Ribosome dynamics during spontaneous frameshifting

by Panagiotis Poulis
Doctoral thesis
Date of Examination:2022-10-04
Date of issue:2022-10-18
Advisor:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Jörg Enderlein
Persistent Address: http://dx.doi.org/10.53846/goediss-9493

 

 

Files in this item

Name:221010_Panos_thesis_final_revised.pdf
Size:11.7Mb
Format:PDF

The following license files are associated with this item:

Abstract

English

During protein synthesis, the ribosome moves along the mRNA in steps of three nucleotides at a time to maintain the translational reading frame. However, on some mRNA sequences that are denoted as slippery, ribosomes may shift by one or several nucleotides to the 5’ direction in a process called spontaneous –1 frameshifting. The result is the production of aberrant peptides with a different length and amino acid sequence than the 0-frame peptide. Frameshifting is a major potential source of translation errors, which compromise the fitness of the cell. It is thus important to understand the molecular mechanism that leads to spontaneous frameshifting. In this study, we use single molecule Förster resonance energy transfer (smFRET) to investigate how the tRNAs move through the ribosome during translocation on slippery mRNA. We investigate the internal motions of the ribosome (i.e. ribosome dynamics) and the role of EF-G during translocation on slippery mRNA. Our results show that uncoupling the movements of the two tRNA that are displaced during translocation is a key determinant of spontaneous frameshifting. While the deacylated tRNA moves rapidly from the P to the E site and is released from the ribosome, the translocation of the peptidyl-tRNA from the A to the P site is slow and stalled between intermediate (chimeric, CHI) and posttranslocation states. Delayed translocation in a different stage of translocation, i.e. prior to the formation of chimeric states, does not contribute to frameshifting. During slow translocation of the peptidyl-tRNA, the SSU head domain is in the swiveled conformation, further facilitating the positional uncertainty of the peptidyl-tRNA. This allows sampling of the 0- and –1-frame codons, thus shifting the reading frame. We anticipate our work to be the starting point for further investigation of spontaneous frameshifting, the effect of frameshifted peptides in the cell in physiological and pathological conditions, the respective mechanism of frameshifting in eukaryotes, as well as the emergence of spontaneous frameshifting as a therapeutic target in human disease and infection.
Keywords: ribosome, smFRET, translocation, frameshifting
 
Statistik