Observing Translation Initiation of the Ribosome on rpsO Transcript by smFRET
Date Issued
2015
Date
2015
Author(s)
Chang, Shu-Ya
Abstract
Many mRNAs fold into secondary structures; however, their codons must be in single-stranded form to be translated. Previous research has revealed that the ribosome itself has helicase activity during the translation process. The rpsO gene transcript of Escherichia coli regulates its own translation through the 5’ untranslated region (5’ UTR), which can fold into a pseudoknot or a double-hairpin conformation. The two structures can be interchanged spontaneously, but the ribosome can only bind to the pseudoknot to initiate translation. On the pseudoknot, only the Shine-Dalgarno (SD) sequence but not the AUG start codon is fully exposed, so the ribosome has to unwind part of the secondary structure to complete the initiation. However, it remains unclear in which stage the conformation is opened by the ribosome. In this study, we characterize the conformational change of the rpsO 5’ UTR in the presence of the 30S ribosomal subunit and initiator tRNA (charged formyl-methionyl tRNA) by using single-molecule fluorescence resonance energy transfer (smFRET). Our results show that the population of the pseudoknot form is increased when 30S binds to the RNA. 30S would begin to search for the AUG start codon after binding to the SD sequence of the pseudoknot by partially unwinding the local structures. In the presence of the initiator tRNA, 30S may completely unwind a stem of the pseudoknot and form the pre-initiation complex. These results demonstrate that the 30S ribosomal subunit alone can perform its helicase activity during the initiation stage.
Subjects
ribosome
translation initiation
single-molecule
FRET
rpsO gene
Type
thesis
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