The process of translation initiation is the most highly regulated step of protein synthesis. In bacteria, three initiation factors (IF1, IF2 and IF3) play crucial roles during initiation. IF3 acts as an anti-association factor for the two ribosomal subunits. Eubacterial IF3 also permits initiator tRNA (i-tRNA) selection at the P site of the ribosome. Two features of i-tRNA, i. e. the characteristic 3GC base pairs in the anticodon stem and the cognate interaction of the anticodon sequence with the initiation codon of the mRNA contribute to IF3 based selection and/or proofreading. However, the exact mechanism of this discrimination and the contribution of the individual domains towards this process of selection/ proofreading are unclear. Further, there are exceptional instances in the natural world where either the codon-anticodon interaction or the anticodon stem composition deviates from the norm. For instance, in mammalian mitochondria, non-AUG codons such as AUU and AUA are present in the genome although they are notoriously poor initiation codons. In addition, some species of Mycoplasma have i-tRNAs with variations in the typically conserved 3GC base pairs of the anticodon stem. In this study, we have investigated the mechanism of proofreading activity of IF3 of E. coli, mitochondrial and mycoplasmal origins. Part I: Proofreading function of IF3 in E. coli IF3 is composed of N and C terminal domains joined by a flexible linker region. By means of complete and partial IF3 knockouts, we show that the C-terminal domain (CTD) is essential for the survival of E. coli while the N-terminal (NTD) is required for cellular fitness. Using reporter assays, we have established the role of the NTD in proofreading, while polysome profile analyses reaffirm that the CTD alone can bind to the 30S and carry out ribosome anti-association. Therefore, we show that the CTD is the ribosome binding and anti-association domain, while the NTD is the major proofreading domain. Unpublished cryoEM structures from Prof. Ramakrishnan’s lab indicate that the NTD of IF3 pushes the i-tRNA at its elbow and helps in P site accommodation of the i-tRNA. We propose that when the codon-anticodon interaction is non-cognate or if the 3GC base pairs of the anticodon stem are not intact, then the dynamic action of the NTD destabilises the tRNA at the P site and leads to its rejection. Part II: Proofreading function of mitochondrial IF3 (IF3mt) Of the 13 protein-coding genes in mammalian mitochondria, 3 utilise the non-canonical AUA codon and one utilises the non-canonical start codon AUU. Since IF3mt does not possess many of the generally conserved residues implicated in proofreading, we decided to characterise the proofreading function of IF3mt and its role in initiation with non-canonical start codons. Structurally, IF3mt is similar to EcoIF3 with its N and C terminal domains joined by a linker region. However, IF3mt additionally possesses N- and C-terminal extensions which are generally disordered in structure. In vivo studies of mitochondrial translation factors have been mired by the lack of methodologies to manipulate mitochondria. We have developed an E. coli strain to study the proofreading functions of mitochondrial IF3 (IF3mt) with the help of reporter genes. Consistent with its function in mitochondria, IF3mt allowed promiscuous initiation from non-AUG codons. However, IF3mt avoided initiation with i-tRNAs lacking evolutionarily conserved 3GC pairs in anticodon stems. Interestingly, expression of IF3mt N-terminal domain or IF3mt devoid of its typical N-, and C-terminal extensions significantly improved its proofreading activity. Our immunoblot assays from polysome profile fractions indicate that the IF3mt derivative lacking extensions is capable of superior 30S ribosome binding. The two derivatives of IF3mt missing the Next (IF3mtΔNext) or both the Next and Cext (IF3mtΔNextCext) display an affinity for the 50S ribosome. We propose that the extensions of IF3mt may have evolved to reduce the affinity of IF3mt to the ribosome and thereby permit initiation with non-canonical start codons like AUU and AUA. Our studies suggest that E. coli provides an excellent heterologous model to study distinctive features of mitochondrial factors. Part III: Fidelity of translation initiation in mycoplasma One of the many singular features of mycoplasma is the presence of many anticodon stem variants of the i-tRNA across different species. In general, i-tRNAs are characterized by the presence of the typical feature of the conserved 3 consecutive GC base pairs (GC/GC/GC) in the anticodon stem. However, many mycoplasmal species have i-tRNAs with AU/GC/GC, GC/GC/GU or AU/GC/GU sequences. Interestingly, the mycoplasmal species which harbour the AU/GC/GU i-tRNA are also human pathogens. Therefore, we decided to investigate whether these organisms possess any unique features to accommodate the i-tRNA variants, by investigating the usage of Shine Dalgarno sequences and by carrying out multiple sequence alignments of genes encoding initiation factors, ribosomal proteins S9 and S13 and 16S rRNA. Since IF3 plays a crucial role in i-tRNA selection, we carried out computational analysis of mycoplasmal IF3 sequences, which revealed many interesting features. Most striking amongst them was the variation of the highly conserved R at position 131 in some species. Interestingly, these were the very mycoplasmal species which possessed the anticodon stem variant AU/GC/GU, suggesting a strong correlation between these two features. It is known that the R131P mutation of EcoIF3 is characterised by an enormous loss of proofreading activity. It seemed unusual that such compromised proofreading would be tolerated in the cell, so we decided to investigate other components of the translational machinery as well. The C-terminal SKR tail of the ribosomal protein S9, which contacts the P-site tRNA, is highly conserved across bacteria. Analysis of the C-terminal sequences of S9 proteins in various mycoplasmal species revealed a surprising variation- the presence of a TKR tail in strains with the AU/GC/GU tRNA. In this study we have investigated the co-occurrence of S9 and IF3 variations in i-tRNA selection in E. coli. We see that the R131P polymorphism of IF3 leads to a tremendous loss of proofreading, but this loss is significantly tempered by the presence of the S9 TKR variation. Our bioinformatics studies revealed that the mycoplasmal species which are sustained on AU/GC/GU i-tRNAs also tend to use a higher percentage of non-AUG codons. By means of our reporter assays in E. coli, we have shown once again that the R131P polymorphism of IF3 leads to a tremendous increase in initiation with the non-canonical start codon AUA, but this increase is significantly tempered by the presence of the S9 TKR variation.