Localization of L11 protein on the ribosome and elucidation of its involvement in EF-G-dependent translocation

doi:10.1006/jmbi.2001.4907

Journal of Molecular Biology

Volume 311, Issue 4, 24 August 2001, Pages 777-787

https://doi.org/10.1006/jmbi.2001.4907 Get rights and content

Abstract

L11 protein is located at the base of the L7/L12 stalk of the 50 S subunit of the Escherichia coli ribosome. Because of the flexible nature of the region, recent X-ray crystallographic studies of the 50 S subunit failed to locate the N-terminal domain of the protein. We have determined the position of the complete L11 protein by comparing a three-dimensional cryo-EM reconstruction of the 70 S ribosome, isolated from a mutant lacking ribosomal protein L11, with the three-dimensional map of the wild-type ribosome. Fitting of the X-ray coordinates of L11-23 S RNA complex and EF-G into the cryo-EM maps combined with molecular modeling, reveals that, following EF-G-dependent GTP hydrolysis, domain V of EF-G intrudes into the cleft between the 23 S ribosomal RNA and the N-terminal domain of L11 (where the antibiotic thiostrepton binds), causing the N-terminal domain to move and thereby inducing the formation of the arc-like connection with the G′ domain of EF-G. The results provide a new insight into the mechanism of EF-G-dependent translocation.

Introduction

L11 is a highly conserved, 14.8 kDa ribosomal protein that is associated with the functional center, the GTPase-associated region (GAR), of the Escherichia coli ribosome. Early immuno-electron microscopy (EM) experiments placed L11 in one of the structurally most flexible regions, the L7/L12 stalk region, at the base of the stalk formed by proteins L10 and L7/L121 of the 50 S ribosomal subunit. L11-minus mutants of both E. coli and Bacillus megaterium are viable but very sick2 and show much reduced rates of protein synthesis,3 suggesting that this protein is very important but not essential for function. Both protein L11 and antibiotic thiostrepton bind cooperatively the highly conserved 58 nucleotide long target segment (nucleotides 1051-1108 region) of the 23 S RNA of the ribosome.4, 5 In addition to L11 and the above-mentioned segment of 23 S RNA, the GAR contains another highly conserved stretch of rRNA, the α-sarcin/ricin stem-loop.6 The 58 nucleotide long segment of the 23 S RNA in complex with protein L11 is referred to as the L11-RNA complex.

During the elongation cycle of protein synthesis, two elongation factors (EFs), EF-Tu and EF-G, alternate in binding to overlapping sites on the ribosome (see Agrawal et al.7). The former, complexed with aminoacyl-tRNA and GTP (ternary complex), is responsible for the delivery of aminoacyl-tRNA to the A site, while the latter, complexed with GTP, is instrumental in catalyzing the translocation reaction, in the course of which A and P-site tRNAs move into the P and E sites, respectively, and mRNA advances by one codon. Both reactions are accompanied by GTP hydrolysis, which requires an interaction of the elongation factors with the GAR region of the large ribosomal subunit.8 The overlapping binding sites of the two factors have been determined by chemical protection6 and hydroxyl radical probing experiments,9 and by direct imaging of binding ligands with the ribosome by cryo-electron microscopy (EF-G;10, 11, 12, 13 EF-Tu ternary complex12, 14). These findings support the notion of structural molecular mimicry,15, 16, 17 which was prompted by the observation of the striking similarity between the X-ray structures of EF-G18, 19, 20 and the EF-Tu ternary complex.15

Recently, the crystal structures of the GAR, the L11 C-terminal domain (CTD) of Bacillus stearothermophilus complexed with the 58 nt 23 S RNA segment21 and the entire L11 (including the N-terminal domain, NTD) of Thermotoga maritima complexed with the same RNA fragment22 have been solved. The picture that emerged from these studies is of a large compact CTD that forms a tight association with the RNA, and a loosely bound NTD. The apparent ability of the NTD domain to change its position and its proximity to a sensitive region of RNA implicated in GTP hydrolysis have given rise to the hypothesis that it might constitute a molecular switch that controls either the accessibility or the conformation of the GAR.22 Clearly, the questions raised in this hypothesis and the complex results of mutation and protection studies of thiostrepton4, 5, 23, 24 and micrococcin25 make it necessary to look at the structural context of GAR within the ribosome. These thiazole class antibiotics are known to inhibit the elongation factor (both EF-Tu and EF-G)-dependent reactions.8 For this reason, the structural basis for the interactions of the L11-23 S RNA complex with these antibiotics and with the elongation factors is of wide medical interest.

Ban and co-workers26 were able to place the RNA portion of the L11-RNA complex convincingly into their 5 Å resolution density map of the ribosome from Haloarcula marismortui. However, the region where L11 was expected on account of the fitting of the L11-RNA complex structure was only partially filled in the X-ray density map, with the NTD region notably absent. A possible reason is the disorder of peripheral regions affected by crystal packing, since both the L1 and L7/L12 stalks are also truncated or absent in this map. A similar placement, inferred from cryo-EM of EF-G bound to the ribosome, has been presented for the E. coli ribosome,12, 27 and in that case the entire mass of the protein could be accounted for. The most detailed visualization of the L11 region is contained in the recent cryo-EM study of the E. coli ribosome at 11.5 Å resolution.28 Interestingly, the L11 region is absent from a recent cryo-EM map (E. coli;29 also see Müller et al.30), and in the 2.4 Å resolution X-ray map (H. marismortui31) of the 50 S subunit.

