SUMMARY Leaderless mRNAs lack a 5’ UTR and SD sequence. Such mRNAs are common in Bacteria and Archaea, with Mycobacteria being the most striking example, as about one-third of mycobacterial mRNAs are naturally leaderless. Leaderless translation is part of a stress response and adaptation mechanism in bacteria. In contrast to the well-understood Shine Dalgarno-containing mRNAs, very little is known about the initiation mechanism of leaderless mRNAs. Biochemical, experimental, and systematic analysis of leaderless mRNA expression suggested that leaderless mRNAs are translated and regulated via alternative mechanisms. However, how this initiation is occurring and regulated remains unknown. Thus, to understand bacterial physiology and antibiotic resistance, we need to reveal the molecular mechanism of leaderless translation. This proposal aims to address this need. We will use single-molecule methods that are particularly well suited for the dissection of dynamic processes, such as translation. We developed a single-molecule fluorescence system that allows us to observe the entire initiation process. We can directly follow mRNA, ribosomal subunits, and translation factors, thus defining how initiation is occurring and regulated. In aim 1, we will determine how leaderless mRNAs are recruited to the ribosomes and how the start codon is recognized. We will directly test for the existence of the proposed alternative initiation mechanisms. Namely, we will define if mRNAs are directly recruited to the 70S ribosomes. We will then determine if start codon recognition is concurrent with mRNA recruitment, as was previously proposed. We will also determine the role of initiation factors in regulation of leaderless translation. In Aim 2, we will focus on the roles of mRNA sequence and structure. Translation efficiency of leaderless mRNAs is regulated by the start codon sequence, presence of nucleotides upstream of the start codon, and mRNA secondary structure. We will use our single-molecule toolkit to determine how these elements regulate translation. In particular, we are interested in how they affect alternative initiation pathways.