At the ribosome’s E (exit) site, deacylated tRNA is released back into the cell’s cytoplasm, completing its role in translation. The tRNA arrives at the E site empty, having already donated its amino acid to the growing protein chain, and it exits the ribosome shortly after. But the journey from the neighboring P site to the E site and then out of the ribosome involves a surprisingly complex sequence of molecular movements.
How tRNA Reaches the E Site
The ribosome has three tRNA binding sites: the A (aminoacyl) site where new amino acid-carrying tRNAs arrive, the P (peptidyl) site where the tRNA holding the growing protein chain sits, and the E (exit) site. After each amino acid is added to the protein, the tRNAs need to shift one position over: A to P, and P to E. This shift is called translocation.
Translocation doesn’t happen in one clean jump. It occurs in two stages, and the tRNA actually occupies an in-between “hybrid” position along the way. Right after the peptide bond forms, the growing amino acid chain transfers from the P-site tRNA onto the A-site tRNA. This leaves the P-site tRNA empty (deacylated). Almost immediately, the top portion of this empty tRNA swings into the E site on the ribosome’s large subunit, while its bottom portion (the anticodon end) stays put in the P site on the small subunit. This split position is called the P/E hybrid state, and the tRNA’s upper end moves about 40 ångströms during this transition.
Completing the move requires an enzyme called EF-G in bacteria (or eEF2 in eukaryotes). EF-G binds to the ribosome and uses energy from GTP to drive the small subunit’s head domain through a rotation of 18 to 21 degrees. This rotation carries the tRNA’s anticodon end from the P site into the E site, completing the full E/E position. Throughout the process, a flexible arm of the ribosome called the L1 stalk swings more than 60 ångströms to grab onto the tRNA and escort it through each intermediate position.
Why Only Empty tRNAs Fit in the E Site
The E site is physically too small to hold a tRNA that still carries an amino acid. X-ray crystal structures of the ribosome show that the pocket where the tRNA’s tail end (nucleotide A76) docks is tightly fitted around the bare end of a deacylated tRNA. The terminal adenosine of the tRNA slots between two ribosomal RNA bases that are splayed apart, and it’s locked into place by hydrogen bonds on multiple sides. There simply isn’t room for an amino acid to be attached. This structural constraint acts as a built-in filter, ensuring only tRNAs that have already done their job end up in the E site.
What Triggers Release From the E Site
Once the tRNA reaches the E/E position, it doesn’t stay long, but the exact trigger for its departure has been debated for decades. The leading explanation involves a long-range communication between the A site and the E site, a phenomenon called negative cooperativity. When a new amino acid-carrying tRNA binds to the now-empty A site, it sends a signal across the ribosome that weakens the E site’s grip on its tRNA, pushing it out.
Single-molecule fluorescence experiments have provided direct evidence for this. During the early cycles of protein building, tRNAs are effectively trapped in the E site until the A site becomes occupied. Once a new tRNA locks into the A site, the E-site tRNA is allosterically released. The effect works in the other direction too: a tRNA sitting in the E site lowers the A site’s affinity for incoming tRNAs. This back-and-forth between the two sites helps coordinate the pace of translation, preventing the ribosome from accepting a new tRNA before it has properly ejected the old one.
That said, E-site tRNAs can also dissociate spontaneously without waiting for A-site binding, especially later in elongation or under certain ionic conditions. The balance between allosteric and spontaneous release likely depends on the stage of translation and the cellular environment.
The E Site’s Role in Translation
The E site might seem like a simple dumping ground, but it plays an active role in keeping translation running smoothly. By holding onto the deacylated tRNA temporarily, the E site maintains the reading frame. The tRNA in the E site is still touching the mRNA codon it was paired with, and this contact helps prevent the mRNA from slipping forward or backward by the wrong number of nucleotides. A frameshift error would scramble the entire protein from that point on.
The coordinated communication between the E and A sites also functions as a quality-control checkpoint. Because E-site occupancy reduces A-site affinity, it creates a brief pause that gives the ribosome time to ensure the correct tRNA is being selected at the A site. This negative cooperativity effectively spaces out the steps of elongation so they happen in the right order.
After the tRNA Leaves
Once released from the E site, the deacylated tRNA drifts into the cytoplasm. It is then “recharged” by an enzyme called aminoacyl-tRNA synthetase, which attaches the correct amino acid back onto it. The recharged tRNA can then pair with a new elongation factor, forming a complex that delivers it back to the A site of a ribosome that displays its matching codon. The whole cycle, from E-site release to recharging to re-entry at the A site, allows each tRNA molecule to be used many times over during the cell’s lifetime.