Monitoring the late events of translation initiation in real-time

by Akanksha Goyal
Doctoral thesis
Date of Examination:2015-11-30
Date of issue:2016-10-19
Advisor:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Heike Krebber
Referee:Prof. Dr. Heinz Neumann
Referee:Prof. Dr. Ralf Ficner
Referee:Prof. Dr. Markus Bohnsack
Referee:Prof. Dr. Wolfgang Wintermeyer
Persistent Address: http://dx.doi.org/10.53846/goediss-5910

 

 

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Abstract

English

In bacteria, translation initiation is promoted by three initiation factors (IF1, IF2 and IF3) and entails three distinct steps. First, the small ribosomal subunit (30S) recruits the initiation factors, initiator tRNA (fMet-tRNAfMet), mRNA and GTP to form a 30S pre-initiation complex (IC). During the next step, the complex matures into a 30S IC upon start codon-anticodon recognition between the mRNA and fMet-tRNAfMet. Finally, the large ribosomal subunit (50S) associates with the 30S IC giving rise to a 70S IC which can participate in the first peptide bond formation. Here, the timing and control of reactions that occur after subunit joining are studied with the help of rapid kinetic techniques and fluorescence-labeled translation components. A detailed kinetic model, based on global fitting of time courses, obtained with ten different reporters, is presented. The interplay between IF1 and IF3, as well as the role of IF2-dependent GTP hydrolysis in promoting 70S IC formation is studied. We observe that IF1 and IF3 together affect the rate of subunit joining, but not the following steps of 70S IC maturation. GTP hydrolysis does not regulate IF3 dynamics but is required for the efficient dissociation of fMet-tRNAfMet from IF2. The absence of GTP hydrolysis also prevents the dissociation of IF1 and IF2 from the 70S ribosome and the equilibrium is shifted towards a stable 70S–IF1–IF2–mRNA–fMet-tRNAfMet complex, suggesting that GTP hydrolysis guides the irreversible transition of the 70S complex into an elongation-competent state.
Keywords: ribosome; translation
 
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