The initiation factor 3 (IF3) residues interacting with initiator tRNA elbow modulate the fidelity of translation initiation and growth fitness in Escherichia coli

Abstract

Initiation factor 3 (IF3) regulates the fidelity of bacterial translation initiation by debarring the use of non-canonical start codons or non-initiator tRNAs and prevents premature docking of the 50S ribosomal subunit to the 30S pre-initiation complex (PIC). The C-terminal domain (CTD) of IF3 can carry out most of the known functions of IF3 and sustain Escherichia coli growth. However, the roles of the N-terminal domain (NTD) have remained unclear. We hypothesized that the interaction between NTD and initiator tRNAfMet (i-tRNA) is essential to coordinate the movement of the two domains during the initiation pathway to ensure fidelity of the process. Here, using atomistic molecular dynamics (MD) simulation, we show that R25A/Q33A/R66A mutations do not impact NTD structure but disrupt its interaction with i-tRNA. These NTD residues modulate the fidelity of translation initiation and are crucial for bacterial growth. Our observations also implicate the role of these interactions in the subunit dissociation activity of CTD of IF3. Overall, the study shows that the interactions between NTD of IF3 and i-tRNA are crucial for coupling the movements of NTD and CTD of IF3 during the initiation pathway and in imparting growth fitness to E. coli.

RNase-mediated reprogramming ofYersiniavirulence

“…Our RNA-Seq analysis comparing the transcription profiles of the wildtype and the Δ rnc mutant revealed that the transcript levels of 11 ribosomal proteins (r-proteins), as well as several translation initiation factors and rRNA/tRNA-modifying enzymes, were significantly altered with a log 2 fold change of +/- ≤1 (Table S1 ). Among them are (i) the r-protein transcripts of rpsJ (S10, log 2 = 3.059) and rplC (L3, 2.784) encoded by the S10 operon involved in rRNA and r-protein regulation [65]; (ii) the translational initiation factor IF-3 (InfC) (log 2 of 1.02), an essential bona fide factor that promotes 30S initial complex formation, accurate tRNA selection, and fidelity of bacterial translation initiation [66][67], (iii) the 16S rRNA methyltransferase RsmG/GidB with the ability to enhance the loading of r-proteins and 30S ribosome assembly in the presence of the 5’-leader [68], and (iv) the 16S rRNA-processing protein RimM assisting in the assembly of the 30S subunit, and promoting translation efficiency [69][70]. The higher abundance of these translation factors in the Δ rnc mutant could also increase (or assist CsrA-mediated activation of) lcrF translation and thus lcrF mRNA stability.…”

“…It is also possible that other translation-relevant factors that were shown to be differrentially expressed in the Δ rnc mutant support this CsrA function or enhance translation initiation separately, which could increase ribosome coverage and thus lcrF transcript stability. Among these factors are 11 ribosomal proteins, and the essential bona fida translation initiation factor IF-3 (InfC) promoting 30S initial complex formation [66][67]. Moreover, transcripts of the 16S rRNA methyltransferase RsmG/GidB and the 16S rRNA-processing protein RimM were significantly increased in the absence of RNase III which both also have a positive influence on translation initiation, e.g.…”

Fidelity of translation initiation in bacteria: an initiator tRNA-centric view

“…Our in vivostudies on the individual domains led to characterization of a crucial role of the NTD in the fidelity i-tRNA selection (Ayyub et al 2017). Subsequently, we showed that IF3 NTD interactions with the elbow region of i-tRNA are crucial for the movements of the two domains of IF3 and in the fidelity of P-site binding of i-tRNA (Singh et al 2022). Other studies revealed that the genetic interactions between uS12 and IF3 also play a role in i-tRNA selection (Datta, Singh, et al 2021).…”

“…More recently, based on the analysis of lamotrigine toxicity (which targets IF2), we showed that this role of i-tRNA in maturation of 16S rRNA is mediated through IF2 and i-tRNA complex bound to the 30S. Also, lamotrigine mediated inhibition of ribosome biogenesis led to an increased accumulation of ribosome binding factor A (RbfA), a late stage ribosome biogenesis factor, on 30S (Singh et al 2023).…”

Engineering Ribosomal Machinery for Noncanonical Amino Acid Incorporation

“…Although all initiation factors can randomly interact with the 30S ribosomal subunit independently, there is a kinetically favored sequence of events: IF-3, IF-2, IF-1, and then specific binding of fMet-tRNA fMet . IF-3 acts as a gatekeeper in choosing the starting tRNA fMet from among other elongator aatRNAs as well as ensuring accurate interactions with the start codon on the mRNA, while IF-2 is a GTPase (like many quality control factors) that recruits the initiator fMet-tRNA fMet and ensures docking of the large (50S) ribosomal subunit with the appropriately loaded PIC. , IF-1 binds to the A-site of a small subunit to prevent aatRNA loading as well as stabilize both IF-2 and IF-3. , When the start codon on the mRNA is recognized, an induced conformation change stabilizes fMet-tRNA fMet binding and leads to dissociation of all initiation factors, which in turn leads to recruitment of the 50S ribosomal subunit and ultimately formation of the mature 70S initiation complex. , …”