In the current work, the position of L11 is obtained by comparing the cryo-EM structure of the 70 S ribosome isolated from an E. coli mutant lacking L11 with that of a wild-type ribosome. Fitting of the X-ray structure of L11 (that was solved as part of the L11-RNA complex)22 into the additional mass in the wild-type reconstruction gives the position of the L11 protein in the ribosome. This independent observation supports the placement of CTD in the X-ray study26 and enables us to identify the mass corresponding to NTD. By molecular modeling, using cryo-EM maps of functional complexes of EF-G and ribosome, we are able to identify the NTD with part of the arc- like connection (ALC) observed in the fusidic acid-stalled ribosome·EF-G·GDP complex,10, 11 providing a new insight into the translocation mechanism.

Section snippets

Localization of protein L11

We isolated the 70 S ribosome from mutants lacking ribosomal protein L11, i.e. from an E. coli strain AM68,32 and obtained a 3D cryo-EM map at 18 Å resolution (Figure 1(a)). The map was amplitude-corrected using low-angle X-ray solution scattering data (see Gabashvili et al.28). The comparison of the 3D map with that of the control ribosome,33 which was amplitude-corrected and filtered to the same resolution (Figure 1(b)), clearly shows absence of density (marked by arrows in Figure 1, Figure 3)

Position of L11 and inter-domain rearrangement

The position of L11 derived from this study is in line with its placement in earlier studies,26, 28 except that the position of the NTD has been ascertained for the first time. Our results confirm (Figure 3) a recent hydroxyl radical probing study,38 which indicated that the NTD lies close to the α-sarcin/ricin loop. In particular, its amino acid residue 19 lies within 20 Å from the nucleotide present at the tip of the α-sarcin/ricin loop. The NTD is required for the cooperative interaction

Preparation of the L11-depleted ribosomes

The 70 S ribosomes were isolated from the E. coli strain AM68,32 lacking gene for the ribosomal protein L11. Cells were grown in 3×100 ml of LB medium with 2% (w/v) glucose. The growth was very slow with a generation time of 140 minutes. The cells were harvested at a cell density of A₅₆₀=0.51. All the following steps were performed at 4°C. The cells were pelleted with a slow-speed centrifugation, and the resulting 1.7 g of cells was mixed with 3.4 g of alumina and ground for two minutes in a

Acknowledgements

We thank Robert A. Grassucci for help with electron microscopy, Christian M. Spahn for comments on the manuscript, and Amy B. Heagle and Yu Chen for help with the preparation of illustrations. The work was supported by grants NIH R37 GM29169, R01 GM55440, P41 RR01219, NSF BIR 9219043 to J.F., and R01 GM61576 to R.K.A.

References (54)

W.P. Tate et al.
The NH2-terminal domain of Escherichia coli ribosomal protein L11. Its three-dimensional location and its role in the binding of release factors 1 and 2
J. Biol. Chem.
(1984)
E. Cundliffe et al.
Ribosomes in thiostrepton-resistant mutants of Bacillus megaterium lacking a single 50 S subunit protein
J. Mol. Biol.
(1979)
G. Stöffler et al.
Mutants of Escherichia coli lacking ribosomal protein L11
J. Biol. Chem.
(1980)
P.C. Ryan et al.
Recognition of the highly conserved GTPase center of 23 S ribosomal RNA by ribosomal protein L11 and the antibiotic thiostreptan
J. Mol. Biol.
(1991)
K.S. Wilson et al.
Mapping the position of translational elongation factor EF-G in the ribosome by directed hydroxyl radical probing
Cell
(1998)
H. Stark et al.
Large-scale movement of elongation factor G and extensive conformational change of the ribosome during translocation
Cell
(2000)
J. Nyborg et al.
Structure of the ternary complex of EF-Tumacromolecular mimicry in translation
Trends Biochem. Sci.
(1996)
K. Abel et al.
A complex profile of protein elongationtranslating chemical energy into molecular movement
Structure
(1996)
S. Al-Karadaghi et al.
The structure of elongation factor G in complex with GDPconformational flexibility and nucleotide exchange
Structure
(1996)
B.T. Wimberly et al.
A detailed view of a ribosomal active sitethe structure of the L11-RNA complex
Cell
(1999)

M. Carson

Ribbons

Acta Grystallog. sect. B

(1997)

J. Thompson et al.

Binding of thiostrepton to a complex of 23-S rRNA with ribosomal protein L11

Eur. J. Biochem.

(1979)

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¹: Edited by W. Baumeister

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Journal of Molecular Biology

Regular articleLocalization of L11 protein on the ribosome and elucidation of its involvement in EF-G-dependent translocation1

Abstract

Introduction

Section snippets

Localization of protein L11

Position of L11 and inter-domain rearrangement

Preparation of the L11-depleted ribosomes

Acknowledgements

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

J. Mol. Biol.

Cell

Cell

Trends Biochem. Sci.

Structure

Structure

Cell

J. Mol. Biol.

J. Mol. Biol.

Biophys. J.

Cell

Structure

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Mol. Cell

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Struct. Biol.

Ultramicroscopy

J. Mol. Biol.

Acta Grystallog. sect. B

Binding of thiostrepton to a complex of 23-S rRNA with ribosomal protein L11

Eur. J. Biochem.

Regular article
Localization of L11 protein on the ribosome and elucidation of its involvement in EF-G-dependent translocation¹