Kinetic Dissection of Translation Initiation in Prokaryotes.

Abstract

Ribosomes are nucleoprotein complexes which synthesize proteins in the cells. The process of translation can be subdivided into several phases: translation initiation, elongation, termination and ribosome recycling. The prokaryotic initiation system requires the small and large ribosomal subunits (30S and 50S subunits, respectively), initiator tRNA (fMet-tRNAfMet), mRNA, and three initiation factors IF1, IF2, IF3. There are two main steps in initiation. During the first step, fMet-tRNAfMet and mRNA bind the 30S subunit with the help of initiation factors forming a 30S initiation complex (IC). In the second step, the 50S subunit binds the 30S IC, whereas IF1 and IF3 dissociate from the complex in a stepwise manner. The docking of the 50S subunit triggers GTP hydrolysis by IF2 which leads to the dissociation of the latter. The resulting complex is able to progress to elongation. During the first part of the present work GTPase activity of IF2 was studied. We measured the rate of GTP hydrolysis by IF2 using a novel fluorescence-based assay. These measurements, in conjunction with the experiments on dissociation of IF1 and IF3, allowed for placing the GTPase reaction in the model of late events in translation initiation. The rate of GTP did not depend on either substrate, or the 50S subunit concentration. Based on these observations we suggest that IF2 undergoes conformational rearrangements after the 50S subunit docking possibly involving the GTPase activation. In the second part of the present work influence of mRNA regulatory elements (Shine-Dalgarno (SD) sequence, initiation codon) on the mRNA binding to the 30S subunit was studied. Our work shows that the primary arrival of mRNA to the ribosome is independent from its TIR structure. TIR was shown to play an important role on the later steps of mRNA stabilization on the 30S IC. We also found that the presence of initiation factors and/or fMet-tRNAfMet in the 30S IC does not influence the primary docking of the mRNA. We conclude that during the assembly of 30S PIC mRNA binds to the complex in parallel but independent on the other components. We also showed that there is an affinity switch upon start codon recognition. mRNAs with AUG codon in the P site are stabilized in the 30S PIC if fMet-tRNAfMet is present. Thus, on the basis of kinetic constants determined in the current work, we identified potential checkpoint for mRNA selection during translation initiation. The aim of the third part of the present project was to crystallize translation initiation complex which consists of 70S ribosome, IF2-GDPNP, mRNA, fMet-tRNAfMet. To date there is no structure of the prokaryotic 70S IC solved by X-ray analysis. It is known from cryo-EM studies that IF2 undergoes structural rearrangements on the ribosome after 50S subunit joining to 30S IC and after the event of GTP hydrolysis. Solving the structure of 70S IC by X-ray analysis would provide important information about the conformation and contacts of IF2 with the ribosome during the dynamic process of translation initiation. In the present work we reconstituted the translation initiation system from thermophilic bacterium Thermus thermophilus and developed the procedure for 70S IC purification for crystallization purposes.