# Regulation of Leaderless mRNA Translation in Bacteria
## 2 Mechanisms for Bacterial Initiation of Translation  
Before turning our attention to the translation of lmRNAs, we will briefly introduce the canonical or Shine-Dalgarno (SD)-led initiation of translation (Figure 1, left), which will be used as a reference throughout this text. In bacteria, translation usually begins with the formation of a complex between the ribosome binding site (RBS) on the mRNA, the small subunit of the ribosome (30S), and the initiator tRNA charged with \(N\)-formylmethionine (fMet-tRNA(Met)). The RBS is recognized through interactions between a sequence located at the ribosomal \(3^{\prime}\) end (anti-SD (aSD) sequence) and a complementary sequence located upstream of the coding sequence (SD sequence). Moreover, the binding of the initial codon to the anticodon of the initiator tRNA and an unfolded region where the 30S particle can bind mRNA are of utmost importance to allow the correct recognition of the RBS [5; 6; 7]. In addition to the role played by the aSD sequence and the initiator tRNA, the bS1 protein (previously S1 [8]) assists in canonical translation. In _E. coli_, this large ribosomal protein of the small subunit is composed of six \"S1\" domains, each one presenting an OB fold that is usually associated with RNA binding. In fact, most of these domains in bS1 bind to RNA and participate in the unfolding of structured RNA. This unfolding is usually required to increase the accessibility of the SD and initiation codon, essential for SD-led translation. Additionally, the RNA-binding domains of bS1 assist in mRNA recognition by binding enhancer sequences located upstream of the SD. Consequentially, bS1 is essential for canonical translation in proteobacteria, particularly when the \(5^{\prime}\) UTR is strongly structured. However, in other groups, such as low G/C content Gram-positive bacteria (e.g., _Bacillus_), bS1 is absent from the ribosomes [9; 10; 11; 12]. Finally, in addition to the role of the \(5^{\prime}\) UTR in RBS recognition, sequences downstream of the starting codon may also affect ribosome binding [13]. After the formation of a 30S-mRNA complex, the large subunit (50S) can bind to form a complete ribosome (70S) at the RBS, allowing translation to begin with the entrance of the first elongator aa-tRNA and peptide bond formation. The entire process is accompanied and enhanced by initiation factors (IF) 1, 2, and 3, which ensure that translation initiates only if a canonical RBS and initiator tRNA have bound to the 30S. For example, IF2 recruits the fMet-tRNA(Met) initiator tRNA, while IF3 enhances the selection of initiator over elongator tRNAs, interferes with the large subunit's association, and helps to discriminate against sites that differ from the canonical RBS. IF1 enhances the activity of the two other initiation factors. The dissociation or displacement of all these factors is required to allow the binding of the 50S subunit and formation of a complete ribosomal particle [1]. In addition to the canonical mechanism of translation initiation, bacteria can initiate translation through at least two other mechanisms [1]. One is 70S scanning, also called translational coupling or readthrough (Figure 1, center). In this mechanism, the ribosome does not dissociate to 30S and 50S subunits after finishing the translation of a cistron. Instead, it continues to be associated with the mRNA and slides downstream until it reaches a canonical RBS where it can initiate translation. In addition to allowing initiation of genes downstream of canonical genes, 70S scanning allows the initiation of genes located at the beginning of a transcript, as long as the \(5^{\prime}\) UTR is unstructured and there are 70S particles available for initiation. Similar to canonical initiation, IF3 is required for this process. By contrast, IF1 is not essential, although it is strongly stimulatory [4; 14]. Finally, translation can also initiate from lmRNAs (Figure 1, right), which are mRNAs that lack a canonical RBS.