Fluorescence studies on the location of L7/L12 relative to L10 in the 50S ribosome of Escherichia coli

Zantema, A.; Maassen, J. Antonie; Kriek, Jan; Moeller, Wim

doi:10.1021/bi00256a007

Cited by 29 publications

(30 citation statements)

References 28 publications

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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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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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“…The stalkless subunits containing one L7/L12 dimer is also prepared by a biochemical approach (61). These lines of evidence suggest that there are two locations of the L7/L12 dimer on the ribosome: the CTD of one dimer makes the stalk, and that of the other dimer turns inward to the base region of the stalk (3), and that the former dimer binds to the particle with lower affinity than the latter (58). It is likely that the compact form of L7/L12 stabilized by the NTD-CTD contacts ( Figure 5, left) corresponds to the latter dimer located in the stalk base.…”

Section: Discussionmentioning

confidence: 99%

A Point Mutation in Ribosomal Protein L7/L12 Reduces Its Ability to Form a Compact Dimer Structure and to Assemble into the GTPase Center

et al. 2003

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An Escherichia coli mutant, LL103, harboring a mutation (Ser15 to Phe) in ribosomal protein L7/L12 was isolated among revertants of a streptomycin-dependent strain. In the crystal structure of the L7/L12 dimer, residue 15 within the N-terminal domain contacts the C-terminal domain of the partner monomer. We tested effects of the mutation on molecular assembly by biochemical approaches. Gel electrophoretic analysis showed that the Phe15-L7/L12 variant had reduced ability in binding to L10, an effect enhanced in the presence of 0.05% of nonionic detergent. Mobility of Phe15-L7/L12 on gel containing the detergent was very low compared to the wild-type proteins, presumably because of an extended structural state of the mutant L7/L12. Ribosomes isolated from LL103 cells contained a reduced amount of L7/L12 and showed low levels (15-30% of wild-type ribosomes) of activities dependent on elongation factors and in translation of natural mRNA. The ribosomal activity was completely recovered by addition of an excess amount of Phe15-L7/L12 to the ribosomes, suggesting that the mutant L7/L12 exerts normal functions when bound on the ribosome. The interaction of Ser15 with the C-terminal domain of the partner molecule seems to contribute to formation of the compact dimer structure and its efficient assembly into the ribosomal GTPase center. We propose a model relating compact and elongated forms of L7/L12 dimers. Phe15-L7/L12 provides a new tool for studying the functional structure of the homodimer.

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“…each L7/L12 dimer contains on the average 1.4 FAPB moieties. On the basis of our previous analyses at least 89 % of these FAPB groups is located at Lys-51 and the remaining FAPB groups are bound elsewhere on the polypeptide chain [12]. FAPB-L7/L12 was reconstituted into 50s core particles.…”

Section: Discussionmentioning

confidence: 99%

“…Reconstitution of 50s cores with FAPB-modified L7/L12 was performed by incubating 50s cores with eight equivalents of FAPB-L7/L12 or 3H-labeled FAPB-L7/L12 for 10 min at 37 "C in GTPase buffer, after which the ribosomes were isolated by centrifugation through a 15 % sucrose cushion in GTPase buffer [12].…”

Section: Abbreviations Fapb-imidate 4-(6-formyl-3-azidophenoxy)butyr-mentioning

confidence: 99%

Localization of Lys‐51 of L7/L12 on 50S ribosomes from Escherichia coli

Maassen

Schop

Möller

1984

European Journal of Biochemistry

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Ribosomal protein L7/L 12 from Escherichia coli was modified specifically at Lys-51 with 4-(6-formyl-3-azidophen0xy)butyrimidate.Reconstitution of ribosomal cores, lacking L7/L12, with imidate-modified L7/L12 resulted in back formation of 50s particles which were fully active in elongation-factor-dependent processes. By use of the formylazidophenoxy moiety as hapten, the position of Lys-51 of L7/L12 on the 50s ribosome was determined by immune electron microscopy. The results show that an L7/L12 dimer is present in the L7/L3 2 stalk in such a way that Lys-51 is located at the far cytoplasmic end of the stalk. The experimental data are discussed in relation to a proposed model for the L7iL12 dimer.During protein synthesis, the ribosome performs a number of discrete steps which are modulated by factors. In both prokaryotic and eukaryotic organisms, the 'acidic proteins' from the large ribosomal subunit are involved in the functioning of many of these factors [l].Whereas most ribosomal proteins are present as singlecopy proteins the large ribosomal subunit seems in general to contain several copies of the acidic proteins [I -31. In the case of ribosomes from Escherichia coli, where the acidic proteins are designated L7/L12, it has been demonstrated that the large subunit contains four copies of L7/L12, probably in form of two dimers [4 -61. Electron microscope studies have shown that L7/L12 is involved in the formation of a morphological feature on the 50s subunit, the so-called L7/L12 stalk [7]. Whether both L7/L12 dimers are located in this stalk is controversial (cf. [8] and [9]).In order to get more detailed information on the localization of L7/L 12 on the ribosome we have modified L 7/L 12 specifically at Lys-51 with 4-(6-formyl-3-azidophenoxy)butyrimidate (FAPB-imidate). The FAPB moiety was used as hapten for the induction of anti-FAPB antibodies. Making use of these antibodies we have mapped the position of Lys-53 on the 50s ribosomal subunit. Our results show that at the most two IgG molecules can bind to the L7/L12 stalk, at a position which is distal from the ribosomal body. MATERIALS AND METHODSRibosomes were isolated from Escherichia coli MRE 600 according to Gesteland [lo] and separated at low magnesium concentration on a sucrose gradient in a zonal rotor [lo]. The ribosomes were stored in GTPase buffer: 20 mM Tris/HCl, pH7.6; 60mM NH4Cl; 10mM MgCl, and 6 m M 2-mercaptoethanol.L 7/L 12, modified with 4-(6-formyl-3-azidophenoxy)butyrimidate (FAPB-imidate) at Lys-51, was prepared and characterized as described previously [Il, 121. Abbreviations. FAPB-imidate, 4-(6-formyl-3-azidophenoxy)butyr-imidate; NaC1/Pi, phosphate-buffered saline.3H-labeled FAPB-L7/L12 was obtained by reduction of the aldehyde group in the FAPB moiety with [3H]NaBH4 (Amersham, GB) as described [ll].Reconstitution of 50s cores with FAPB-modified L7/L12 was performed by incubating 50s cores with eight equivalents of FAPB-L7/L12 or 3H-labeled FAPB-L7/L12 for 10 min at 37 "C in GTPase buffer, after which the ribosomes were isolated by...

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Fluorescence studies on the location of L7/L12 relative to L10 in the 50S ribosome of Escherichia coli

Cited by 29 publications

References 28 publications

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

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

A Point Mutation in Ribosomal Protein L7/L12 Reduces Its Ability to Form a Compact Dimer Structure and to Assemble into the GTPase Center

Localization of Lys‐51 of L7/L12 on 50S ribosomes from Escherichia coli

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