Cleavage of the A Site mRNA Codon during Ribosome Pausing Provides a Mechanism for Translational Quality Control  Christopher S Hayes, Robert T Sauer  Molecular Cell  Volume 12, Issue 4, Pages 903-911 (October 2003) DOI: 10.1016/S1097-2765(03)00385-X

Figure 1 Ribosome Stalling Leads to A Site mRNA Cleavage (A) The plasmid-borne ybeL-PP gene is transcribed from a promoter (φ10) for T7 RNA polymerase (which itself is transcribed under IPTG control) and has a T7 transcriptional terminator (Tφ). The gene and protein sequences near the UAA stop codon are shown, as are the positions of DNA oligonucleotide probes used for Northern blotting and S1 mapping experiments, an amber mutation used in some experiments, and the 3′ terminus of a truncated control ybeL-PP transcript, which terminates after the second base of the stop codon, synthesized by T7 RNA polymerase in vitro. (B) Northern blot of ybeL-PP mRNA purified from wild-type cells containing tmRNA (ssrA+) or lacking tmRNA (ssrA−). The positions of the full-length transcript and a control transcript that ends after the second base of the normal stop codon are indicated. Both the truncated experimental and control ybeL transcripts hybridized to a probe 5′ to the termination codon but not to a probe 3′ to the termination codon. (C) S1 mapping of the truncated ybeL-PP transcript from cells shows a pattern similar to that of the truncated control in vitro transcript, consistent with a major site of cleavage within the UAA stop codon. A strain in which expression of T7 RNA polymerase was not induced with IPTG showed no detectable S1 protection. Oligonucleotide standards of different lengths were generated by annealing the 3′-labeled S1 probe to a complementary DNA oligonucleotide and cleaving with the restriction enzymes HgaI, AlwI, NlaIV, and BamHI. The sites of cleavage relative to the stop codon are shown in (A). Molecular Cell 2003 12, 903-911DOI: (10.1016/S1097-2765(03)00385-X)

Figure 2 Suppression or Enhancement of A Site Cleavage (A) Northern blots (5′ probe) of mRNAs from the ybeL-PP gene and from the ybeL-PP-am128 gene, which contains an amber codon in place of the Glu128 codon. (B) Northern blots (5′ probe) of mRNAs transcribed from the ybeL-DP or ybeL-PP genes in proline auxotrophs grown in the presence of proline (Pro) or the proline analog azetidine-2-carboxylic acid (Aze). (C) Northern blots were quantified by phosphorimaging to determine the fraction of ybeL mRNA represented by the A site cleavage product in different strains. Overproduction of release factor 1 (RF1++), growth in the presence of azetidine, changing the C-terminal residues of YbeL to Glu-Ala (EA), and introduction of the amber mutation all suppress A site cleavage. (D) A site cleavage in the ybeL-EP gene is increased in strains lacking release factor 3 (ΔRF3) and in strains in which the UAA termination codon of the ybeL gene is replaced with a UGA stop codon. All experiments were performed in ΔtmRNA strains. In (C) and (D), there were no significant changes in the level of total ybeL mRNA. Molecular Cell 2003 12, 903-911DOI: (10.1016/S1097-2765(03)00385-X)

Figure 3 Toxin Deleted Strains Exhibit A Site Cleavage and ssrA Tagging (A) E. coli strains deleted for five known toxin/antitoxin systems were assayed for levels of A site cleavage of ybeL-PP mRNA by Northern blotting using the 5′ probe. (B) The same strains shown in (A) were assayed for ssrA tagging of YbeL-PP protein by SDS-PAGE of whole-cell lysates followed by staining with Coomassie blue. A tmRNA variant encoding a nondegradable ssrA-His6 tag was used to permit accumulation of the ssrA-tagged YbeL protein. Molecular Cell 2003 12, 903-911DOI: (10.1016/S1097-2765(03)00385-X)

Figure 4 Model Showing Possible Outcomes When a Ribosome Pauses in the Midst of Translation These fates are: (1) continuation of normal translation, (2) aberrant translation, (3) A site cleavage, and (4) other mRNA processing events. The tmRNA system functions in protein tagging and ribosome rescue for the A site cleavage pathway and for a subset of errors resulting from the aberrant translation pathway. Some aberrant translation events result in the ribosome reading to the 3′ end of the full-length mRNA because stop codons are misread as sense codons (readthrough) or because in-frame stop codons are not encountered as a result of frameshift errors. In other instances, however, readthrough or frameshift errors may lead to termination of translation at other stop codons and therefore fail to engage the tmRNA system. Other mRNA cleavage events may also result in opportunities for tmRNA tagging or ribosome rescue. Molecular Cell 2003 12, 903-911DOI: (10.1016/S1097-2765(03)00385-X)