Abstract
RNA messenger stability is in uenced by how well it is protected from ribonuclase degradation. The level and distribution of translating ribosomes on the messenger has an effect on messenger stability, which is tuned partly by the nature of the ribosome binding site and partly by the codon composition of the coding sequence of a gene.
This thesis focussed on illuminating the impact of ribosome binding sites on the functional messenger half-life using a range of ribosome binding sites with altered binding strength. Furthermore, the additive in uence of the transcription terminator, Rho, on messenger stability was examined. Depending on the translation initiation frequency, the chance of an initial ribosome trailing the RNA polymerase get better for better initiation sites, thus protecting transcription from termination by Rho. A polarity assay in which the activity of the downstream lacA in the lac operon was measured, demonstrated that Rho dependent transcriptional pre-termination correlated with ribosome translation initiation frequency. It was further shown that Rho terminates RNA polymerases during transcription on messengers normally considered stable, suggesting Rho transcriptional pre-termination to be a global phenomena in gene regulation.
The influence of codon usage in the early coding region on messenger stability was examined, in order to establish how fast or slow the ribosome has to decode the sequence for it to protect the messenger from degradation. The experiments demonstrated that very fast translation in the beginning of the coding region results in fast messenger degradation; hence the ribosome should not be too fast in clearing the region. A proposed model in which the anti-termination protein NusG has not yet bound to the RNA polymerase before the ribosome catches up with it and possibly pulls out the nascent transcript.
Finally this thesis also focussed on the cell stress caused by an artificial and very strong RNA psudoknot used in a fusion gene. The technique of ribosomal foot printing and whole transcriptome deep sequencing was employed to facilitate an overview of the changes in gene expression upon induction of the pseudoknot construct. It was demonstrated that typical genes associated with the heat stress response were upregulated -a response that suggested the pseudoknot construct possibly conferred misfolding of the fusion gene or were unable to release ribosomes for reinitiation in the translation cycle.
This thesis focussed on illuminating the impact of ribosome binding sites on the functional messenger half-life using a range of ribosome binding sites with altered binding strength. Furthermore, the additive in uence of the transcription terminator, Rho, on messenger stability was examined. Depending on the translation initiation frequency, the chance of an initial ribosome trailing the RNA polymerase get better for better initiation sites, thus protecting transcription from termination by Rho. A polarity assay in which the activity of the downstream lacA in the lac operon was measured, demonstrated that Rho dependent transcriptional pre-termination correlated with ribosome translation initiation frequency. It was further shown that Rho terminates RNA polymerases during transcription on messengers normally considered stable, suggesting Rho transcriptional pre-termination to be a global phenomena in gene regulation.
The influence of codon usage in the early coding region on messenger stability was examined, in order to establish how fast or slow the ribosome has to decode the sequence for it to protect the messenger from degradation. The experiments demonstrated that very fast translation in the beginning of the coding region results in fast messenger degradation; hence the ribosome should not be too fast in clearing the region. A proposed model in which the anti-termination protein NusG has not yet bound to the RNA polymerase before the ribosome catches up with it and possibly pulls out the nascent transcript.
Finally this thesis also focussed on the cell stress caused by an artificial and very strong RNA psudoknot used in a fusion gene. The technique of ribosomal foot printing and whole transcriptome deep sequencing was employed to facilitate an overview of the changes in gene expression upon induction of the pseudoknot construct. It was demonstrated that typical genes associated with the heat stress response were upregulated -a response that suggested the pseudoknot construct possibly conferred misfolding of the fusion gene or were unable to release ribosomes for reinitiation in the translation cycle.
| Original language | English |
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| Publisher | Department of Biology, Faculty of Science, University of Copenhagen |
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| Number of pages | 134 |
| Publication status | Published - 2015 |