Indefinite. Restriction lift date: 10000-01-01
Genetic decoding dynamics: from its exploitation to enhance or rescue gene expression to a search for relevance for early evolution
University College Cork
The genetic code and its readout were initially thought to be ‘frozen’ in the sense of being a fixed entity who’s decoding invariably involved standard rules. Recoding involves exceptions to these standard rules that are embedded in mRNA sequences. Elements in the mRNA can alter the meaning of specific codons or cause ribosomes to shift frame or with particular relevance to this thesis, result in ribosomes bypassing long untranslated regions. 30 – 50% of ribosomes successfully bypass a fifty-nucleotide insertion in phage T4 gene 60 mRNA. This coding gap begins with an in-frame stop codon and is flanked by matching GGA glycine codons. Bypassing is stimulated by a mixture of signals including a nascent peptide and a mRNA stem loop which includes the take-off codon at the start of the coding gap. An additional stem loop whose 3’ end is nine nucleotides 5’ of the take-off codon, was recently demonstrated to be important for bypassing in vitro. I investigated the effect of this structure in vivo. The structure is important in vivo when the number of ribosomes translating gene 60 is very small. There is good evidence that the 5’ stem loop functions to provide forward momentum for bypassing ribosomes in the 5’ region of the coding gap, and it may also possibly have a role in preventing backward movement. No effect for the 5’ stem loop was observed in high expression systems and this was deduced to be due to the following ribosome performing what is normally carried out by this structure, giving bypassing ribosomes forward momentum. Prior work claimed that mRNA structure 3’ of the site of coding resumption influenced bypassing efficiency. In vivo data reported here does not support this under the conditions tested with either multiple or few ribosomes translating the gene 60 sequence. While half to one third of ribosomes successfully bypass the non-coding sequence, earlier experiments provided strong evidence that the efficiency of peptidyl-tRNA dissociation from the take-off codon at the start of the coding gap is 100%. Therefore, the ability of ribosomes to recognise the glycine landing codon and resume translation is key for efficient bypassing. The identity of the matching codons is important for efficient bypassing with combinations of matched codons other than GGA permitting bypassing. Variants at the resume codon position had not previously been tested. The present work shows that slow-to-decode codons had the largest effect on bypassing when present as the resume codon. Bypassing and frameshifting ribosomes enter a non-canonical state during the respective recoding events, potentially affecting their ability to resume translation immediately afterwards. When bypassing ribosomes resume translation, we showed that they decode several codons before fully regaining standard translational properties, consistent with earlier more indirect inferences. Productive translational bypassing was first identified twenty-eight years ago, yet new cases of bypassing have not been abundant. Recently, 81 potential cases of bypassing were discovered in the yeast Magnusiomyces capitatus with 3 cases being experimentally validated. We have identified new bypassing candidates in Streptomyces coelicolor. These bacteria are proposed to take advantage of the presence of a rare UUA codon to stimulate bypassing. Others have provided suggestive evidence that certain Streptomyces phages also utilize translational bypassing. Extensive and costly searches for life on Mars are underway. However, no such search for radical new life has been undertaken here on Earth. The likely theory of the RNA World postulates that an RNA-organism predated the emergence of DNA and protein catalysts. We undertook a multidisciplinary search of deep earth cores and pockets of ancient water that have been isolated from modern genetic organisms. We initiated a search for niches that might contain RNA in the absence of ribosomal RNA, and much more extensive further investigations are merited.
Recoding , Gene 60 , Translational bypassing
Coakley A. J. 2016. Genetic decoding dynamics: from its exploitation to enhance or rescue gene expression to a search for relevance for early evolution. PhD Thesis, University College Cork.