Yeast ribosomal P0 protein has two separate binding sites for P1/P2 proteins

Krokowski, Dawid; Boguszewska, Aleksandra; Abramczyk, Dariusz; Liljas, Anders; Tchórzewski, Marek; Grankowski, Nikodem

doi:10.1111/j.1365-2958.2006.05117.x

Cited by 78 publications

(108 citation statements)

References 77 publications

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“…By sucrose gradient centrifugation, the hybrid particle composed of E. coli 50 S core and the archaeal stalk complex was collected. The labeled Ph-L12 protein included was quantified by measurement of the specific radioactivity of 32 P-Ph-L12 (42 cpm/pmol protein), and the value was related to the amounts of 50 S particles estimated by A 260 (Fig. 3).…”

Section: Resultsmentioning

confidence: 99%

“…We tested this point by deletion of the third stalk dimer from the stalk complex derived using the C-terminal truncation mutants of Ph-P0. We assayed the functions by using a hybrid ribosome system, which we recently developed (32,34). Unexpectedly, the results showed only a slight effect of deletion of the third stalk dimer in the C-terminal region on factor accessibility (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Three Binding Sites for Stalk Protein Dimers Are Generally Present in Ribosomes from Archaeal Organism

Maki

Hashimoto

Zhou

et al. 2007

Journal of Biological Chemistry

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Ribosomes have a characteristic protuberance termed the stalk, which is indispensable for ribosomal function. The ribosomal stalk has long been believed to be a pentameric protein complex composed of two sets of protein dimers, L12-L12, bound to a single anchor protein, although ribosomes carrying three L12 dimers were recently discovered in a few thermophilic bacteria. Here we have characterized the stalk complex from Pyrococcus horikoshii, a thermophilic species of Archaea. This complex is known to be composed of proteins homologous to eukaryotic counterparts rather than bacterial ones. In truncation experiments of the C-terminal regions of the anchor protein Ph-P0, we surprisingly observed three Ph-L12 dimers bound to the C-terminal half of Ph-P0, and the binding site for the third dimer was unique to the archaeal homologs. The stoichiometry of the heptameric complex Ph-P0(Ph-L12) 2 (Ph-L12) 2 (Ph-L12) 2 was confirmed by mass spectrometry of the intact complex. In functional tests, ribosomes carrying a single Ph-L12 dimer had significant activity, but the addition of the second and third dimers increased the activity. A bioinformatics analysis revealed the evidence that ribosomes from all archaeal and also from many bacterial organisms may contain a heptameric complex at the stalk, whereas eukaryotic ribosomes seem to contain exclusively a pentameric stalk complex, thus modifying our view of the stalk structure significantly.The ribosomal stalk proteins at the "GTPase-associated center" in the large subunits play a central role in the interaction between the ribosomes and GTP-bound translation factors (1-4). In bacteria, the stalk protein is L7/L12 and is usually present in four copies in the form of two stable dimers bound to the C-terminal regions of L10 (5, 6). The L10⅐L7/L12 complex, together with another protein, L11, binds to the highly conserved domain around 1070 (Escherichia coli numbering is used throughout) of 23 S rRNA through the interaction with the L10 moiety (7,8). The protein complex constitutes a highly flexible domain in the ribosome (9 -15), and the detailed structure within the ribosome has not been resolved by x-ray crystallography (16 -21). It has been found that an E. coli ribosome mutant carrying only one L7/L12 dimer retains significant translation activity (6), and more recently, that there are a few bacterial species whose ribosomes have three L7/L12 dimers in a heptameric complex, L10(L7/L12) 2 (L7/L12) 2 (L7/L12) 2 (22, 23). These findings therefore bring the question as to the significance of multi-stalk dimers in the ribosome into focus.In eukaryotic ribosomes, two related phosphoproteins P1 and P2 are counterparts of bacterial L7/L12 (24 -26). They form heterodimers (27-29), and the two P1-P2 dimers bind to neighboring sites within the C-terminal half of the L10-like stalk base protein P0 (30 -32). This pentameric P0⅐P1-P2 stalk complex can be substituted for E. coli L10⅐L7/L12 complex in the 50 S subunit and makes the ribosome accessible to eukaryotic elongation factors (33)....

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Three Binding Sites for Stalk Protein Dimers Are Generally Present in Ribosomes from Archaeal Organism

Maki

Hashimoto

Zhou

et al. 2007

Journal of Biological Chemistry

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“…Multiple copies of the acidic ribosomal protein, or so-called stalk protein, are key components of this functional center (5-9). The stalk proteins form homo-or heterodimers, and two or three dimers bind to the ribosome through an anchor protein, L10 in bacteria and P0 in eukaryotes (7,(9)(10)(11)(12).In the case of bacteria, the structure and function of the stalk protein L7/L12 (termed L12 hereafter) is well established. The L12 protein is composed of an N-terminal dimerization domain and a globular C-terminal domain, which is connected by a flexible hinge region and has a wide range of movement (7, 13).…”

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confidence: 99%

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Archaeal ribosomal stalk protein interacts with translation factors in a nucleotide-independent manner via its conserved C terminus

