Localization of two domains of a mutant form of Escherichia coli protein L7/L12 that binds the large ribosomal subunit as a single dimer

Wiggers, Robert J.; Hadian, Hoora; Traut, Robert R.; Oleinikov, Andrew V.; Glitz, Dohn G.

doi:10.1016/s0300-9084(97)80031-4

Cited by 5 publications

(5 citation statements)

References 30 publications

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“…Therefore, the stalk may be at least transiently composed of only a single dimer while another may take over dynamic functions during translation [16,111,165]. In agreement, some researchers suggested that the stalk comprises a low-affinity binding site for only a single L12 dimer [145,150]. These conclusions are corroborated by a re ce nt ma pping of the E. c oli L12 C TD s on the 70S particles by cryo-EM [55].…”

Section: Diversity In the Ribosomal Locationmentioning

confidence: 75%

“…The L10 sequence lacks an internal symmetry and it will be interesting to see how the two implicit dimer binding sites are distinguished. In agreement with two unequal dimer binding sites on L10, it has been observed that there are a strong and a weak binding site of L12 on the ribosome [150]. Recent L10 deletion analyses by Traut's group showed that the C-terminal 20 amino acids of L10 are important for both dimer binding sites while removal of the last 10 amino acids affected only one site [151].…”

Section: Complexes With L10/p0mentioning

confidence: 79%

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Structure and Function of the Acidic Ribosomal Stalk Proteins

Wahl

Möller²

2002

curr protein pept sci

105

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The acidic L7/L12 (prokaryotes) and P1/P2 (eukaryotes) proteins are the only ribosomal components that occur in more than one, specifically four, copies in the translational machinery. These ribosomal proteins are the only ones that do not directly interact with ribosomal RNA but bind to the particles via a protein, L10 and P0, respectively. They constitute a morphologically distinct feature on the large subunit, the stalk protuberance. Since a long time proteins L7/L12 have been implicated in translation factor binding and in the stimulation of the factor-dependent GTP-hydrolysis. Recent studies reproduced such activities with the isolated components and L7/L12 can therefore in retrospect be regarded as the first GTPase activating proteins identified. GTP-hydrolysis induces a drastic conformational change in elongation factor (EF) Tu, which enables it to dissociate from the ribosome after having successfully delivered aminoacylated tRNA into the A-site. It is also used as a driving force for translocation, mediated by EF-G. The in vitro stimulation of translation-uncoupled EF-G-dependent GTP-hydrolysis seems to be an intrinsic property of the ribosome that is dependent on L7/L12, reaches a maximum with four copies of the proteins per particle, and reflects the in vivo hydrolysis rate during translation. It is much larger than the analogous activity observed for EF-Tu, which is correlated with the in vitro polypeptide synthesis rate. Therefore, at least certain stimulatory activities of L7/L12 are controlled by the ribosomal environment, which in the case of EF-Tu senses the successful codon-anticodon pairing. Present knowledge is consistent with a picture in which proteins L7/L12 constitute a "landing platform" for the factors and after rearrangements induce GTP-hydrolysis. The molecular mechanism of the GTPase activation is unknown. While sequence comparisons show a large diversity in the stalk proteins across the kingdoms, a conserved functional domain organization and conserved designs of their genetic units are discernible. Consistently, stalk transplantation experiments suggest that coevolution took place to maintain functional L7/L12 EF-G and P-protein EF-2 couples. The acidic proteins are organized into three distinct functional parts: An N-terminal domain is responsible for oligomerization and ribosome association, a C-terminal domain is implicated in translation factor interactions, and a hinge region allows a flexible relative orientation of the latter two portions. The bacterial L7/L12 proteins have long been portrayed as highly elongated dimers displaying globular C-terminal domains, helical N-termini, and unstructured hinges. Conversely, recent crystal structures depict a compact hetero-tetrameric assembly with the hinge region adopting either an alpha-helical or an open conformation. Two different dimerization modes can be discerned in these structures. Models suggest that dimerization via one association mode can lead to elongated dimeric complexes with one helical and one unstructured hinge. The ...

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Section: Diversity In the Ribosomal Locationmentioning

confidence: 75%

Section: Complexes With L10/p0mentioning

confidence: 79%

Structure and Function of the Acidic Ribosomal Stalk Proteins

Wahl

Möller²

2002

curr protein pept sci

105

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“…It should be pointed out that the independent and specific interaction of the two yeast P1/P2 dimers with the P0 protein reported here closely resembles the behaviour of E. coli L7/L12 dimers. Previously, it was found that bacterial dimers exhibit different affinities for binding to the L10 protein (Zantema et al ., 1982; Wiggers et al ., 1997), which has two independent binding sites for two L7/L12 protein pairs (Griaznova and Traut, 2000). Recently, the structural organization of the bacterial stalk has been solved, by using Thermotoga maritima model (Diaconu et al ., 2005).…”

