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Structural and biochemical investigation of viral translational recoding events
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Date
2024
Authors
O'Connor, Kate
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Publisher
University College Cork
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Abstract
In standard translation elongation, sequential non-overlapping codons encode amino acids, with 61 sense codons and 3 stop codons (UGA, UAG, and UAA). Translational recoding involves dynamic changes to this standard readout. One type of recoding, called readthrough, involves stop codons being read as sense codons. Other recoding events involve temporary suspension of the linear, non-overlapping reading of codons. This thesis focuses on these such recoding cases characterised by dissociation of peptidyl-tRNA from the mRNA within the ribosome, ribosome slippage, and tRNA repairing with the mRNA. Frameshifting refers to instances where the tRNA repairs with the mRNA at an overlapping codon, thereby allowing translation to continue downstream in a different reading frame. Bypassing relates to cases where repairing occurs at a non-overlapping codon, which may be in the same or a different reading frame as the upstream translated region. All documented natural instances of bypassing involve repairing at a codon 3’ of the take-off codon.
In chapter 2, the first results chapter, the mechanism of -1 programmed ribosomal frameshifting (-1PRF) in SARS-CoV-2 is investigated. Frameshifting is vital in coronaviral decoding to allow translating ribosome access open reading frame (ORF) 1b which encodes several essential proteins for virus replication. Site-directed mutagenesis is used in this chapter to examine the key features involved in stimulating frameshifting, including of the frameshift stimulatory pseudoknot and the nascent chain. Furthermore, the importance of a zero frame stop codon found 5 codons downstream of the slippery site is investigated. It is found that the stop codon is important for frameshifting when there is a high ribosome load. We hypothesise that it allows ribosomes that do not successfully frameshift to quickly terminate and disassemble from the mRNA, allowing rapid pseudoknot refolding before trailing ribosomes reach the slippery site. Finally, various potential modulators of SARS-CoV-2 frameshifting are assessed, with Merafloxacin emerging as a promising candidate for further investigation.
In Chapter 3, experimental investigations of several bioinformatically predicted novel gene 60 bypassing cases are presented and discussed. The temperature dependency of these cases is compared to that of the well-studied example of phage T4. Among them, two cases stand out. MGS12, which was sequenced from a sample taken from a 50C hot spring, was found to have a higher optimal temperature for bypassing higher than that of the E. coli phage, T4. MGS13 was found to lack temperature dependence between 12C and 37C. This is unlike T4 and the other bypassing cases tested within this temperature range, which exhibited a decrease in bypass efficiency at reduced temperatures. The temperature insensitivity of MGS13 vs T4 can be attributed primarily to the nascent chain sequence. Furthermore, the role of the A-site stem-loop (SL) of T4 is further investigated with a cryo-EM study, revealing two distinct SL structures. Intriguingly, one of these SL conformations potentially functions as a tRNA mimic, aiding the recruitment of EF-G to the ribosome.
Chapter 4 focuses on an intriguing novel gene 60 bypassing case, R13, that lacks the potential to form a tetraloop-capped SL in the ribosomal A-site at take-off. A combination of a cryo-EM structure of the E. coli ribosome stalled at the take-off site and mutational assays measuring bypass efficiency, demonstrate the lack of an A-site mRNA secondary structure. Instead, the UAG codon in the ribosomal A-site at take-off adopts an unusual conformation, with substitutions of UGA or UAA proving detrimental for bypassing. Further differences between T4 and R13 bypassing are identified, including stacking of R13 mRNA bases within the ribosomal mRNA entrance channel. Unlike T4, R13 has higher bypass efficiency under high ribosome load compared to low ribosome load. However, parallels between the two cases also emerge. The nascent chain encoded 5’ of the take-off site, is also crucial for bypassing in R13, with efficient bypassing occurring upon replacement with the nascent chain of T4. A Shine-Dalgarno-like sequence within the coding gap aids in promoting landing at the correct site in the case of R13.
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Keywords
Translational recoding , Frameshifting , Translational bypassing
Citation
O'Connor, K. M. 2024. Structural and biochemical investigation of viral translational recoding events. PhD Thesis, University College Cork.