Studies on the translation initiation factors in bacteria and mitochondria

Abstract

The process of protein synthesis is a fundamental and key event in living organisms. The process of translation initiation is assisted by specific proteins known as initiation factors. In bacteria, three initiation factors—initiation factor 1 (IF1), initiation factor 2 (IF2), and initiation factor 3 (IF3) are universally present. While these protein factors are individually assigned specific functions in the process of protein synthesis, collectively, they play integral roles in facilitating efficient protein synthesis. Among them, IF3 plays a vital role in maintaining the fidelity of translation initiation. Earlier work from the lab demonstrated that the N-terminal domain (NTD) of IF3 is involved in the crucial function of fidelity of initiator tRNA selection and initiation from the correct start codon in Escherichia coli. One part of the work described here extends our knowledge of the role of IF3 as a fidelity factor by investigating the precise mechanism by which its NTD contributes to the accuracy of translation initiation.

The endosymbiotic origin of mitochondria, from bacteria is evident in its translational machinery, which includes several conserved elements including two homologous initiation factors, mtIF2 and mtIF3. Although an independent IF1 homolog is universally absent from the mitochondrial system, IF1-like function has been shown to be carried out by the other initiation factors in the mitochondria of some organisms. However, such a phenomenon remains to be elucidated in the important model organism, Saccharomyces cerevisiae. In the literature, there is evidence that a pentatricopeptide repeat protein, Rmd9p may possess features that may support IF1-like function in S. cerevisiae. Thus, I have investigated the role of Rmd9p in potentially conferring IF1-like activity in S. cerevisiae and extended this study to explore its (Rmd9p) function in 15S rRNA (rRNA in small subunit of the mitochondrial ribosome) biogenesis.

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

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 preinitiation complex (PIC). The C-terminal domain (CTD) of IF3 can carry out most of the known functions of IF3 and sustain E. 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 of IF3 to ensure fidelity of initiation. Further, using atomistic molecular dynamics (MD) simulation runs, it was shown 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. The observations also implicate the role of these interactions in the subunit dissociation activity of IF3CTD. Overall, the study shows that the interactions between IF3NTD 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.

Part II-A: Investigation of the role of Rmd9p in mitochondrial translation in S. cerevisiae.

Mitochondria are essential for energy generation in eukaryotic cells. Mitochondrial translation machinery exhibits similarities with the bacterial one. Out of the three translation initiation factors in bacteria, only two have homologs in mitochondria, and an independent initiation factor 1 (IF1) is universally absent from the mitochondrial system. Due to the universal conservation of decoding nucleotides, the presence of an IF1-like protein in mitochondria seems relevant. Although, IF1 like functions seem to be very diverged as different organisms are reported to have other proteins involved in this function. The identity of such protein in S. cerevisiae is still unknown. Here, we have proposed Rmd9p protein as a potential IF1 candidate in S. cerevisiae based on its sequence alignment with human mitochondrial initiation factor 2 (mtIF2) and identified a stretch of residues involved in this function. Mutation of important residues from this stretch affected the mitochondrial function Rmd9p and caused an imbalance in mitochondrial translation. Further, using E. coli as a model, we showed that this stretch might be involved in IF1-like function in S. cerevisiae.

Part II-B: Role of Rmd9p in 3’-end processing of mitochondrial 15S rRNA in Saccharomyces cerevisiae

Ribosome biogenesis, involving the processing and assembly of rRNAs and ribosomal proteins is a vital process. In S. cerevisiae mitochondria, the ribosomal small subunit comprises 15S rRNA (15S). While the 15S 5'-end processing uses Ccm1p and Pet127p, the mechanisms of the 3'-end processing remain unclear. We reveal the involvement of Rmd9p in safeguarding/processing of 15S 3'-end. Rmd9p deficiency results in cleavage at a position 183 nucleotides upstream of 15S 3’-end and in the loss of the structurally and functionally important 3'-minor domain. Rmd9p binds close to the 3'-end of mature 15S in the 15S precursor, and a genetic interaction between rmd9 and dss1 indicates that Rmd9p regulates/limits mtEXO activity during the 3’-end spacer processing.