 The initiation of Prokaryotic translation commences with the small subunit and is catalyzed by three translation initiation factors called IF1, IF2 and IF3. Each factor facilitates a key step in the initiation process. So, IF1 is the first initiation factor. It prevents tRNAs from binding to the portion of the small subunit that will become part of the A site. IF2 is a GTPase that interacts with three key components of the initiation machinery. That is the small subunit, the IF1 and the charged initiator tRNA which is F-Met tRNA. By interacting with these components, IF2 facilitates the association of F-Met tRNA that is initiator tRNA with the small subunit and prevents other charged tRNAs from associating with the small subunit. IF3 binds to the small subunit and blocks it from re-associating with the large subunit. Because initiation requires a free small subunit, the binding of IF3 is critical for a new cycle of translation. IF3 becomes associated with the small subunit at the end of a previous round of translation when it helps to dissociate the 70s ribosome into its large and small subunits. Each of the initiation factors binds at or near one of the three tRNA binding sites on the small subunit. For example, there are three tRNA binding sites on the small subunit. These initiation factors bind from one or two of these sites. Consistent with its role in blocking the binding of charged tRNA to the A site, IF1 binds directly to the portion of the small subunit that will become the A site. IF2 binds to IF1 and reaches over the A site into the P site to contact the F-Met tRNA. Finally, IF2 is connected to the F-Met tRNA. IF2 is connected to the F-Met tRNA and reaches over the A site into the P site to contact the F-Met tRNA. Finally, IF3 occupies the part of the small subunit that will become the E site. Thus, A site B occupies and E site B occupies. Now, only the P site is free for the initiator tRNA. Thus, of the three potential tRNA binding sites on the small subunit, only the P site is capable of binding a tRNA in the presence of the initiation factors. With all three initiation factors bound, the small subunit is prepared to bind to the mRNA and the initiator tRNA. Now, the mRNA and the initiator tRNA are ready to bind. These two tRNAs can bind in either order and independently of each other. That is, if the mRNA is first binded and the initiator tRNA is then binded, whether they are both simultaneously binded, whether they are vice versa. This is independent. There is no sequence involved. In this, you can see that the start is in the intact ribosome. The large subunit and the small subunit are detached. After this, the IF3 is binded to the E site. After this, the IF1 along with the GTP and IF2 will be binded to the A site. Directly on the A site, the IF1 is attached to the IF1, IF2 is attached to the IF2 and IF2 is attached to the green color GTP. After that, when this is attached, the two RNAs, the mRNA and the FMET tRNA, will be attached to it. Now, this is a 30S initiation complex. That is, the small unit's initiation complex is not attached to the large subunit. In this, you can see that the A site is also occupied and the E site is also occupied. After that, the FMET tRNA will be attached to the P site. After that, this is the larger subunit. And as soon as the IF3 is released, the large subunit can be attached to it. That is, the IF3 was blocked by the large subunit. Now, this is a full complex. After this, this is called a 70S initiation complex, where the small subunit and the large subunit will be joined. Binding FMET tRNA to the small subunit is facilitated by its interaction with IF2 bound to GTP and base pairing between the anticodon and the start codon of the mRNA. Anticodon of tRNA and start codon of mRNA. Similarly, base pairing between FMET tRNA and the mRNA serves to position the start codon in the P site. The last step of initiation involves the association of the large subunit to create the 70S initiation complex. When the start codon and FMET tRNA base pair, the small subunit undergoes a change in conformation. This altered conformation results in the release of IF3. In the absence of IF3, the large subunit is free to bind to the small subunit with its cargo of IF1, IF2, mRNA and FMET tRNA. In particular, IF2 acts as an initial docking site of the large subunit and this interaction subsequently stimulates the GTP-A's activity of the IF2-GTP complex. IF2 bound to GTP has reduced affinity for the ribosome and the initiator tier. In particular, IF2 acts as an initial docking site of the large subunit and this interaction subsequently stimulates the GTP-A's activity of the IF2-GTP complex. IF2 bound to GTP has reduced affinity for the ribosome and the initiator tRNA, leading to the release of IF2-GTP complex as well as IF1 from the ribosome. Thus, the net result of initiation is the formation of an intact 70S ribosome assembled at the start site of mRNA with FMET tRNA in the P site with an empty A site. The ribosome mRNA complex is now poised to accept a charged tRNA into the A site and commence polypeptide synthesis.