Mechanisms of mRNA frame maintenance and its subversion during translation of the genetic code

Abstract

Important viral and cellular gene products are regulated by stop codon readthrough and mRNA frameshifting, processes whereby the ribosome detours from the reading frame defined by three nucleotide codons after initiation of translation. In the last few years, rapid progress has been made in mechanistically characterizing both processes and also revealing that trans-acting factors play important regulatory roles in frameshifting. Here, we review recent biophysical studies that bring new molecular insights to stop codon readthrough and frameshifting. Lastly, we consider whether there may be common mechanistic themes in -1 and +1 frameshifting based on recent X-ray crystal structures of +1 frameshift-prone tRNAs bound to the ribosome.

Keywords: Frameshifting; Protein synthesis; RNA structure; Ribosome; Translocation.

Figure 1. Examples of recoding events in viral and cellular genes

A) MLV stop codon readthrough required for expression of the pol gene. B) An example of −1 frameshifting on the slippery sequence (blue) of the IBV 1a/1b gene (note wild-type sequence is 5’-U UUA AAC-3’) and C) on the slippery sequence (blue) of the dnaX gene. D) +1 frameshifting by frameshift suppressor tRNA^SufA6 on hisD3018 mRNA.

Figure 2. Selected probes used in recent biophysical studies of −1 programmed frameshifting

A) Multiple conformational changes of ribosome components are necessary for mRNA-tRNA translocation during the elongation cycle. Opening and closing of the L1 arm is presumed to be associated with mRNA-tRNA translocation. The small ribosomal subunit body (b), head (h) and platform (pl) domains can move independently of one another while rotation of the small subunit (blue) relative to the large subunit (gray) and an orthogonal swiveling of the head domain are necessary for mRNA-tRNA translocation (30S shoulder domain closing is not shown). B) Radiolabeled methionine along with fluorescently labeled (purple star) tRNA (P-site tRNA is orange; A-site tRNA is yellow), EF-G and ribosomal proteins (S13, purple) were used to follow the mechanistic sub-steps of −1 programmed frameshifting on the Infectious Bronchitis Virus (IBV) pseudoknot frameshift signal (29). C) Fluorescent and fluorescence quencher oligonucleotides were hybridized to 16S rRNA helix 44 and 23S rRNA Helix 101 to follow small subunit rotation. tRNA or elongation factor arrival during translation of the dnaX frameshift signal was also followed by fluorescence (46). D) Qin et al. used multiple fluorescently labeled ribosomal proteins (S6 and L9 depicted in purple) to identify conformational states of the ribosome while programmed with the dnaX frameshift signal (45). E) A study by Kim et al. used smFRET between tRNA and ribosomal protein L1 to follow peptide bond formation and tRNA translocation on the dnaX frameshift signal (44).

Figure 3. Models for −1 programmed frameshifting

It has been proposed that frameshifting occurs A) during accommodation of aminoacyl tRNA on the third codon of the slippery sequence (‘9 Å’ model); B) after tRNA accommodation but before peptide bond formation (‘simultaneous slippage’ model); C) while tRNAs are in the hybrid state, which coincides with rotation of the small subunit (‘dynamic’ model); D) during eEF2 catalyzed formation of the post translocation state (‘mechanical’ model); E) during tRNA accommodation but with E- and P-site tRNAs in a noncanonical post translocation state. The organization of the figure is adapted from Brierley et al. (22) with the large subunit shown in grey, the small subunit in blue and the slippery mRNA sequence in red.

Figure 4. Base pairing at 32–38 of the ASL is important for frame maintenance

Base pairing within the anticodon stem loop of tRNA (stem, purple; anticodon loop, yellow) is important for decoding of the mRNA codon (green). X-ray crystal structures of +1 frameshift-prone tRNAs bound to the 70S ribosome reveal disruption of the 32–38 pair. Cryo-electron microscopy particle reconstructions have shown domain IV of EF-G (red) contacts the ASL in the vicinity of the 32–38 base pair before translocation in the A site, suggesting how the 32–38 base pair may be linked to frameshifting (59).