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, 4 (5), 499-504

Maintenance of the Correct Open Reading Frame by the Ribosome

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Maintenance of the Correct Open Reading Frame by the Ribosome

Thomas M Hansen et al. EMBO Rep.

Abstract

During translation, a string of non-overlapping triplet codons in messenger RNA is decoded into protein. The ability of a ribosome to decode mRNA without shifting between reading frames is a strict requirement for accurate protein biosynthesis. Despite enormous progress in understanding the mechanism of transfer RNA selection, the mechanism by which the correct reading frame is maintained remains unclear. In this report, evidence is presented that supports the idea that the translational frame is controlled mainly by the stability of codon-anticodon interactions at the P site. The relative instability of such interactions may lead to dissociation of the P-site tRNA from its codon, and formation of a complex with an overlapping codon, the process known as P-site tRNA slippage. We propose that this process is central to all known cases of +1 ribosomal frameshifting, including that required for the decoding of the yeast transposable element Ty3. An earlier model for the decoding of this element proposed 'out-of-frame' binding of A-site tRNA without preceding P-site tRNA slippage.

Figures

Figure 1
Figure 1
Illustration of +1 frameshifting, shown in parallel with the process of standard translation. The kinetic requirement for efficient frameshifting is the commensurability of the rates of the two processes. Usually, the rate of standard translation is significantly higher than that of frameshifting. A low concentration of incoming transfer RNA in the initial frame (magenta) makes standard translation slower than usual. Low stability of the initial P-site codon–anticodon complex ('0'), high stability of the equivalent complex in the new frame ('+1'), and high levels of the incoming tRNA corresponding to the A-site codon in the new frame (green), increase the rate of frameshifting. As a result, a high input of several stimulating factors can compensate for the lack of one of them. In Ty3, it is the lack of stable P-site codon–anticodon duplexes in the +1 shifted frame that is compensated for by three other factors.
Figure 2
Figure 2
+1 frameshifting at the gene encoding release factor 2, wherethe CUU shift-site is replaced by GUU or AAG. (A) Constructs and their products. XYZ represents the position of CUU, which was replaced in these constructs. (B) Pulse–chase experiment. Electrophoretic separation of glutathione-S-transferase (GST)-tagged proteins on an SDS gel. The samples electrophoresed on the gel were from constructs containing a Shine–Dalgarno sequence (SD) or lacking this sequence (ΔSD). (C) Protein analysis. Mass spectra in the 67,500–71,500 Da range are shown for GST-tagged proteins that were purified from strains expressing constructs containing an SD. The major peaks are at 69,479 Da (GUU) and 69,508 Da (AAG), corresponding to frameshift products with expected masses of 69,479.6 Da (GUU) and 69,508.6 Da (AAG). A minor peak with a mass corresponding to the major product lacking one methionine is indicated (−M). An adduct of the major product is apparent in the AAG spectrum (*). (D) Codon–anticodon pairing in the +1 frame. Possible Watson–Crick (bar) and wobble (circle) base pairs are indicated. FS, product of +1 frameshifting in the insert; mRNA, messenger RNA; Term, product resulting from termination at UGA in the insert; tRNA, transfer RNA.
Figure 3
Figure 3
Illustration of how ribosomes prevent out-of-frame binding of incoming transfer RNA. (A) Details of the A site and P site. The A-site transfer RNA is shown in yellow; helix 6 from the neighbouring ribosome molecule that mimics P-site tRNA is shown in dark blue. (B) Details of the ribosomal components surrounding the A-site tRNA; adenosines 1,492 and 1,493 are labelled. (C) Schematic illustration of out-of-frame binding at the A site. Magenta arrows indicate that the discriminating adenosines do not sense a correct base pair; green arrows indicate that the discriminating adenosines recognize a correct base pair. Whereas standard translation and P-site tRNA slippage lead to normal recognition at the A site, out-of-frame binding does not. The illustrations in (A,B) are abstracted with permission from Ogle et al. (2001) © (2001) American Association for the Advancement of Science. ASL, anticodon stem–loop; mRNA, messenger RNA.

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