The P site (peptidyl site) of a ribosome holds the tRNA that carries the growing protein chain. During active translation, this is a peptidyl-tRNA, a transfer RNA molecule with the elongating polypeptide attached to it. But the P site’s occupant changes depending on the stage of translation, and understanding those shifts is key to understanding how proteins get made.
The Initiator tRNA Arrives First
Before a ribosome begins building a protein, the P site receives a special molecule: the initiator tRNA. This tRNA carries the amino acid methionine and is structurally distinct from all other tRNAs in the cell. It bypasses the usual delivery route that elongation tRNAs use and instead binds directly to the P site with the help of initiation factors. In eukaryotic cells, it arrives as part of a complex with the initiation factor eIF2 and GTP. This complex lands on the small ribosomal subunit, forming what’s called a pre-initiation complex, which then scans along the messenger RNA until it finds the start codon (AUG).
When the start codon is recognized, GTP is hydrolyzed, eIF2 releases, and the initiator tRNA is left sitting in the P site with its anticodon base-paired to the AUG on the mRNA. The large ribosomal subunit then joins, and the ribosome is ready to start elongation. The initiator tRNA’s structure is specifically tuned for P site binding. Researchers have found that no single structural feature accounts for this preference; rather, a combination of features across the molecule gives it a unique affinity for the P site over the A site.
During Elongation: Peptidyl-tRNA
Once translation is underway, the P site holds the peptidyl-tRNA, the tRNA carrying the growing chain of amino acids. This is the ribosome’s “holding” position. The neighboring A site (aminoacyl site) accepts each new incoming tRNA carrying a single amino acid. The chemistry happens when the amino group on the A site tRNA attacks the bond connecting the peptide chain to the P site tRNA. This transfers the entire growing chain onto the A site tRNA, making it one amino acid longer. The P site tRNA, now stripped of its cargo, is called a deacylated tRNA.
So within a single catalytic step, the P site goes from holding a peptidyl-tRNA to holding a deacylated (empty) tRNA. That deacylated tRNA then shifts into the E site (exit site) and leaves the ribosome entirely.
How tRNAs Move Into the P Site
After each new amino acid is added, the tRNAs and the mRNA need to shift by one codon so the next round can begin. This movement, called translocation, is powered by an elongation factor called EF-G in bacteria (eEF-2 in eukaryotes), which uses GTP hydrolysis as an energy source. The peptidyl-tRNA that just received the growing chain in the A site needs to move into the P site.
Structurally, this is a surprisingly short trip. The midsections of tRNAs in the A and P sites are separated by only about 2 to 10 angstroms, and the full translocation movement covers less than 30 angstroms (roughly 3 nanometers). EF-G facilitates this by physically occupying space in the A site after the small ribosomal subunit rotates, acting as a steric block that prevents the tRNA from sliding back. A specific structural domain of EF-G (domain IV) pushes the tRNA forward into the P site as the subunit rotates back into its resting position. The result is that the new peptidyl-tRNA now sits in the P site, the A site is empty and ready for the next delivery, and the deacylated tRNA has moved to the E site.
What the P Site tRNA Does With mRNA
Inside the P site, the tRNA’s anticodon loop forms base pairs with the mRNA codon, anchoring the reading frame. In a standard tRNA, three anticodon nucleotides pair with three mRNA nucleotides. The ribosomal RNA itself helps stabilize this pairing: a specific nucleotide in the 16S ribosomal RNA (C1400) stacks against nucleotide 34 of the tRNA anticodon, while another ribosomal RNA residue contacts the tRNA backbone near the characteristic U-turn of the anticodon loop. These contacts help lock the mRNA into the correct reading frame and ensure that each codon is read accurately as translation proceeds.
At Termination: Release Factors Replace tRNA
When the ribosome reaches a stop codon, no tRNA binds the A site. Instead, a release factor protein recognizes the stop codon and enters the A site. The release factor then triggers hydrolysis of the bond between the finished protein and the P site tRNA. It does this in two ways: it opens up the ribosome’s catalytic center to allow water molecules in, and it uses a conserved amino acid motif (found in both bacterial and eukaryotic release factors) to precisely position a water molecule for the reaction. The water molecule breaks the ester bond linking the completed polypeptide to the P site tRNA, freeing the finished protein.
At this final stage, the P site still holds a tRNA, but it’s now deacylated, with no amino acid or peptide chain attached. The ribosome then disassembles with the help of recycling factors.
Antibiotics That Target the P Site
Several antibiotics work by interfering with normal P site function, which is why the P site matters beyond basic biology. Edeine, a cationic peptide antibiotic, binds directly to the P site on the small ribosomal subunit and prevents the initiator tRNA from entering, blocking translation before it even starts. GE81112, a tetrapeptide antibiotic, also occupies the small subunit P site but works differently: it distorts the shape of the initiator tRNA’s anticodon loop so it can’t properly read the start codon.
On the large subunit side, blasticidin S sits in the P site and wedges itself between nucleotides at the business end of the P site tRNA, displacing a critical base and disrupting peptide bond formation. Bactobolin A binds in a similar location and pushes the tRNA’s working end toward the A site, preventing normal chemistry. Pleuromutilins, a class that includes the clinically used antibiotic lefamulin, bind the catalytic center and interfere with the positioning of both A site and P site tRNAs simultaneously. Streptogramin A antibiotics span both the A site and P site regions, allowing tRNAs to bind but preventing their working ends from reaching the correct position to form peptide bonds.
These drugs illustrate how essential proper P site binding is. Even small distortions in tRNA positioning within the P site are enough to shut down protein production entirely.