Reprogrammed Genetic Decoding in Cellular Gene Expression Olivier Namy, Jean-Pierre Rousset, Sawsan Napthine, Ian Brierley  Molecular Cell  Volume 13, Issue 2, Pages 157-168 (January 2004) DOI: 10.1016/S1097-2765(04)00031-0 Copyright © 2004 Cell Press Terms and Conditions

Figure 1 Autoregulation of RF2 Synthesis by +1 Frameshifting The frameshift signal of the prfB gene is depicted, with the internal termination codon (UGA) highlighted (STOP) and the number of amino acids to the termination codon in the +1 frame shown. As RF2 becomes limiting (blue arrow) due to termination at this site, +1 slippage of tRNALeu occurs from the zero-frame codon CUU (underlined) to the overlapping +1 frame codon (UUU), enhanced by the upstream SD-like sequence (purple). However, as RF2 accumulates (red arrow), translation termination begins to predominate. Molecular Cell 2004 13, 157-168DOI: (10.1016/S1097-2765(04)00031-0) Copyright © 2004 Cell Press Terms and Conditions

Figure 2 Regulation of Cellular Polyamine Levels Using Antizyme +1 Frameshifting as a Sensor (A) High polyamine levels stimulate +1 frameshifting required for the synthesis of functional antizyme 1 (AZ1). AZ1 binds ornithine decarboxylase (ODC) and triggers its degradation by the 26S proteasome, being itself recycled. As ODC catalyzes the first step of the polyamine biosynthesis pathway, its degradation leads to a decrease in polyamine levels, which in turn reduces frameshifting efficiency. (B) Key features of the AZ1 frameshift signal are shown. The site of frameshifting is a “shifty stop” (5′ UCC UGA 3′, in blue and red, respectively) and the process is enhanced both by a stimulatory RNA pseudoknot just 3′ of the slippery sequence and a poorly defined 5′ stimulatory sequence (?). The mechanism of frameshifting is not known. Currently an occlusion model is favored, with the pseudoknot and/or upstream stimulator modifying the structure of the A site (when occupied by the termination codon UGA) such that the U is occluded and the +1 frame GAU codon can be decoded out-of-frame by tRNAAsp (Atkins et al., 2001). How polyamines stimulate the frameshift event remains to be determined. Molecular Cell 2004 13, 157-168DOI: (10.1016/S1097-2765(04)00031-0) Copyright © 2004 Cell Press Terms and Conditions

Figure 3 Conservation of EST3 Organization among Saccharomyces Species EST3 expression requires a +1 frameshift to fuse the products of ORF1 (93 amino acids) and ORF 2 (92 amino acids). The percentage amino acid identity between the sequences identified in S. cerevisiae and related Saccharomyces species is shown. The “missing” portion of ORF2 in S. bayanus (*) reflects the absence of sequence information (contig. end). The slippery sequences identified in the EST3 homologs, which are identical, are indicated with the stop codon of ORF1 in red. Molecular Cell 2004 13, 157-168DOI: (10.1016/S1097-2765(04)00031-0) Copyright © 2004 Cell Press Terms and Conditions

Figure 4 Examples of –1 Frameshifting in Prokaryotic Genes (A and B) Frameshifting in the E. coli dnaX gene. (A) Shows the structural organization of DNA polymerase III during the synthesis of the leading and lagging strands. The τ (normal translation) and γ subunits (–1 frameshifting) are derived from the dnaX gene. (B) Organization of the dnaX frameshift signal. Frameshifting occurs at the slippery sequence A AAA AAG (blue), enhanced by an upstream SD-like element (purple) and a downstream stem-loop structure. The stop codon of the γ subunit, in the –1 frame, is shown in red. Panel A of Figure 4 was reprinted from Leu, F., Georgescu, R. and O'Donnell, M. (2003). Mechanism of E. coli τ processivity. Switch during lagging strand synthesis. Molecular Cell 11, 315-327. Copyright (2003) with permission from Elsevier. (C) Ribosomal frameshifting in the B. subtilis cdd gene. Frameshifting occurs at the slippery sequence CGA AAG by an unusual single-tRNALys slippage event in the ribosomal A-site, from AAG to the overlapping AAA codon in the –1 frame. An SD-like sequence (purple) can stimulate the frameshift process or translation initiation at the start codon of the overlapping bex gene (orange). The cdd stop codon is indicated in red. Molecular Cell 2004 13, 157-168DOI: (10.1016/S1097-2765(04)00031-0) Copyright © 2004 Cell Press Terms and Conditions

Figure 5 Incorporation of Unusual Amino Acids at Stop Codons (A) Insertion of selenocysteine at UGA codons requires a selenocysteine insertion element (SECIS). In eubacteria, a specialized translational elongation factor SelB binds both the bSECIS element, located immediately downstream of the recoded UGA codon (in red), and the selenocysteine-incorporating tRNA(Ser)Sec. In eukaryotes, the SECIS is located in the 3-untranslated region. Association of the eukaryotic SelB ortholog mSelB (also known as eEFsec) requires an adaptor protein SBP2, the SECIS binding factor. (B) Provides examples of SECIS elements from eubacteria (E. coli fdnG) and eukaryotes (H. sapiens selZ). Unconventional base pairs within the selZ SECIS known to be essential for function are indicated in purple and the recoded UGA in red. Alongside the SECIS elements are putative PYLIS elements (pyrrolysine insertion elements) located downstream of the recoded UAG codons (in red) of the mtmB1 and B2 genes of various Methanosarcini strains. As mfold-derived structures (http://www.bioinfo.rpi.edu/applications/mfold/old/rna/form1.cgi), unconventional base pairs of the kind seen in SECIS elements would not be identified. Molecular Cell 2004 13, 157-168DOI: (10.1016/S1097-2765(04)00031-0) Copyright © 2004 Cell Press Terms and Conditions