The Study of Ribosome Pausing by Frameshift-stimulating RNA Sequence

• Main Authors: Chieh Han, 韓捷
• Other Authors: Jin-Der Wen
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/99255670142862151453

碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 103 === In E. coli, the transcript of dnaX gene is translated into two subunits of DNA polymerase III, gamma (γ) and tau (τ), through the regulation of programmed -1 ribosomal frameshifting. There are three important elements on RNA that lead to -1 frameshifting: an internal Shine-Dalgarno (SD) sequence, a slippery sequence and a secondary structure. When a ribosome translates to the slippery sequence, 16S rRNA may interact with the internal SD. At the same time, the secondary structure may form a road block to the ribosome. These two factors may cause the ribosomes to pause and undergo frameshifting. In order to study the correlation between ribosome pausing and these two factors, we used both in vivo and in vitro methods to conduct the experiments. In our study, we used the dnaX -1 frameshifting motif as a model system. We mutated the slippery sequence to abolish frameshifting to directly observe the influence of internal SD and hairpin on ribosome pausing. To prevent polyribosome formation, we shortened the distance between the start codon (AUG) and the secondary structure to ensure that no ribosomes can initiate at the start codon when a ribosome is paused by the structure. Our results suggest that both the internal SD and secondary structure led to ribosome pausing and the secondary structure was the dominant element. Moreover, if two or more ribosomes can accommodate between the AUG and secondary structure, the frequency of ribosome pausing was decreased considerably. We also discovered the ribosome could bind to the internal SD and inhibit initiation of ribosomes on the start codon. The internal SD-bound ribosomes may drop off spontaneously or be displaced by the upstream, newly initiated ribosomes.