Topology of the secondary structure elements of ribosomal protein L7/L12 from <i>E. coli</i> in solution

Bocharov, Eduard V.; Gudkov, A.T.; Arseniev, A.S.

doi:10.1016/0014-5793(95)01531-0

Cited by 38 publications

(56 citation statements)

References 22 publications

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“…An earlier study of Escherichia coli L12 by x-ray crystallography revealed the structure of the CTD to be comprised of three ␣ helices (␣4, ␣5, and ␣6) and a central three-stranded ␤ sheet (10). This structure was confirmed by a recent crystallographic study of L12 from Thermotoga maritima (30) and also by NMR analyses of E. coli L12 (16). More recently, the crystal structure of the complex between the N-terminal domain (helices ␣1 and ␣2) of L12 and L10 from T. maritima was elucidated using a reconstituted sample (6).…”

supporting

confidence: 56%

“…7) and its binding to L10 (for review see Ref. 8), the globular C-terminal domain (CTD) (9,10), which interacts with the elongation factors (11)(12)(13)(14), and a hinge region, which connects the two domains (15,16). It was generally accepted that, in the bacterial ribosomal large subunit, two L12 dimers bind to L10 to form a pentameric complex, L10⅐(L12) 2 ⅐(L12) 2 (7,17).…”

mentioning

confidence: 99%

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Engineering and Characterization of the Ribosomal L10·L12 Stalk Complex

Miyoshi

Nomura

Uchiumi

2009

Journal of Biological Chemistry

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The ribosomal stalk protein L12 is essential for events dependent on the GTP-binding translation factors. It has been recently shown that ribosomes from Thermus thermophilus contain a heptameric complex L10⅐(L12) 2 ⅐(L12) 2 ⅐(L12) 2 , rather than the conventional pentameric complex L10⅐(L12) 2 ⅐(L12) 2 . Here we describe the reconstitution of the heptameric complex from purified L10 and L12 and the characterization of its role in elongation factor G-dependent GTPase activity using a hybrid system with Escherichia coli ribosomes. The T. thermophilus heptameric complex resulted in a 2.5-fold higher activity than the E. coli pentameric complex. The structural element of the T. thermophilus complex responsible for the higher activity was investigated using a chimeric L10 protein (Ec-Tt-L10), in which the C-terminal L12-binding site in E. coli L10 was replaced with the same region from T. thermophilus, and two chimeric L12 proteins: Ec-Tt-L12, in which the E. coli N-terminal domain was fused with the T. thermophilus C-terminal domain, and Tt⅐Ec-L12, in which the T. thermophilus N-terminal domain was fused with the E. coli C-terminal domain. High GTPase turnover was observed with the pentameric chimeric complex formed from E. coli L10 and Ec-Tt-L12 but not with the heptameric complex formed from Ec-Tt-L10 and Tt⅐Ec-L12. This suggested that the C-terminal region of T. thermophilus L12, rather than the heptameric nature of the complex, was responsible for the high GTPase turnover. Further analyses with other chimeric L12 proteins identified helix ␣6 as the region most likely to contain the responsible element.

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supporting

confidence: 56%

mentioning

confidence: 99%

Engineering and Characterization of the Ribosomal L10·L12 Stalk Complex

Miyoshi

Nomura

Uchiumi

2009

Journal of Biological Chemistry

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“…NMR studies of L7/L12 in solution predicted that dimers of the ribosomal protein L7/L12 from Escherichia coli had a parallel (head-to-head) orientation and that the N-terminal domain had no contacts with the C-terminal domain. The hinge region was predicted as an unordered structure (Bocharov et al, 1996). However, the 3D high resolution structure of L7/L12 as a dimer linked to two proteolysed fragments confirmed the existence of the three predicted domains but revealed a totally different organisation ( Fig.…”

Section: Prokaryotic Equivalents Of the P-proteinsmentioning

confidence: 92%

The puzzling lateral flexible stalk of the ribosome

Gonzalo

Reboud

2003

Biology of the Cell

127

131

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The lateral flexible stalk of the large ribosomal subunit is made of several interacting proteins anchored to a conserved region of the 28S (26S) rRNA termed the GTPase-associated domain or thiostrepton loop. This structure is demonstrated to adopt puzzling changes of conformation following the different steps of the elongation cycle. Some of these proteins termed the P-proteins in eukaryotes and L10 and L7/L12 in bacteria, present little structural similarities between Eubacteria on one side and Archae and Eukaryotes on the other side. However, up to now, these proteins seem to present a similar macromolecular organisation and they have been involved in the same functions. Convincing evidence attests that these proteins participate in elongation factor binding to the ribosome, and it has been suggested that these proteins might be evolved in a GTP hydrolysis activating protein activity. Involvement of these proteins in the translational mechanism is discussed. Moreover, in eukaryotes, small P-proteins are also found as isolated proteins in a cytoplasmic pool that exchanges with the ribosome-associated P-proteins. Moreover, a part of the ribosomal proteins is phosphorylated (hence their P-protein names). The biological signification of these particularities is discussed.

