Changed in translation: mRNA recoding by -1 programmed ribosomal frameshifting

Abstract

Programmed -1 ribosomal frameshifting (-1PRF) is an mRNA recoding event commonly utilized by viruses and bacteria to increase the information content of their genomes. Recent results have implicated -1PRF in quality control of mRNA and DNA stability in eukaryotes. Biophysical experiments demonstrated that the ribosome changes the reading frame while attempting to move over a slippery sequence of the mRNA--when a roadblock formed by a folded downstream segment in the mRNA stalls the ribosome in a metastable conformational state. The efficiency of -1PRF is modulated not only by cis-regulatory elements in the mRNA but also by trans-acting factors such as proteins, miRNAs, and antibiotics. These recent results suggest a molecular mechanism and new important cellular roles for -1PRF.

Keywords: decoding; gene expression; mRNA reading frame maintenance; protein synthesis; ribosome; translation.

Figure I

Three major types of recoding events. Read-through entails the insertion of an unusual amino acid (gold) at the position of a stop codon. Bypassing connects two parts of a discontinuous reading frame (green and blue linear segments) resulting in a single protein (green-blue). Frameshifting may be used to regulate the synthesis of a single protein or to produce two proteins (peptide ORF1 and peptide ORF1+2) from a single mRNA with two overlapping open reading frames (ORFS; green and blue segments). The black arrows indicate the direction of ribosome movement.

Figure 1

Frameshifting stimulatory signals on mRNA. (A) Left panel, schematic of the modified frameshifting sequence of the avian infectious bronchitis virus (IBV) 1a/1b mRNA . The original IBV slippery sequence (U UUA AAC) was changed to ensure maximum frameshifting efficiency in Escherichia coli. The amino acids incorporated upon translation in the 0- or −1-frame are indicated above the mRNA sequence. Middle and right panels, structure and surface representation of the frameshifting pseudoknot. (B) Left panel, schematic of the programmed −1 ribosomal frameshifting (−1PRF) regulatory elements in dnaX from E. coli. In addition to the slippery sequence and stimulatory stem-loop, frameshifting on dnaX is modulated by a Shine–Dalgarno (SD)-like sequence upstream of the slippery site . Middle and right panels, structure and surface representation of the frameshifting stem-loop. Images were prepared using PyMOL (http://www.pymol.org/).

Figure 2

Codon walk over the frameshifting sequence of avian infectious bronchitis virus (IBV). (A) Schematic of the experiments. Purified translation components are rapidly mixed in a quench-flow device. After the desired time the reactions are stopped by rapid mixing with a strong base (quencher) and the peptide products are analyzed by HPLC, measuring [³H]Met radioactivity. (B) Example of a time-course of peptide synthesis on a frameshifting mRNA. Monitored peptides are fMetTyr (MY), fMetTyrLeu (MYL), fMetTyrLeuLys (MYLK), fMetTyrLeuLysPhe (MYLKF), and fMetTyrLeuLysVal (MYLKV). The time-courses are used to calculate the rates and efficiencies of insertion into peptide of each amino acid in 0- and −1-frame. (C) Comparison of the amino acid incorporation rates (s⁻¹) for frameshifting (dark colors) and non-frameshifting (light colors) mRNAs. The frameshifting mRNA contained both a slippery sequence and pseudoknot, the non-frameshifting mRNA had no stimulatory elements for programmed −1 ribosomal frameshifting (−1PRF). (D) Contributions of the regulatory elements in the mRNA. SS and PK indicate the presence of a slippery site or a pseudoknot, respectively, in the mRNA .

Figure 3

Kinetic model of programmed −1 ribosomal frameshifting (−1PRF). Frameshifting occurs during translocation of the two tRNAs bound to the slippery sequence (tRNA^Leu in the P site and MYLK-tRNA^Lys in the A site). Recruitment of EF-G (step 1) to the pre-translocation (PRE) complex facilitates rapid tRNA movement (step 2) into a state where translocation on the 50S subunit is completed; however, the following steps are inhibited by the presence of the pseudoknot. tRNA^Leu moves on the 50S subunit while the distance to the 30S subunit is not changed (steps 3 and 6 in 0-frame and −1-frame, respectively). Then, tRNA^Leu and the 30S subunit move apart (steps 4 and 7). Steps 3 and 4 are particularly slow for the tRNA that remains in 0-frame, which limits the decoding rate in the 0-frame by Phe-tRNA^Phe (step 5). By contrast, tRNA^Leu movement on those ribosomes that shift to the −1-frame is faster (step 6), followed by dissociation of tRNA^Leu from the 30S subunit, 30S head rotation, dissociation of EF-G (step 7), and binding of Val-tRNA^Val (step 8). Reproduced, with permission, from .

Figure I

Schematic of translocation. Figure modified from .