Section: Discussionmentioning

confidence: 99%

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

Krokowski

Boguszewska

Abramczyk

et al. 2006

Molecular Microbiology

100

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SummaryThe ribosome has a distinct lateral protuberance called the stalk; in eukaryotes it is formed by the acidic ribosomal P-proteins which are organized as a pentameric entity described as P0-(P1-P2) 2 . Bilateral interactions between P0 and P1/P2 proteins have been studied extensively, however, the region on P0 responsible for the binding of P1/P2 proteins has not been precisely defined. Here we report a study which takes the current knowledge of the P0 -P1/P2 protein interaction beyond the recently published information. Using truncated forms of P0 protein and several in vitro and in vivo approaches, we have defined the region between positions 199 and 258 as the P0 protein fragment responsible for the binding of P1/P2 proteins in the yeast Saccharomyces cerevisiae . We show two short amino acid regions of P0 protein located at positions 199-230 and 231-258, to be responsible for independent binding of two dimers, P1A-P2B and P1B-P2A respectively. In addition, two elements, the sequence spanning amino acids 199-230 and the P1A-P2B dimer were found to be essential for stalk formation, indicating that this process is dependent on a balance between the P1A-P2B dimer and the P0 protein.

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“…In E. coli, one L12 dimer is more tightly associated with L10 than the other (Wiggers et al 1997). This observation agrees with the present structure where the proximal L12 NTD dimer in each of the three tmaL10:(L12NTD) 6 complexes engages in interactions with the L10 NTD, which are not seen for the distal dimers.…”

Section: Detailed Insights Into the L10-l12 Interactionsupporting

confidence: 91%

“…coli, one of the L12 dimers is more tightly bound to L10 than the other (Wiggers et al 1997). Indeed, the proximal dimer of L12 in the tmaL10:(L12 NTD) 6 structure engages in interactions with the L10 NTD, which are not observed for the other two dimers.…”

Section: The Tmal10:(l12 Ntd) 6 Complexmentioning

confidence: 96%

Structural Analysis of the 50S Ribosomal Stalk

Stefania¹

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Protein biosynthesis represents a dynamic process that takes place on the ribosome and is driven by translation factors. Some of these factors are GTP binding proteins. They possess a limited inherent GTPase activity that is stimulated by interactions with the ribosome in a region located on the large ribosomal subunit (GTPase associated region).This site comprises several 23S rRNA elements (L10/L11 rRNA binding region and sarcin-ricin loop) and r-proteins, such as L6, L11, L14, and the L7/L12 stalk. The latter corresponds to an extended feature of the 50S ribosomal subunit, encompassing multiple copies of protein L12 that are linked to the ribosomal RNA via L10. Numerous lines of evidence indicated that L12 is essential for both translation factor binding and stimulation of their GTPase activities. Functionally, L12 can be divided into an N-terminal domain (NTD) responsible for dimerization and interaction with L10, a C-terminal domain (CTD) necessary for factor-related functions, and an intervening flexible hinge.Crystallographic studies of 50S subunits and 70S ribosomes hitherto failed to disclose the structure of the L7/L12 stalk, most probably due to the high mobility of the L12 hinge region. Thus, a complex anticipated to exhibit less flexibility was designed. It encompassed L10 and the NTD of L12 from the hyperthermophilic bacterium Thermotoga maritima. In the three crystal structures obtained, L10 displayed a globular NTD connected by a flexible loop to a long C-terminal α-helix. The latter displayed different orientations relative to the L10 NTD in different crystal forms and harbored three consecutive binding sites for the L12 NTD dimers. Such a 1:6 (L10:L12) stoichiometry was unexpected, as a 1:4 ratio was well established in E. coli. The L12 NTDs formed dimers that fitted to a mode of dimerization reported for the protein in isolation, both in solution (Bocharov et al. 2004;Moens et al. 2005) and in crystalline environment (Wahl et al. 2000a). In the crystal structure of isolated T. maritima L12, the hinge region of one protomer exhibited an α-helical shape, folded onto the L12 NTDs of the dimer, while in tmaL10:(L12 NTD) 6 , the hinge was found replaced by the C-terminal α-helix of L10. Thus, it is likely that in complex with L10, the L12 hinges are flexible and unstructured, in agreement with several studies of this protein in solution.In addition to obtaining the structure of tmaL10:(L12 NTD) 6 , attempts to solve the crystal structure of the full-length L10:L12 complex were also undertaken. While the 1 Summary crystallization of the complex from the hyperthermophilic bacterium Aquifex aeolicus proved to be unsuccessful, the corresponding complex from Thermotoga maritima yielded crystals that diffracted to 3.5 Å. The structure could be solved by molecular replacement using the tmaL10:(L12 NTD) 6 complex as a search model. No electron density could be detected for the L12 hinges and CTDs, consistent with a degradation of L12 during crystallization, as revealed by SDS-PAGE analysis of dissolved cryst...

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Localization of two domains of a mutant form of Escherichia coli protein L7/L12 that binds the large ribosomal subunit as a single dimer

Cited by 5 publications

References 30 publications

Structure and Function of the Acidic Ribosomal Stalk Proteins

Structure and Function of the Acidic Ribosomal Stalk Proteins

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

Structural Analysis of the 50S Ribosomal Stalk

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