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2A-induced ribosome stallingOdon, Valèrie M. N. January 2014 (has links)
Originally 2A was characterised in foot-and-mouth disease virus. Site directed mutagenesis identified a C-terminus consensus motif [D(V/I)ExNPGP] and it is proposed that 2A interacts with the exit tunnel of the ribosome in a way that a specific peptide bond is skipped between the last glycine of 2A and the proline of 2B, thus providing a discontinuity in translation, resulting in release of discrete proteins from one single ORF. 2A was also identified in other picornaviruses, positive, single and double-stranded RNA insect viruses and mammalian rotaviruses. A motif present at the C-terminus of the 2A oligopeptide [D(V/I)ExNPGP] is very highly, though not completely conserved . The sequence upstream of this motif shows, however, no apparent conservation between 2As of different viruses. In this study, extensive site-directed mutagenesis were performed on several 2A sequences and a series of ‘hybrid' 2As comprising different consensus motifs juxtaposed with different upstream contexts were created as part of a detailed analysis of the mechanism of 2A-mediated ribosome stalling. The results demonstrated that a minimal region of twenty to twenty-three amino acids interacts with the exit tunnel of the ribosome to bring about a pause in processivity, alter the peptidyl transferase centre geometry and restrict the ribosome A site via two distinctive stalling mechanisms. Other molecular analyses tested here will require further optimisations or alternative methods: a visual method to explore the dynamics of re-initiation of translation from proline codon, purification of the translation-regulating factors and structural resolution of 2A sequences. Previously, cellular 2As were identified in non-LTR retrotransposons of trypanosomes. It is reported here as part of two other cellular organisms Saccoglossus kowalevskii (acorn worm) and Branchiostoma floridae (amphioxus). In the acorn worm, the nucleotides sequences corresponding to 2A motifs were part of the untranslated genome. In amphioxus, three 2A elements were identified in hypothetical proteins, and at the N-terminus of twenty non-LTR retrotransposons.
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Genetic Analysis of Ribosome Stalling and RescueTanner, Douglas Ray 22 May 2009 (has links) (PDF)
In eubacteria, ribosome stalling on broken messenger RNA transcripts can lead to cell death. The trans-translation quality control mechanism rescues many of these stalled ribosomes. In this process, tmRNA enters stalled ribosomes by mimicking a transfer RNA, accepting the stalled nascent peptide. The ribosome then releases the broken mRNA and resumes translation on a coding region within tmRNA itself. Translation of tmRNA marks the nascent peptide for destruction by the addition of a short proteolysis tag and the ribosome is released at a stop codon within the tmRNA open reading frame. An intriguing aspect of trans-translation is that the ribosome synthesizes one protein from two RNA templates. How is the proper site chosen on tmRNA to resume translation? Do the conserved pseudoknot structures help set the reading frame? Using a genetic selection to assay libraries of tmRNA mutants, we found that stable hairpin structures can functionally replace pseudoknot 1. We conclude that the role of pseudoknot 1 in tmRNA function is purely structural. Our results demonstrate that the inactivity of an RNA mutant designed to destroy a given structure should not be interpreted as proof that the structure is necessary for RNA function. Such mutations may only destabilize a global fold that could be formed equally well by an entirely different, stable structure. Broken mRNAs are not the only cause of ribosome stalling; stalling can also result from nascent peptide interactions with the ribosomal exit tunnel that inhibit peptidyl-transferase activity. SecM, TnaC, and ErmCL all stall ribosomes to regulate the expression of downstream genes. What other peptide sequences can cause ribosome stalling? We modified our tmRNA-based selection to screen libraries of random peptides and identified a number of novel stalling peptides, including the sequence FxxYxIWPP. This sequence interacts with the exit tunnel differently than SecM and TnaC as seen in studies using mutant ribosomes. Like SecM, stalling occurs on this sequence with the next aminoacyl tRNA trapped in the A site but unable to react with the nascent peptide. These results show that a variety of peptides can interact in the exit tunnel and peptidyl-transferase center to regulate ribosome activity.
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