The Acidic Protein Binding Site Is Partially Hidden in the Free <i>Saccharomyces cerevisiae</i> Ribosomal Stalk Protein P0

Pérez-Fernández, Jorge; Remacha, Miguel; Ballesta, Juan P. G.

doi:10.1021/bi047332r

Cited by 25 publications

(30 citation statements)

References 53 publications

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“…After completion of this manuscript, Diaconu et al (36) reported the crystal structure of the L10⅐L7/L12 complex in Thermotoga martima, and the results surprisingly revealed three repetitive binding sites for L7/L12 dimers on the discrete C-terminal helix ␣-8. In yeast, the regions including residues 230 -290 (25), 212-262 (26), and 213-250 (27) within the C-terminal half of P0 have been identified as sites for P1/P2 binding (Fig. 8A), although individual sites for two P1-P2 dimers could not be resolved.…”

Section: Discussionmentioning

confidence: 99%

“…Current biochemical and genetic evidence indicates that P1 and P2 proteins form the heterodimer (16,(21)(22)(23)(24) and P1-P2 dimers bind to a specific region within the C-terminal domain of P0 (25)(26)(27). On the other hand, the rRNA binding site seems to be located within the N-terminal region comprising about 200 amino acids (25), although direct evidence has not been shown.…”

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confidence: 97%

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A Mode of Assembly of P0, P1, and P2 Proteins at the GTPase-associated Center in Animal Ribosome

Hagiya

Naganuma

Maki

et al. 2005

Journal of Biological Chemistry

108

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Ribosomal P0, P1, and P2 proteins, together with the conserved domain of 28 S rRNA, constitute a major part of the GTPase-associated center in eukaryotic ribosomes. We investigated the mode of assembly in vitro by using various truncation mutants of silkworm P0. When compared with wild type (WT)-P0, the C-terminal truncation mutants C⌬65 and C⌬81 showed markedly reduced binding ability to P1 and P2, which was offset by the addition of an rRNA fragment covering the P0⅐P1-P2 binding site. The mutant C⌬107 lost the P1/P2 binding activity, whereas it retained the rRNA binding. In contrast, the N-terminal truncation mutants N⌬21-N⌬92 completely lost the rRNA binding, although they retained P1/P2 binding capability, implying an essential role of the N terminus of P0 for rRNA binding. The P0 mutants N⌬6, N⌬14, and C⌬18-C⌬81, together with P1/P2 and eL12, bound to the Escherichia coli core 50 S subunits deficient in L10⅐L7/L12 complex and L11. Analysis of incorporation of 32 P-labeled P1/P2 into the 50 S subunits with WT-P0 and C⌬81 by sedimentation analysis indicated that WT-P0 bound two copies of P1 and P2, but C⌬81 bound only one copy each. The hybrid ribosome with C⌬81 that appears to contain one P1-P2 heterodimer retained lower but considerable activities dependent on eukaryotic elongation factors. These results suggested that two P1-P2 dimers bind to close but separate regions on the C-terminal half of P0. The results were further confirmed by binding experiments using chimeric P0 mutants in which the C-terminal 81 or 107 amino acids were replaced with the homologous sequences of the archaebacterial P0.The ribosomal large subunits from all organisms contain an active site termed the "GTPase-associated center" that is responsible for the GTPase-related events in protein biosynthesis. This active site is composed of the two highly conserved domains around 1070 and 2660 (Escherichia coli numbering is used throughout) of 23 S/28 S rRNA and the ribosomal proteins bound to the 1070 region (1-3). The protein components of this site in prokaryotic ribosomes constitute a characteristic pentameric complex, L10(L7/L12) 2 (L7/L12) 2 (4, 5), in which two L7/L12 homodimers bind to the C-terminal regions of L10 (6) and constitute a highly flexible and functionally important lateral protuberance, the so-called "stalk" (7). Although the ribosomal stalk is observed by cryo-electron microscopy (8), the detailed structure of this pentameric complex has not been resolved by x-ray crystallography of ribosomes (9 -11). The chemical features of protein-protein and proteinrRNA interactions in the GTPase-associated center remain to be clarified.The animal ribosomal phosphoproteins P0 and P1/P2 (P proteins) are counterparts of prokaryotic L10 and L7/L12, respectively, although P1 and P2 are related but different proteins, unlike prokaryotic L7/L12 (12-14). In yeast cells, there are two P1-type proteins, P1␣ and P1␤, and two P2-type proteins, P2␣ and P2␤ (15). It is believed that P proteins constitute a pentameric complex, designated here as...

