The mouse mammary tumor virus (MMTV) gag-pro frameshifting pseudoknot is an H-type RNA pseudoknot that contains an unpaired adenosine (A14) at the junction of the two helical stems required for efficient frameshifting activity. The thermodynamics of folding of the MMTV vpk pseudoknot have been compared with a structurally homologous mutant RNA containing a G[bull ]U to G-C substitution at the helical junction (U13C RNA), and an A14 deletion mutation in that context (U13CΔA14 RNA). Dual wavelength optical melting and differential scanning calorimetry reveal that the unpaired adenosine contributes 0.7 (±0.2) kcal mol−1 at low salt and 1.4 (±0.2) kcal mol−1 to the stability (ΔG°37) at 1 M NaCl. This stability increment derives from a favorable enthalpy contribution to the stability ΔΔH = 6.6 (±2.1) kcal mol−1 with ΔΔG°37 comparable to that predicted for the stacking of a dangling 3′ unpaired adenosine on a G-C or G[bull ]U base pair. Group 1A monovalent ions, NH4+, Mg2+, and Co(NH3)63+ ions stabilize the A14 and ΔA14 pseudoknots to largely identical extents, revealing that the observed differences in stability in these molecules do not derive from a differential or specific accumulation of ions in the A14 versus ΔA14 pseudoknots. Knowledge of this free energy contribution may facilitate the prediction of RNA pseudoknot formation from primary nucleotide sequence (Gultyaev et al., 1999, RNA 5:609–617).

A new reporter system has been developed for measuring translation coupling efficiency of recoding mechanisms such as frameshifting or readthrough. A recoding test sequence is cloned in between the renilla and firefly luciferase reporter genes and the two luciferase activities are subsequently measured in the same tube. The normalized ratio of the two activities is proportional to the efficiency with which the ribosome “reads” the recoding signal making the transition from one open reading frame to the next. The internal control from measuring both activities provides a convenient and reliable assay of efficiency. This is the first enzymatic dual reporter assay suitable for in vitro translation. Translation signals can be tested in vivo and in vitro from a single construct, which allows an intimate comparison between the two systems. The assay is applicable for high throughput screening procedures. The dual-luciferase reporter system has been applied to in vivo and in vitro recoding of HIV-1 gag-pol, MMTV gag-pro, MuLV gag-pol, and human antizyme.

Superantigens (SAgs) are microbial proteins which have potent effects on the immune system. They are presented by major histocompatibility complex (MHC) class II molecules and interact with a large number of T cells expressing specific T cell receptor Vβ domains. Encounter of a SAg leads initially to the stimulation and subsequently to the clonal deletion of reactive T cells. SAgs are expressed by a wide variety of microorganisms which use them to exploit the immune system to their own advantage. Bacterial SAgs are exotoxins which are linked to several diseases in humans and animals. A classical example is the toxic shock syndrome in which the massive release of cytokines by SAg-reactive cells is thought to play a major pathogenic role. The best characterized viral SAg is encoded by mouse mammary tumour virus (MMTV) and has proved to have a major influence on the viral life cycle by dramatically increasing the efficiency of viral infection. In this paper, we review the general properties of SAgs and discuss the different types of microorganisms which produce these molecules, with a particular emphasis on the role played by the SAg-induced immune response in the course of microbial infections.