Shape properties of proteins L7 and L12 from E. coli ribosomes

Wong, K.P.; Paradies, Hasko H.

doi:10.1016/0006-291x(74)90550-6

Cited by 60 publications

(9 citation statements)

References 12 publications

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“…Subsequently, crosslinking studies with the E. coli protein detected dimers [127]. Small-angle X-ray scattering saw E. coli L12 in solution as a tightly associated and highly elongated dimer (~180Å in length) [127] in agreement with results from sedimentation and viscosity measurements [128,129]. Sedimentation equilibrium studies also indicated dimers for the isolated acidic r-proteins from the other lines of decent [130] and the dimeric state of L12-like proteins in solution has now been widely accepted [102,127,131].…”

Section: Oligomerization and Shapesupporting

confidence: 52%

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: Oligomerization and Shapesupporting

confidence: 52%

Structure and Function of the Acidic Ribosomal Stalk Proteins

Wahl

Möller²

2002

curr protein pept sci

105

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“…As expected from the reduced content of L7/L12, LL103 50S subunits showed a low level, about 15% of wild-type 50S subunits, of EF-Gdependent GTPase activity ( Figure 1B) that was nonetheless higher than that of the L7/L12-deficient core particles prepared by the ethanol treatment. A similar level of activity 16.5% SDS-polyacrylamide gel electrophoresis. The gel was stained with fluorescent regent, SYPRO Orange, and amounts of L7/L12 in the 50S subunits were estimated, as described in Materials and Methods.…”

Section: Properties Of 50s Ribosomal Subunits From E Coli Ll103mentioning

confidence: 88%

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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“…L7/L12 was initially characterized as a rigid, highly elongated dimer based on low-angle X-ray scattering, sedimentation velocity, and viscosimetry experiments (O ¨sterberg et al, 1976;Wong & Paradies, 1974;Luer & Wong, 1979; these studies reported axial ratios in the range of 8:1 to 19:1. More recently, neutron scattering studies reported an axial ratio of 6.5:1 for L7/L12 associated with the 50S ribosomal subunit (Nowotny et al, 1994).…”

mentioning

confidence: 99%

Rotational and Conformational Dynamics of Escherichia coli Ribosomal Protein L7/L12

et al. 1996

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Fluorescence methods were utilized to study dynamic aspects of the 24 kDa dimeric Escherichia coli ribosomal protein L7/L12. Oligonucleotide site-directed mutagenesis was used to introduce cysteine residues at specific locations along the peptide chain, in both the C-terminal and N-terminal domains, and various sulfhydryl reactive fluorescence probes (iodoacetamido) fluorescein, IAEDANS, pyrenemethyl iodoacetate) were attached to these residues. In addition to the full-length proteins, a hinge-deleted variant and variants corresponding to the C-terminal fragment and the N-terminal fragment were also studied. Both steady-state and time-resolved fluorescence measurements were carried out, and the results demonstrated that L7/L12 is not a rigid molecule. Specifically, the two C-terminal domains move freely with respect to one another and with respect to the dimeric N-terminal domain. Removal of the hinge region, however, significantly reduces the mobility of the C-terminal domains. The data also show that the rotational relaxation time monitored by the fluorescent probe-depends upon the probe's excited state lifetime. This observation is interpreted to indicate that a hierarchy of motions exists in the L7/L12 molecule including facile motions of the C-terminal domains and dimeric N-terminal domain, in addition to the overall tumbling of the protein. Probes attached to the N-terminal domain exhibit global rotational relaxation times consistent with the molecular mass of the dimeric N-terminal fragment. Upon reconstitution of labeled L7/L12 with ribosomal cores, however, the motion associated with the dimeric N-terminal domain is greatly diminished while the facile motion of the C-terminal domains is almost unchanged.

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Shape properties of proteins L7 and L12 from E. coli ribosomes

Cited by 60 publications

References 12 publications

Structure and Function of the Acidic Ribosomal Stalk Proteins

Structure and Function of the Acidic Ribosomal Stalk 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

Rotational and Conformational Dynamics of Escherichia coli Ribosomal Protein L7/L12

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