Multiplication of Ribosomal P-Stalk Proteins Contributes to the Fidelity of Translation

Abstract

The P-stalk represents a vital element within the ribosomal GTPaseassociated center, which represents a landing platform for translational GTPases. The eukaryotic P-stalk exists as a uL10-(P1-P2) 2 pentameric complex, which contains five identical C-terminal domains, one within each protein, and the presence of only one such element is sufficient to stimulate factor-dependent GTP hydrolysis in vitro and to sustain cell viability. The functional contribution of the P-stalk to the performance of the translational machinery in vivo, especially the role of P-protein multiplication, has never been explored. Here, we show that ribosomes depleted of P1/P2 proteins exhibit reduced translation fidelity at elongation and termination steps. The elevated rate of the decoding error is inversely correlated with the number of the P-proteins present on the ribosome. Unexpectedly, the lack of P1/P2 has little effect in vivo on the efficiency of other translational GTPase (trGTPase)-dependent steps of protein synthesis, including translocation. We have shown that loss of accuracy of decoding caused by P1/P2 depletion is the major cause of translation slowdown, which in turn affects the metabolic fitness of the yeast cell. We postulate that the multiplication of P-proteins is functionally coupled with the qualitative aspect of ribosome action, i.e., the recoding phenomenon shaping the cellular proteome.KEYWORDS ribosomal proteins, ribosomal stalk, ribosome A t the expense of energy from GTP hydrolysis, translational GTPases (trGTPases) confer the unidirectional trajectory for the translational apparatus, providing at the same time unique timing for individual steps (1, 2). The main landing platform for trGTPases is situated on the large ribosomal subunit called the GTPase-associated center (GAC), and it represents a universally conserved ribosomal element where stimulation of trGTPase catalytic activity takes place (3). The GAC consists of two main elements, a conserved fragment of rRNA called the sarcin-ricin loop (SRL) and a ribosomal stalk composed of ribosomal proteins, which form an oligomeric protein complex (4). The protein part of GAC, the ribosome stalk, can be divided into two functionally and evolutionarily distinct parts, the base of the stalk and its lateral elements. The stalk base is composed of conserved ribosomal proteins uL11 (former names L11 and L12 for prokaryotes and eukaryotes, respectively) and uL10 (former names L10 and P0), which anchor the stalk to the rRNA (5, 6). The lateral part of the stalk is built of dimeric complexes P1-P2 in eukaryotes/archaea or (bL12) 2 in prokaryotes (4, 7). Despite the lack of amino acid sequence conservation, the lateral stalk fulfils the same functions and has a similar structural architecture across all domains of life (8). P1/P2 and bL12 proteins are built of two domains. The globular N-terminal domain (NTD) is responsible for dimerization, whereas the highly acidic C-terminal domain (CTD) interacts with trGTPases (9, 10). However, the structure of the CTD in euk...

Phosphorylation of the N‐terminal domain of ribosomal P‐stalk protein uL10 governs its association with the ribosome

“…In our analyses, the dynamics of the C-terminal uL10 variants, irrespective of the modifications tested, resembled that of the wild-type, showing that this region has no role in the interplay of uL10 with the ribosome. In line with this observation are published data showing that this C terminus represents a critical part of the ribosomal stalk, which governs the interaction with translational GTPases or RIPs [31,70,71] and plays a decisive role in the biological activity of P-proteins [25]. Importantly, our recent analyses indicate that the phosphorylation of the C terminus significantly influences the kinetics of the interaction between stalk proteins and external factors (personal communication from M. Tchorzewski, 2020).…”

“…The primary role of the P-stalk is interaction with translational GTPases and stimulation of their GTPase activity during each step of translation [18][19][20][21][22][23][24]. The phenomenon of multiplication of P-stalk proteins was functionally linked with the decoding event, thus associating the stalk also with translational fidelity [25]. The stalk proteins were named P-proteins [26], because they are phosphorylated by CK2 protein kinase at their conserved C termini [27][28][29], that is, a protein element that is regarded as a functional region responsible for direct interactions with translational factors or ribosome-inactivating proteins [23,24,30,31].…”

Systematic Review of Plant Ribosome Heterogeneity and Specialization

“…During cytosolic LSU maturation in yeast, a RLP24 placeholder protein is replaced by RP eL24, then RP uL16 is added and P-stalk assembly is initiated in parallel to or after Rei1 action ( Meyer et al., 2010 ). The P-Stalk is a pentameric uL10-(P1-P2) 2 complex in yeast ( Wawiórka et al., 2017 ), with additional P3 components in plants, that assists translation associated GTPases. For P-stalk assembly, Yvh1 mediates the release of Mrt4, a placeholder for uL10, and enables substitution by functional uL10 ( Zhou et al., 2019 ).…”

The C-terminal helix of ribosomal P stalk recognizes a hydrophobic groove of elongation factor 2 in a novel fashion

“…N-domain (rRNA-anchoring domain) anchors the arm proteins at GTPase-associated center in the large subunit of ribosome, whereas C-domain is a helical spine to bind multiple copies of arm proteins. Each arm protein hands translational GTPases and recruits them to the ribosome, and it was reported that copies of arm protein involve in both efficiency and fidelity of translation ( 10 , 14 ). Although the rRNA-anchoring domains of P0 and L10 have similar structures, the helical spines have different conformations, which are consistent with the structural difference between aP1 (P1•P2) and L12.…”