Role of uL16 P site loop

Introduction

Ribosomes: Structure and Function

Ribosomes are vital molecular machines responsible for synthesizing proteins in all living cells. They achieve this by translating genetic information carried by messenger RNA (mRNA) into functional proteins, catalyzing the polymerization of amino acids into polypeptide chains. Structurally, ribosomes are ribonucleoprotein complexes composed of two subunits: the small subunit—40S in eukaryotes and 30S in prokaryotes—which decodes the mRNA by pairing it with transfer RNA (tRNA) anticodons, and the large subunit—60S in eukaryotes and 50S in prokaryotes—which catalyzes the formation of peptide bonds between amino acids. Each subunit contains ribosomal RNA (rRNA) and ribosomal proteins (r-proteins), with the large subunit housing the peptidyl transferase center (PTC), a critical catalytic site primarily composed of rRNA. Protein synthesis, or translation, occurs in three main phases: initiation, where the ribosome assembles at the mRNA's start codon; elongation, during which amino acids are added one by one to the growing chain; and termination, which happens when a stop codon is reached. During elongation, the ribosome has three key tRNA binding sites: the A site (Aminoacyl-tRNA site) where charged tRNAs enter, the P site (Peptidyl-tRNA site) which holds the tRNA with the growing polypeptide, and the E site (Exit site) where tRNAs exit after transferring their amino acid. The process begins as a charged tRNA enters the A site, followed by the ribosome forming a peptide bond between the new amino acid and the polypeptide in the P site. The ribosome then translocates, moving the tRNA from the A to the P site and from the P to the E site for release. This precise and efficient movement is regulated by rRNAs and r-proteins, which ensure the fidelity of protein synthesis (Figure 1).

Figure 1: Overview of protein translation, illustrating the key stages of initiation, elongation, and termination.

Role of uL16 (RPL10) in Translation

uL16, known as RPL10 in eukaryotes, is a highly conserved ribosomal protein located in the large (60S) subunit of the ribosome, near the peptidyl transferase center (PTC). It plays a central role in maintaining the efficiency and fidelity of translation. Positioned at a critical junction of the ribosome, RPL10 directly contributes to tRNA accommodation by interacting with the elbow regions of tRNAs in both the A and P sites, stabilizing their orientation for accurate peptide bond formation (Figure 2a-c). Additionally, RPL10 is involved in the subunit rotation or "ratcheting" motion that enables translocation of tRNAs from the A site to the P site and eventually to the E site. Beyond structural support, RPL10 interfaces with key elongation factors—such as eEF2 in eukaryotes—to facilitate their engagement with the ribosome during the elongation phase of protein synthesis. These multifaceted roles underscore RPL10’s importance in orchestrating the dynamic events of translation.

RPL10 is evolutionarily conserved across all domains of life, emphasizing its essential role in protein synthesis. In bacteria, its homolog is known as L10, while in archaea and eukaryotes, both the sequence and structure of the protein remain remarkably similar (Figure 2d). Structural studies, particularly cryo-electron microscopy (cryo-EM), have shown that RPL10 consistently occupies a homologous position within the large ribosomal subunit and forms conserved interactions with tRNAs. This level of conservation highlights RPL10’s critical function in aligning and stabilizing tRNAs at the PTC during translation elongation.

Figure 2. Role of uL16 in translation. (a) 80S ribosome with Rpl10 and A & P-site tRNAs (PDB:8UTI); (b) localization of Rpl10 near the PTC interacting with A & P site tRNAs (PDB:8UTI); (c) interaction of P site loop of Rpl10 [aa 102-112] interacting with the acceptor arms of tRNAs (PDB:6qt0), (d) Multiple sequence alignment of uL16 in various organisms shows high conservation of the loop.

High-resolution cryo-EM analyses of the 80S ribosome (e.g., PDB: 8UTI) have revealed the strategic positioning of RPL10 in direct contact with A- and P-site tRNAs, reinforcing its role in facilitating accurate catalysis. A particularly important structural feature of RPL10 is the P-site loop (amino acids 102–112), which extends toward the acceptor stems of the tRNAs and contributes to their stabilization within the catalytic center. This loop appears to serve as a dynamic scaffold, interacting with both rRNA and tRNA to help coordinate conformational changes during translation and ensure translational fidelity.

Importantly, mutations in RPL10, especially within or near the P-site loop, have been linked to several human diseases, including cancers and ribosomopathies. One of the most studied mutations, R98S, located adjacent to the P-site loop, is recurrently observed in T-cell acute lymphoblastic leukemia (T-ALL). Such mutations may disrupt normal tRNA positioning or ribosome dynamics, leading to translational errors that alter the expression of proteins involved in cell proliferation, differentiation, or apoptosis. Additionally, RPL10 mutations have been associated with intellectual disabilities and autism spectrum disorders, although the precise molecular mechanisms are not yet fully understood. Given the P-site loop’s role in tRNA interaction and ribosomal conformational transitions, alterations in this region could impair elongation efficiency or translocation fidelity, resulting in broader translational dysregulation. These clinical associations highlight the importance of RPL10’s structural integrity and its potential as a biomedical target.

Despite significant advances, several aspects of RPL10’s function remain poorly understood. Current structural data offer static snapshots, but the dynamic behavior of RPL10—especially the P-site loop—during different phases of translation is still largely inferred. How this loop changes conformation in response to various tRNA states (e.g., pre- versus post-peptidyl transfer) is unknown, as is its precise role during translocation and ribosome recycling. Moreover, the molecular specificity of its interaction with tRNA acceptor stems—whether governed by sequence recognition, structural complementarity, or both—remains unclear. It is also an open question whether the P-site loop plays an active catalytic role or serves solely as a structural stabilizer. Addressing these knowledge gaps will require high-resolution, time-resolved structural studies and biochemical investigations to uncover the full mechanistic repertoire of RPL10 in translation.

In this study, we aim to examine how deletion of the P-site loop in Rpl10 affects ribosome function. Specifically, we assess the impact of this structural alteration on ribosome activity, translational efficiency, and overall translation dynamics. To achieve this, we performed ribosome profiling on mutant ribosomes lacking the P-site loop, enabling us to capture ribosome positions on mRNA and analyze how this mutation alters translation.

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