Abstract
Substitution of the conserved Histidine 448 present in one of the three consensus elements characterizing the guanosine nucleotide binding domain (IF2 G2) of Escherichia coli translation initiation factor IF2 resulted in impaired ribosome-dependent GTPase activity which prevented IF2 dissociation from the ribosome, caused a severe protein synthesis inhibition, and yielded a dominant lethal phenotype. A reduced IF2 affinity for the ribosome was previously shown to suppress this lethality. Here, we demonstrate that also a reduced IF2 affinity for fMet-tRNA can suppress this dominant lethal phenotype and allows IF2 to support faithful translation in the complete absence of GTP hydrolysis. These results strengthen the premise that the conformational changes of ribosome, IF2, and fMet-tRNA occurring during the late stages of translation initiation are thermally driven and that the energy generated by IF2-dependent GTP hydrolysis is not required for successful translation initiation and that the dissociation of the interaction between IF2 C2 and the acceptor end of fMet-tRNA, which represents the last tie anchoring the factor to the ribosome before the formation of an elongation-competent 70S complex, is rate limiting for both the adjustment of fMet-tRNA in a productive P site and the IF2 release from the ribosome.
Highlights
Initiation factor IF2 is a multi-domain GTPase belonging to the subfamily of translational GTPases [1] containing a GTP/GDP/ppGpp binding domain (IF2G-2) comprising the same highly conserved elements implicated in guanosine nucleotide binding and hydrolysis found in other ribosomal GTPases such as EF-Tu, EF-G, and RF3 [2,3,4]
We show that an IF2 mutant totally lacking GTPase activity and with a strongly reduced affinity for fMet-tRNA can suppress the dominant lethal phenotype associated with the GTPase inactivation and can promote the formation of a productive 70S EC and faithful mRNA translation
Binding experiments carThe capacity of IF2 mutants to promote the binding of fMet-tRNA to mRNA-prograried out with mant-GTP showed that upon incubation of this ligand with IF2 wt and with mmed 30S ribosomal subunits in the presence of IF1 and IF3, thereby forming 30S initiation complex (30S IC), all IF2 mutants there is an increase of the amplitude of mant-GTP fluorescence
Summary
Initiation factor IF2 is a multi-domain GTPase belonging to the subfamily of translational GTPases [1] containing a GTP/GDP/ppGpp binding domain (IF2G-2) comprising the same highly conserved elements implicated in guanosine nucleotide binding and hydrolysis found in other ribosomal GTPases such as EF-Tu, EF-G, and RF3 [2,3,4].IF2 binds to both 30S and 50S ribosomal subunits and plays an essential function in all the initiation steps of translation. The nature of the events occurring during the long (~200 ms) time lag, which follows GTP hydrolysis and precedes 70S EC formation, has been the object of numerous structural [3,4,8,9,10,11,12,13,14,15] and kinetic [5,6,16,17,18,19,20,21,22,23] investigations The results of these studies demonstrate that before an “initiation dipeptide” (i.e., the peptide made between fMet and the amino acid corresponding to the second mRNA codon) is formed and IF2 is recycled off the ribosomes, a number of conformational changes of the ribosome and of its ligands (fMet-tRNA and IF2) must take place. There is universal consensus that the structure of IF2-GTP is substantially different from that of IF2-GDP [3,8,24] and that the presence of IF2-bound
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