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Section: Discussionmentioning

confidence: 99%

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confidence: 97%

A Mode of Assembly of P0, P1, and P2 Proteins at the GTPase-associated Center in Animal Ribosome

Hagiya

Naganuma

Maki

et al. 2005

Journal of Biological Chemistry

108

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“…More recently, using Y2H and deletion mutant strains, the P0 region between positions 213 and 260 has been involved in the interaction with the P1/P2 proteins in S. cerevisiae. In contrast, mutation of the putative interacting leucine residues in this region did not impair the binding of P1 and P2 proteins (Pérez-Fernández et al, 2005). The crystal structure of Pyrococcus horikoshii stalk complex has been recently solved (Naganuma et al, 2010).…”

Section: Introductionmentioning

confidence: 97%

Interaction map of the Trypanosoma cruzi ribosomal P protein complex (stalk) and the elongation factor 2

Smulski¹,

Longhi²,

Ayub³

et al. 2010

J of Molecular Recognition

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The large subunit of the eukaryotic ribosome possesses a long and protruding stalk formed by the ribosomal P proteins. This structure is involved in the translation step of protein synthesis through interaction with the elongation factor 2 (EF-2). The Trypanosoma cruzi stalk complex is composed of four proteins of about 11 kDa, TcP1α, TcP1β, TcP2α, TcP2β and a fifth TcP0 of about 34 kDa. In a previous work, a yeast two-hybrid (Y2H) protein-protein interaction map of T. cruzi ribosomal P proteins was generated. In order to gain new insight into the assembly of the stalk, a complete interaction map was generated by surface plasmon resonance (SPR) and the kinetics of each interaction was calculated. All previously detected interactions were confirmed and new interacting pairs were found, such as TcP1β-TcP2α and TcP1β-TcP2β. Moreover P2 but not P1 proteins were able to homo-oligomerize. In addition, the region comprising amino acids 210-270 on TcP0 was identified as the region interacting with P1/P2 proteins, using Y2H and SPR. The interaction domains on TcP2β were also mapped by SPR identifying two distinct regions. The assembly order of the pentameric complex was assessed by SPR showing the existence of a hierarchy in the association of the different P proteins forming the stalk. Finally, the TcEF-2 gene was identified, cloned, expressed and refolded. Using SPR analysis we showed that TcEF-2 bound with similar affinity to the four P1/P2 ribosomal P proteins of T. cruzi but with reduced affinity to TcP0.

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“…In Saccharomyces cerevisiae, the P1 and P2 groups are further subdivided into four P-proteins (P1A, P1B, P2A, and P2B), forming two heterodimers (P1A-P2B and P1B-P2A) (24). They are bound to the highly specific, helical regions of uL10 within a C-terminal polypeptide, called the P-domain (25)(26)(27), and form the pentameric organization uL10-(P1A-P2B)(P1B-P2A) (28). It should be pointed out that the presence of the P-domain alone on the eukaryotic uL10 on the ribosome is sufficient to sustain ribosome functioning, even in the complete absence of P1/P2 proteins (19,29).…”

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confidence: 99%

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

Wawiórka

Molestak

Szajwaj

et al. 2017

Molecular and Cellular Biology

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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...

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The Acidic Protein Binding Site Is Partially Hidden in the Free Saccharomyces cerevisiae Ribosomal Stalk Protein P0

Cited by 25 publications

References 53 publications

A Mode of Assembly of P0, P1, and P2 Proteins at the GTPase-associated Center in Animal Ribosome

A Mode of Assembly of P0, P1, and P2 Proteins at the GTPase-associated Center in Animal Ribosome

Interaction map of the Trypanosoma cruzi ribosomal P protein complex (stalk) and the elongation factor 2

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

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