed by using FELIX 95.0 (Biosym Technologies, San Diego). One-dimensional (1D) jump-return experiments were done at various temperatures (Fig. 2). All of th...

The effects of four types of fullerene compounds (C60, C60−OH, C60−COOH, C60−NH2) were examined on two model microorganisms (Escherichia coli W3110 and Shewanella oneidensis MR-1). Positively charged C60−NH2 at concentrations as low as 10 mg/L inhibited growth and reduced substrate uptake for both microorganisms. Scanning electron microscopy (SEM) revealed damage to cellular structures. Neutrally charged C60 and C60−OH had mild negative effects on S. oneidensis MR-1, whereas the negatively charged C60−COOH did not affect either microorganism's growth. The effect of fullerene compounds on global metabolism was further investigated using [3-13C]l-lactate isotopic labeling, which tracks perturbations to metabolic reaction rates in bacteria by examining the change in the isotopic labeling pattern in the resulting metabolites (often amino acids). − The 13C isotopomer analysis from all fullerene-exposed cultures revealed no significant differences in isotopomer distributions from unstressed cells. This result indicates that microbial central metabolism is robust to environmental stress inflicted by fullerene nanoparticles. In addition, although C60−NH2 compounds caused mechanical stress on the cell wall or membrane, both S. oneidensis MR-1 and E. coli W3110 can efficiently alleviate such stress by cell aggregation and precipitation of the toxic nanoparticles. The results presented here favor the hypothesis that fullerenes cause more membrane stress − than perturbation to energy metabolism.