Kinetic models of sequential initiation events upon polysome formation

by Irena Andreeva
Doctoral thesis
Date of Examination:2016-05-09
Date of issue:2017-09-08
Advisor:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Heinz Neumann
Referee:Prof. Dr. Holger Stark
Persistent Address: http://dx.doi.org/10.53846/goediss-6471

 

 

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Abstract

English

Polysomes are active protein-synthesizing units consisting of multiple ribosomes assembled along one mRNA. The frequency of ribosome loading on the mRNA defines the number of ribosomes per polysome, which is an important regulator of the proteomic output from the transcriptome. The mechanisms that regulate the frequency of ribosome loading on the mRNA, and thus protein abundance are largely unknown. Here we have studied ribosome loading in real-time in vitro employing fluorescence resonance energy transfer (FRET), rapid kinetics, and global fitting approaches. We monitored how the first ribosome in a polysome moves along the mRNA away from the initiation site, whereas the second ribosome starts the initiation on the same mRNA. We established novel approaches for monitoring mRNA recruitment by efficient FRET between the 5ʹ-end of model mRNAs and proteins from the initiation machinery. We recapitulated in vitro the overall mRNA loading frequency reported in vivo and explored how the rate of clearance of the initiation site from the first ribosome and the secondary structures around the ribosome binding site (RBS) affected ribosome loading. We compared the mechanisms of translation initiation of the first and the second ribosomes and measured the initiation and elongation rate constants. We showed that the recruitment of the second ribosome happened co-translationally and was RBS specific. We obtained a set of elemental rates for each model mRNA in the process of the second ribosome recruitment. The analysis revealed that the 30S PIC recruitment followed two kinetically different mechanisms depending on the mRNA used. These results suggested that ribosome recruitment into a polysome may comprise an important regulatory step that defines the frequency of translation of a given mRNA. Taken together, this study provides quantitative new insights into the interactions of the 30S PIC with various mRNAs in the first step of polysome formation
Keywords: Ribosomes
 
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