On programmed ribosomal frameshifting: the alternative proteomes

We describe a dynamic programming algorithm for predicting optimal RNA secondary structure, including pseudoknots. The algorithm has a worst case complexity of O(N6) in time and O(N4) in storage. The description of the algorithm is complex, which led us to adopt a useful graphical representation (Feynman diagrams) borrowed from quantum field theory. We present an implementation of the algorithm that generates the optimal minimum energy structure for a single RNA sequence, using standard RNA folding thermodynamic parameters augmented by a few parameters describing the thermodynamic stability of pseudoknots. We demonstrate the properties of the algorithm by using it to predict structures for several small pseudoknotted and non-pseudoknotted RNAs. Although the time and memory demands of the algorithm are steep, we believe this is the first algorithm to be able to fold optimal (minimum energy) pseudoknotted RNAs with the accepted RNA thermodynamic model. Copyright 1999 Academic Press.

Tying the pseudoknot Ribosomal frameshifting is a translational mechanism involved in protein synthesis during the replication of many viral pathogens and in cellular genes more generally. A new set of images of an 80S ribosome stalled at an mRNA pseudoknot shows how the pseudoknot manipulates the ribosome into a different reading frame. Supplementary information The online version of this article (doi:10.1038/nature04735) contains supplementary material, which is available to authorized users.

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.