Initiating translation is complex because it involves multiple molecules. Initiator tRNA, ribosomal subunits, and eukaryotic initiation factors (eIFs) are all required to assemble on the initiation codon of mRNA. This process consists of several steps that are mediated by different eIFs.

First, the initiator tRNA must be selected from the pool of elongator tRNAs by eukaryotic initiation factor 2 (eIF2). The initiator tRNA (Met-tRNAi) has conserved sequence elements including modified bases at specific positions. This makes Met-tRNAi different from other

tRNAs carrying Methionine.

Next, the eIF2/GTP/Met-tRNAi ternary complex and other eIFs bind to the small ribosomal subunit to form a 43S preinitiation complex. Before the preinitiation complex binds the mRNA, to make sure that a correctly processed mRNA is translated, the cell uses initial recognition of the 5’ cap of the mRNA by the eIF4E subunit of eIF4F.

Most eukaryotic mRNAs are monocistronic, that is, they encode only a single protein. Once the preinitiation complex is bound to the mRNA, the complex moves forward to search for the first AUG triplet, which is usually 50–100 nucleotides downstream of the 5′-terminal cap.

During this scan, the nucleotides adjacent to the start codon affect the efficiency of codon recognition. If the recognition site is substantially different from the consensus recognition sequence (5ʹ-ACCAUGG-3ʹ), the preinitiation complex may skip over the first AUG triplet in the mRNA and continue scanning to the next AUG. This phenomenon is known as “leaky scanning.” Several viruses, such as the human papillomavirus, use leaky scanning as the predominant mechanism during translation. Other cells also frequently use this to produce multiple proteins from the same mRNA molecule.

Bacterial ribosomes can assemble directly on a start codon that lies several nucleotides downstream of the Shine-Dalgarno sequence. So, a single molecule of bacterial mRNA can code for several different proteins, which makes bacterial mRNAs polycistronic.