Nomura

Honda

Baba

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Protein synthesis on the ribosome requires translational GTPase factors to bind to the ribosome in the GTP-bound form, take individual actions that are coupled with GTP hydrolysis, and dissociate, usually in the GDP-bound form. The multiple copies of the flexible ribosomal stalk protein play an important role in these processes. Using biochemical approaches and the stalk protein from a hyperthermophilic archaeon, Pyrococcus horikoshii, we here provide evidence that the conserved C terminus of the stalk protein aP1 binds directly to domain I of the elongation factor aEF-2, irrespective of whether aEF-2 is bound to GTP or GDP. Site-directed mutagenesis revealed that four hydrophobic amino acids at the C terminus of aP1, Leu-100, 103, 106, and Phe-107, are crucial for the direct binding. P1 was also found to bind to the initiation factor aIF5B, as well as aEF-1α, but not aIF2γ, via its C terminus. Moreover, analytical ultracentrifugation and gel mobility shift analyses showed that a heptameric complex of aP1 and aP0, aP0ðaP1Þ 2 ðaP1Þ 2 ðaP1Þ 2 , can bind multiple aEF-2 molecules simultaneously, which suggests that individual copies of the stalk protein are accessible to the factor. The functional significance of the C terminus of the stalk protein was also shown using the eukaryotic proteins P1/P2 and P0. It is likely that the conserved C terminus of the stalk proteins of archaea and eukaryotes can bind to translation factors both before and after GTP hydrolysis. This consistent binding ability of the stalk protein may contribute to maintaining high concentrations of translation factors around the ribosome, thus promoting translational efficiency.GTPase-associated center | ribosome protein P0 | ribosome protein P1 | hyperthermophilic archaeon M ajor dynamic steps in translational initiation, elongation, and termination are promoted by the actions of several translational GTPase factors (1-3). During the elongation step, two elongation factors bind alternately to the ribosome in their GTP-bound states. After they have carried out their respective functions, which are linked to GTP hydrolysis, they then dissociate from the ribosome. The large ribosomal subunit contains an active center, termed the GTPase-associated center or factorbinding center, which interacts with the elongation factors (4). The GTPase-associated center plays a crucial role in the recruitment of translation factors, GTP hydrolysis, and the release of inorganic phosphate, which is required for the subsequent dissociation of the factor. Multiple copies of the acidic ribosomal protein, or so-called stalk protein, are key components of this functional center (5-9). The stalk proteins form homo-or heterodimers, and two or three dimers bind to the ribosome through an anchor protein, L10 in bacteria and P0 in eukaryotes (7,(9)(10)(11)(12).In the case of bacteria, the structure and function of the stalk protein L7/L12 (termed L12 hereafter) is well established. The L12 protein is composed of an N-terminal dimerization domain and a globular C-terminal doma...

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“…In eukaryotes, the stalk occurs in a pentameric configuration P0-(P1/P2) 2 (14,15), where P0 constitutes the base of the stalk and anchors two P1/P2 dimers (16). The N-terminal domain of the P0 protein is responsible for attachment of the stalk to the large rRNA, and the P-domain anchors the P1/P2 proteins at two separate binding sites (17)(18)(19). The P1/P2 proteins form a heterodimer (20 -22), with the N-terminal domain responsible for dimerization and linking the dimer to the P0 protein (23,24), whereas the Cterminal part is regarded as a functional element involved in factor recruitment (19).…”

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Pentameric Organization of the Ribosomal Stalk Accelerates Recruitment of Ricin A Chain to the Ribosome for Depurination

Grela

Krokowski

et al. 2010

Journal of Biological Chemistry

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Ribosome inactivating proteins (RIPs) depurinate a universally conserved adenine in the ␣-sarcin/ricin loop (SRL) and inhibit protein synthesis at the translation elongation step. We previously showed that ribosomal stalk is required for depurination of the SRL by ricin toxin A chain (RTA). The interaction between RTA and ribosomes was characterized by a twostep binding model, where the stalk structure could be considered as an important interacting element. Here, using purified yeast ribosomal stalk complexes assembled in vivo, we show a direct interaction between RTA and the isolated stalk complex. Detailed kinetic analysis of these interactions in real time using surface plasmon resonance (SPR) indicated that there is only one type of interaction between RTA and the ribosomal stalk, which represents one of the two binding steps of the interaction with ribosomes. Interactions of RTA with the isolated stalk were relatively insensitive to salt, indicating that nonelectrostatic interactions were dominant. We compared the interaction of RTA with the full pentameric stalk complex containing two pairs of P1/P2 proteins with its interaction with the trimeric stalk complexes containing only one pair of P1/P2 and found that the rate of association of RTA with the pentamer was higher than with either trimer. These results demonstrate that the stalk is the main landing platform for RTA on the ribosome and that pentameric organization of the stalk accelerates recruitment of RTA to the ribosome for depurination. Our results suggest that multiple copies of the stalk proteins might also increase the scavenging ability of the ribosome for the translational GTPases.

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Yeast ribosomal P0 protein has two separate binding sites for P1/P2 proteins

Cited by 78 publications

References 77 publications

Three Binding Sites for Stalk Protein Dimers Are Generally Present in Ribosomes from Archaeal Organism

Three Binding Sites for Stalk Protein Dimers Are Generally Present in Ribosomes from Archaeal Organism

Archaeal ribosomal stalk protein interacts with translation factors in a nucleotide-independent manner via its conserved C terminus

Pentameric Organization of the Ribosomal Stalk Accelerates Recruitment of Ricin A Chain to the Ribosome for Depurination

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