The Study of Polyribosome Effect on -1 Ribosomal Frameshifting Efficiency
Date Issued
2014
Date
2014
Author(s)
Wang, Hao-Che
Abstract
In Esherichia coli, DNA polymerase III holoenzyme includes γ and τ subunits that are synthesized by regulation of the -1 frameshifting translation on mRNA of dnaX. Frameshifting efficiency can be determined by some elements on mRNA, which are the slippery sequence, secondary structure and Shine-Dalgarno sequence (SD sequence). Another element is polyribosomes, which occur at high frequency of initiation and cause the decrease of frameshifting efficiency. The reason that polyribosome decreases the frameshifting is the downstream ribosome blocks the refolding of secondary structures and prevents the upstream ribosome from interacting with the secondary structures.
In our study, we use the dnaX frameshifting motif as a model system to study the influence of the density of polyribome to frameshifting efficiency in vivo and in vitro. In these studies, we control the induction time and concentration of Isopropyl β-D-1-thiogalactopyranoside (IPTG) for the in vivo experiments; in in vitro experiments, we change the translation time, the concentration of DNA plasmid or mRNA, and the fourth nucleotide of the stop codon. Our results show that with longer IPTG induction time or higher IPTG concentrations, the frameshifting efficiency was higher in in vivo experiment. For in vitro experiments, we observed that higher frameshifting efficiency comes from longer translation times or higher concentrations of the DNA plasmid or mRNA. Both results can be explained by the polyribosome effect; when mRNA concentration is increased, the ratio of ribosome to mRNA will be decreased, thus the density of polyribosome decreases to make frameshifting efficiency increased. Furthermore, the fourth nucleotide of the stop codon can influence termination efficiency; when the efficiency is too low, then the ribosome will pause longer to cause polyribosome effect and reduce frameshifting efficiency.
Subjects
dnaX
轉譯
框架位移
聚核醣體
終止密碼子
Type
thesis
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