Contributions of the N- and C-Terminal Domains of Initiation Factor 3 to Its Functions in the Fidelity of Initiation and Antiassociation of the Ribosomal Subunits

Initiation factor 3 (IF3) is one of the three conserved prokaryotic translation initiation factors essential for protein synthesis and cellular survival. Bacterial IF3 is composed of a conserved architecture of globular N-and C-terminal domains (NTD and CTD) joined by a linker region. IF3 is a ribosome antiassociation factor which also modulates selection of start codon and initiator tRNA. All the functions of IF3 have been attributed to its CTD by in vitro studies. However, the in vivo relevance of these findings has not been investigated. By generating complete and partial IF3 (infC) knockouts in Escherichia coli and by complementation analyses using various deletion constructs, we show that while the CTD is essential for E. coli survival, the NTD is not. Polysome profiles reaffirm that CTD alone can bind to the 30S ribosomal subunit and carry out the ribosome antiassociation function. Importantly, in the absence of the NTD, bacterial growth is compromised, indicating a role for the NTD in the fitness of cellular growth. Using reporter assays for in vivo initiation, we show that the NTD plays a crucial role in the fidelity function of IF3 by avoiding (i) initiation from non-AUG codons and (ii) initiation by initiator tRNAs lacking the three highly conserved consecutive GC pairs (in the anticodon stem) known to function in concert with IF3. IMPORTANCE Initiation factor 3 regulates the fidelity of eubacterial translation initiation by ensuring the formation of an initiation complex with an mRNA bearing a canonical start codon and with an initiator tRNA at the ribosomal P site. Additionally, IF3 prevents premature association of the 50S ribosomal subunit with the 30S preinitiation complex. The significance of our work in Escherichia coli is in demonstrating that while the C-terminal domain alone sustains E. coli for its growth, the N-terminal domain adds to the fidelity of initiation of protein synthesis and to the fitness of the bacterial growth

Antimicrobial activity of fusidic acid in Escherichia coli is dependent on the relative levels of ribosome recycling factor and elongation factor G

During protein synthesis, elongation factor G (EFG) participates at the steps of translocation and ribosome recycling. Fusidic acid (FA) is a bacteriostatic antibiotic, which traps EFG on ribosomes, stalling them on mRNAs. How the bacterial susceptibility to FA is determined, and which of the two functions of EFG (translocation or ribosome recycling) is more vulnerable, has remained debatable. The in vivo studies addressing these aspects of FA mediated inhibition of protein synthesis are lacking. Here, we used a system of Escherichia coli strains and their complementation/supplementation with the plasmid borne copies of the inducible versions of EFG and ribosome recycling factor (RRF) genes. Additionally, we investigated FA sensitivity in a strain with increased proportion of stalled ribosomes. We show that the cells with high EFG/RRF (or low RRF/EFG) ratios are more susceptible to FA than those with low EFG/RRF (or high RRF/EFG) ratios. Our in vivo observations are consistent with the recent in vitro reports of dependence of FA susceptibility on EFG/RRF ratios, and the notion that an overriding target of FA is the translocation function of EFG. An applied outcome of our in vivo study is that FA mediated growth inhibition could be facilitated by depletion or inactivation of cellular RRF

Coevolution of the translational machinery optimizes initiation with unusual initiator tRNAs and initiation codons in mycoplasmas

Initiator tRNAs (i-tRNAs) are characterized by the presence of three consecutive GC base pairs (GC/GC/GC) in their anticodon stems in all domains of life. However, many mycoplasmas possess unconventional i-tRNAs wherein the highly conserved sequence of GC/GC/GC is represented by AU/GC/GC, GC/GC/GU or AU/GC/GU. These mycoplasmas also tend to preferentially utilize non-AUG initiation codons. To investigate if initiation with the unconventional i-tRNAs and non-AUG codons in mycoplasmas correlated with the changes in the other components of the translation machinery, we carried out multiple sequence alignments of genes encoding initiation factors (IF), 16S rRNAs, and the ribosomal proteins such as uS9, uS12 and uS13. In addition, the occurrence of Shine-Dalgarno sequences in mRNAs was analyzed. We observed that in the mycoplasmas harboring AU/GC/GU i-tRNAs, a highly conserved position of R131 in IF3, is represented by P, F or Y and, the conserved C-terminal tail (SKR) of uS9 is represented by the TKR sequence. Using the Escherichia coli model, we show that the change of R131 in IF3 optimizes initiation with the AU/GC/GU i-tRNAs. Also, the SKR to TKR change in uS9 was compatible with the R131P variation in IF3 for initiation with the AU/GC/GU i-tRNA variant. Interestingly, the mycoplasmas harboring AU/GC/GU i-tRNAs are also human pathogens. We propose that these mycoplasmas might have evolved a relaxed translational apparatus to adapt to the environment they encounter in the host

Coevolution of the translational machinery optimizes initiation with unusual initiator tRNAs and initiation codons in mycoplasmas

<p>Initiator tRNAs (i-tRNAs) are characterized by the presence of three consecutive GC base pairs (GC/GC/GC) in their anticodon stems in all domains of life. However, many mycoplasmas possess unconventional i-tRNAs wherein the highly conserved sequence of GC/GC/GC is represented by AU/GC/GC, GC/GC/GU or AU/GC/GU. These mycoplasmas also tend to preferentially utilize non-AUG initiation codons. To investigate if initiation with the unconventional i-tRNAs and non-AUG codons in mycoplasmas correlated with the changes in the other components of the translation machinery, we carried out multiple sequence alignments of genes encoding initiation factors (IF), 16S rRNAs, and the ribosomal proteins such as uS9, uS12 and uS13. In addition, the occurrence of Shine-Dalgarno sequences in mRNAs was analyzed. We observed that in the mycoplasmas harboring AU/GC/GU i-tRNAs, a highly conserved position of R131 in IF3, is represented by P, F or Y and, the conserved C-terminal tail (SKR) of uS9 is represented by the TKR sequence. Using the <i>Escherichia coli</i> model, we show that the change of R131 in IF3 optimizes initiation with the AU/GC/GU i-tRNAs. Also, the SKR to TKR change in uS9 was compatible with the R131P variation in IF3 for initiation with the AU/GC/GU i-tRNA variant. Interestingly, the mycoplasmas harboring AU/GC/GU i-tRNAs are also human pathogens. We propose that these mycoplasmas might have evolved a relaxed translational apparatus to adapt to the environment they encounter in the host.</p