# Regulation of Leaderless mRNA Translation in Bacteria
## 2 Mechanisms for Bacterial Initiation of Translation
In this case, the initiation codon is at or very near to the 5\({}^{\prime}\) end of the mRNA. Thus, these RNAs are practically devoid of the leader or 5\({}^{\prime}\) untranslated region (5\({}^{\prime}\) UTR) and completely lack an SD or other sequence from the 5\({}^{\prime}\) UTR that guides 30S binding for the canonical initiation of translation. In effect, in the presence of IF3, 30S does not bind to model lmRNAs in vitro [19]. Instead, lmRNA translation begins mostly by the direct binding of a 70S particle to an initiation codon that is at or very proximal to the 5\({}^{\prime}\) end of an mRNA [20; 21; 22]. In general, translation initiation factors seem to have a less relevant role in lmRNA translation than that observed for SD-led translation [20; 23]. Perhaps this is because lmRNAs bind directly to 70S particles and do not require IFs to modulate 70S formation. Nevertheless, as discussed below, IFs do alter the behavior of lmRNA translation and are necessary for the translation of at least some lmRNAs. There is limited information about the features that help with recognition of the initiation codon in lmRNA by ribosomes. It is known that, in _E. coli_, lmRNA translation starts at the first AUG site. Alternative codons such as GUG, UUG, and CUG are much less efficient, with some not showing any protein production at all, although this loss of translation efficiency is stronger in artificial [24; 25] than natural [26] lmRNAs. A similar trend is observed in some species, such as _Caulobacter crescentus_[27], while in others, initiation from non-AUG codons is more efficient; for instance, GUG in _Mycobacterium smegmatis_[25], _Saccharomyces erythnaea_[28], and _Streptomyces coelicolor_[29]. How these initiation codons are recognized is still not known. In SD-led translation, interaction between the initiation codon and initiator tRNA is essential for the recognition of the  
Figure 1: Mechanisms of translation initiation in bacteria. In bacteria, translation can be initiated by three mechanisms. Shine-Dalgarno (SD)-led translation (**left**), 70S scanning (**center**), and leaderless initiation (**right**). As shown, although most of the components are similar between these three mechanisms, translation initiation factors (IFs) have different roles. Arrows indicate either movement on mRNA or binding/unbinding of translation machinery components. Figure created with BioRender.com. Model of IF1 is based on pdb 1 AH9 [15] and model of IF2 on pdb 4b48 [16]; model of IF3 was obtained from AlphaFold (POA70) [17; 18]. initiation site. Consequently, an SD-led mRNA where the initiation codon has been replaced by an amber stop codon (UAG) will not allow translation initiation. Nevertheless, if an amber-suppressing initiator tRNA is present, canonical translation can proceed. In contrast to canonical translation, lmRNA translation cannot initiate in a similar setup, even if an amber-suppressing initiator tRNA is present. Thus, the selection of an AUG codon does not depend solely on binding by the initiator tRNA, and the mechanism behind its recognition selectivity is currently not understood [24]. In addition to the initiation codon, other characteristics are also expected to assist in lmRNA recognition by the translation machinery, as the loss of translation efficiency is smaller in natural than in synthetic lmRNAs when the initiation codon is changed from AUG to GUG (see above). For instance, it has been shown that the presence of a phosphate at the 5\({}^{\prime}\) end is required for lmRNA translation [30], and CA repeats downstream and near the start codon are known to strongly enhance translation [13]. By contrast, the distance from the 5\({}^{\prime}\) end to the start codon [27; 31] and the stability of the structures surrounding this codon [27] have been observed to strongly decrease lmRNA translation in some bacterial species.