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“…1C); by contrast, the crosspeaks from the 50S subunit are typical of those of a highly structured protein. Examination of the HSQC spectra in more detail indicates that the large majority of the observable resonances from the 50S correspond to those reported for the interdomain hinge and the CTD (residues 43-120) of free L7͞L12 (11) (Fig. 1B).…”

Section: Hsqcmentioning

confidence: 98%

“…1 H NMR spectroscopy on isolated 50S and 70S ribosomes has provided evidence for regions of local motional averaging, revealing several narrow resonances that have been attributed to L7͞L12 (9,10). 15 N NMR spectroscopy of isolated L7͞L12 proteins shows two apparently independent structured domains linked by a flexible hinge, with the nuclear Overhauser effect patterns indicating an N-terminal domain (NTD) dimer independent of the respective C-terminal domain (CTD) (11). Similar studies of a pentameric complex (the four copies of L7͞L12 along with L10) show that the binding of L10 to the NTD of L7͞L12 causes the disappearance of the resonances of the latter (12).…”

mentioning

confidence: 99%

Heteronuclear NMR investigations of dynamic regions of intact Escherichia coli ribosomes

Christodoulou

Larsson

Fucini

et al. 2004

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

121

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15 N-1 H NMR spectroscopy has been used to probe the dynamic properties of uniformly 15 N labeled Escherichia coli ribosomes. Despite the high molecular weight of the complex (Ϸ2.3 MDa), [ 1 H-15 N] heteronuclear single-quantum correlation spectra contain Ϸ100 well resolved resonances, the majority of which arise from two of the four C-terminal domains of the stalk proteins, L7͞L12. Heteronuclear pulse-field gradient NMR experiments show that the resonances arise from species with a translational diffusion constant consistent with that of the intact ribosome. Longitudinal relaxation time (T1) and T1 15 N-spin relaxation measurements show that the observable domains tumble anisotropically, with an apparent rotational correlation time significantly longer than that expected for a free L7͞L12 domain but much shorter than expected for a protein rigidly incorporated within the ribosomal particle. The relaxation data allow the ribosomally bound C-terminal domains to be oriented relative to the rotational diffusion tensor. Binding of elongation factor G to the ribosome results in the disappearance of the resonances of the L7͞L12 domains, indicating a dramatic reduction in their mobility. This result is in agreement with cryoelectron microscopy studies showing that the ribosomal stalk assumes a single rigid orientation upon elongation factor G binding. As well as providing information about the dynamical properties of L7͞L12, these results demonstrate the utility of heteronuclear NMR in the study of mobile regions of large biological complexes and form the basis for further NMR studies of functional ribosomal complexes in the context of protein synthesis. P rotein synthesis in living systems takes place on the ribosome, a complex macromolecular assembly whose structural and functional properties are rapidly emerging from a powerful combination of electron microscopy (EM) and x-ray crystallography (1, 2). In Escherichia coli, the ribosome is composed of 54 different proteins and three RNA molecules (23S, 16S, and 5S rRNA) This 2.3-MDa complex is termed the 70S ribosome and is made up of two components, the 30S and 50S subunits. The translation of genetic information into functional proteins involves a number of auxiliary factors, many of which are GTPases, including IF2, EF-Tu, elongation factor G (EF-G), and RF3 (2). These molecules bind to overlapping sites on the 50S subunit and regulate the transition of the ribosome through various states on the translational pathway. The binding sites are collectively known as the GTPase-associated region, due to the role of this region in stimulating the GTPase activity of the auxiliary factors.The GTPase-associated region (GAR) includes helices 42-44 and 95 (the ␣-sarcin loop) of 23S RNA, and the proteins L10, L11, and L7͞L12 (1). The latter (L7͞L12) is located on the ribosomal stalk and is unique among the ribosomal proteins, because it is the only protein present in multiple copies (four proteins per ribosomal particle). Although atomic details of much of the GAR have been reve...

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Topology of the secondary structure elements of ribosomal protein L7/L12 from E. coli in solution

Cited by 38 publications

References 22 publications

Engineering and Characterization of the Ribosomal L10·L12 Stalk Complex

Engineering and Characterization of the Ribosomal L10·L12 Stalk Complex

The puzzling lateral flexible stalk of the ribosome

Heteronuclear NMR investigations of dynamic regions of intact Escherichia coli ribosomes

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