The Ribosomal Stalk Binds to Translation Factors IF2, EF-Tu, EF-G and RF3 via a Conserved Region of the L12 C-terminal Domain

Helgstrand, Magnus; Mandava, Chandra Sekhar; Mulder, Frans A. A.; Liljas, Anders; Sanyal, Suparna; Akke, Mikael

doi:10.1016/j.jmb.2006.10.025

Cited by 115 publications

(131 citation statements)

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“…Subsequent studies isolated mutants of L7/L12, which altered the fidelity and speed of protein synthesis in vivo (29). Recent in vitro studies have converged on a small cluster of conserved, positively charged or aliphatic residues of L7/L12, which may interact with the G protein factors EF-Tu, EF-G, RF3, and IF2 (9,10,30).…”

Section: Discussionmentioning

confidence: 99%

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Nechifor

Murataliev

Wilson

2007

Journal of Biological Chemistry

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Protein L7/L12 of the bacterial ribosome plays an important role in activating the GTP hydrolytic activity of elongation factor G (EF-G), which promotes ribosomal translocation during protein synthesis. Previously, we cross-linked L7/L12 from two residues (209 and 231) flanking ␣-helix A G in the G subdomain of Escherichia coli EF-G. Here we report kinetic studies on the functional effects of mutating three neighboring glutamic acid residues (224, 228, and 231) to lysine, either singly or in combination. Two single mutations (E224K and E228K), both within helix A G , caused large defects in GTP hydrolysis and smaller defects in ribosomal translocation. Removal of L7/L12 from the ribosome strongly reduced the activities of wild type EF-G but had no effect on the activities of the E224K and E228K mutants. Together, these results provide evidence for functionally important interactions between helix A G of EF-G and L7/L12 of the ribosome.

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Section: Discussionmentioning

confidence: 99%

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Nechifor

Murataliev

Wilson

2007

Journal of Biological Chemistry

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“…Aliquots were stored at −80°C in 10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, 50 mM ammonium chloride, 6 mM β-mercaptoethanol. EF-G was expressed and purified following a previously described procedure (15). Chemicals were purchased from Sigma unless otherwise stated.…”

Section: Methodsmentioning

confidence: 99%

“…Specifically, ribosomes are uniformly labeled with stable isotopes ( 13 C and 15 N) to provide sufficient mass differences to resolve heterocomplexes formed with wild-type ribosomes. After mixing [ 12 C, 14 N] wild-type ribosomes (l-) with [ 13 C, 15 N]-labeled ribosomes (h-) hetero stalk complexes will form of the composition [l/hL10(lL7/L12) x (hL7/ L12) y ] where x+y = 4. To confirm that isotopic labeling method does not affect the subunit interactions within the ribosome we first recorded mass spectra of h-ribosomes.…”

Section: Isotopically Labeled Ribosomes Maintain Interactionsmentioning

confidence: 99%

Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

et al. 2012

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The ribosomal stalk complex binds and recruits translation factors to the ribosome during protein biosynthesis. In Escherichia coli the stalk is composed of protein L10 and four copies of L7/L12. Despite the crucial role of the stalk, mechanistic details of L7/L12 subunit exchange are not established. By incubating isotopically labeled intact ribosomes with their unlabeled counterparts we monitored the exchange of the labile stalk proteins by recording mass spectra as a function of time. Based on kinetic analysis we proposed a mechanism whereby exchange proceeds via L7/L12 monomers and dimers. We also compared exchange of L7/L12 from free ribosomes with exchange from ribosomes in complex with elongation factor G (EF-G), trapped in the posttranslocational state by fusidic acid. Results showed that binding of EF-G reduces the L7/L12 exchange reaction of monomers by ~27% and of dimers by ~47% compared with exchange from free ribosomes. This is consistent with a model in which binding of EF-G does not modify interactions between the L7/L12 monomers but rather one of the four monomers, and as a result one of the two dimers, become anchored to the ribosome-EF-G complex preventing their free exchange. Overall therefore our results not only provide mechanistic insight into the exchange of L7/L12 monomers and dimers and the effects of EF-G binding but also have implications for modulating stability in response to environmental and functional stimuli within the cell.Protein biosynthesis is carried out by the ribosomal machinery and requires interaction of several translation factors with the ribosomal stalk complex. In bacteria the base of the stalk region, interacting with the rRNA of the 50S large ribosomal subunit, is composed of adjacent proteins L11 and L10 (1). The C-terminal domain (CTD) of L10 is a long and mobile helix which interacts with two dimers of protein L12 (2). Three pairs of L12 are observed in thermophilic bacteria and archaea (3-6) and L12 is the only protein present as multiple copies on the ribosome. The N-terminal domain (NTD) of L12 is responsible for dimerization and for anchoring of the protein to the ribosome (7). In Escherichia coli (E. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts coli), L12 is partially N-acetylated to form protein L7 (8). The ratio of L7 to L12 is known to change throughout the growth phase (8-10) and has been linked to variation in the stability of the stalk complex via hydrogen exchange mass spectrometry (MS) experiments (11). L12 CTD and NTD are connected via a flexible hinge region providing mobility to the highly structured CTD (12, 13). These highly mobile proteins have eluded high resolution X-ray analysis for many years. Only recently the atomic structure of a truncated stalk complex of a thermophilic bacterium revealed the binding mode of the NTD of L12 proteins to L10 (4). The tight antiparallel binding of the L12 NTDs is similar to that observed by NMR for the E. coli L7/L12 dimer in solution (14). The mobility and dynamics o...

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“…However, the interaction between L12 and EF-Tu and its contribution to GTP hydrolysis has not been clarified in the crystal structure of the ribosome•EF-Tu complex (17,18). The analysis of NMR chemical shifts has demonstrated the presence of interactions between the C-terminal domain of purified L12 and the translational GTPases EF-G, EF-Tu, initiation factor 2 (IF2), and release factor 3 (RF3), which implies that L12 interacts with a structural feature shared by these factors (19). The detailed mechanisms of the interactions and their contributions to the functions of the factors remain unclear.…”

mentioning

confidence: 99%

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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The Ribosomal Stalk Binds to Translation Factors IF2, EF-Tu, EF-G and RF3 via a Conserved Region of the L12 C-terminal Domain

Cited by 115 publications

References 53 publications

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

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

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