Crystallographic, Biochemical and Genetic Studies on Halophilic Ribosomes

Franceschi, François; Sagi, Irit; Böddeker, Nina; Evers, Ute; Arndt, Evelyn; Paulke, Christiane; Hasenbank, Renate; Laschever, Miriam; Glotz, Carola; Piefke, Jutta; Müssig, J.; Weinstein, S.; Yonath, Ada

doi:10.1016/s0723-2020(11)80342-5

Cited by 12 publications

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“…All four proteins not observed in the 2.4Å map match the list of proteins that we detached selectively from halophilic ribosomes (20). Furthermore, the low salt conditions used for the stabilization of the crystals are similar to those developed by us for the detachment of the selected proteins, namely the lowering of the salt concentration while adding modest amounts of organic materials.…”

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confidence: 52%

The Search and Its Outcome: High-Resolution Structures of Ribosomal Particles from Mesophilic, Thermophilic, and Halophilic Bacteria at Various Functional States

Yonath

2002

Annu. Rev. Biophys. Biomol. Struct.

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We determined the high-resolution structures of large and small ribosomal subunits from mesophilic and thermophilic bacteria and compared them with those of the thermophilic ribosome and the halophilic large subunit. We confirmed that the elements involved in intersubunit contacts and in substrate binding are inherently flexible and that a common ribosomal strategy is to utilize this conformational variability for optimizing its functional efficiency and minimizing nonproductive interactions. Under close-to-physiological conditions, these elements maintain well-ordered characteristic conformations. In unbound subunits, the features creating intersubunit bridges within associated ribosomes lie on the interface surface, and the features that bind factors and substrates reach toward the binding site only when conditions are ripe. 257 Annu. Rev. Biophys. Biomol. Struct. 2002.31:257-273. Downloaded from www.annualreviews.org Access provided by 54.191.190.102 on 05/12/18. For personal use only. 258 YONATH INTRODUCTIONRibosomes are the universal cellular organelles that catalyze the sequential polymerization of amino acids according to the genetic blueprint encoded in the mRNA. They are built of two subunits that associate for performing this task. The larger subunit creates the peptide bonds and provides the path for the progression of the nascent proteins. The smaller subunit has key roles in the initiation of the process, in decoding the genetic message, in discriminating against non-and near-cognate aminoacylated tRNA molecules, in controlling the fidelity of codon-anti-codon interactions, and in mRNA/tRNA translocation. The prokaryotic large ribosomal subunit (50S) has a molecular weight of 1.5 × 10 6 Dalton and contains two RNA chains with a total of ∼3000 nucleotides and ∼35 proteins. The small ribosomal subunit (called 30S) has a molecular weight of 8.5 × 10 5 Dalton and contains one RNA chain of over 1500 nucleotides and ∼20 proteins.Over two decades ago, we initialized a long and demanding search for the determination of the three-dimensional structure of the ribosome by X-ray crystallography (74). The key to high-resolution data was to crystallize homogenous preparations under conditions similar to their in situ environments or to induce a selected conformation after the crystals were formed. Relatively robust ribosomal particles were chosen, assuming that they would deteriorate less during preparation and therefore provide more homogenous starting materials for crystallization.The first crystals to yield some crystallographic information (e.g., symmetry, unit cell parameters, and resolution) were of the large subunit from Bacillus stearothermophilus (71) and Haloarcula marismortui (H50S) (39,66). Shortly afterward, we characterized crystals of the small subunit from Thermus thermophilus (T30S) (72). Microcrystals of the same source were grown independently at approximately the same time (64).An alternative approach was to design complexes containing ribosomes at defined functional stages, such as o...

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confidence: 52%

The Search and Its Outcome: High-Resolution Structures of Ribosomal Particles from Mesophilic, Thermophilic, and Halophilic Bacteria at Various Functional States

Yonath

2002

Annu. Rev. Biophys. Biomol. Struct.

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“…It should be mentioned that the salts required for functional activity of H50S (2.5-3 M KCl and 0.5 M NH 4 Cl) hamper cryo-EM studies. The only available EM reconstruction of H50S was obtained using a solution of significantly lower salt concentration [28], close to that used at the initial step of H50S crystallization [1,34]. We used this low salt concentration to control the crystallization process because the crystallization terminated after a few hours in a shower of disordered microcrystals when the higher salt concentration necessary for activity was employed.…”

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confidence: 99%

“…Hence, we designed a procedure in which crystallization occurs in solutions with the minimum salt needed for the integrity of the particle (1.2-1.7 M KCl and 0.5 M NH 4 Cl), at which no functional activity was detected. Once the crystals are formed they are transferred to solutions containing the salts required for functional activity [18,34], allowing X-ray measurements under these conditions.…”

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confidence: 99%

Elucidating the medium-resolution structure of ribosomal particles: an interplay between electron cryo-microscopy and X-ray crystallography

et al. 1999

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Single-particle cryo-EM can contribute to the progress of crystallography of non-symmetrical, large and flexible macromolecular assemblies. Besides confirming heavy-atom sites, obtained from flat or overcrowded difference Patterson maps, the cryo-EM reconstructions assisted in elucidating packing arrangements. They also provided tools for the identification of the conformation within the crystals and for the estimation of the level of inherent non-isomorphism.

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“…Protein L11 is involved in elongation factor activities [32], and the absence of protein L1 has a negative effect on the rate of protein synthesis [33,34]. All four proteins are known to be rather flexible [31,[35][36][37] and to be held loosely by the core of the large subunit [37,38]. Interestingly, they match the list of proteins that we detached selectively from halophilic ribosomes [37,38] under conditions similar to those used for obtaining the highresolution structure of H50S.…”

Section: Introductionmentioning

confidence: 81%

“…All four proteins are known to be rather flexible [31,[35][36][37] and to be held loosely by the core of the large subunit [37,38]. Interestingly, they match the list of proteins that we detached selectively from halophilic ribosomes [37,38] under conditions similar to those used for obtaining the highresolution structure of H50S. In addition, almost all the structural features known to be involved in the non-catalytic functional aspects of protein biosynthesis were found to be disordered in the 2.4 Å structure of H50S [11].…”

Section: Introductionmentioning

confidence: 99%

High-Resolution Structures of Large Ribosomal Subunits from Mesophilic Eubacteria and Halophilic Archaea at Various Functional States

Yonath

2002

curr protein pept sci

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Structural analysis of the recently determined high resolution structures of the small and the large ribosomal subunits from three bacterial sources, assisted by the medium resolution structure of a complex of the entire ribosome with three tRNAs, led to a quantum jump in our understanding of the process of the translation of the genetic code into proteins. Results of these studies highlighted dynamic aspects of protein biosynthesis; illuminated the modes of action of several antibiotics; indicated strategies adopted by ribosomes for maximizing their functional activity and revealed a wealth of architectural elements, including long tails of proteins penetrating the particle s cores and stabilizing the intricate folds of the RNA chains. Binding of substrate analogues showed that the decoding and the peptide-bond formation are accomplished mainly by RNA. However, several proteins may be functionally relevant in directing the mRNA and in mediating the proper orientation of the tRNA molecules within the ribosomal rRNA frame. Elements involved in intersubunit contacts or in substrate binding are inherently flexible, but maintain well-ordered characteristic conformations in unbound particles. The ribosomes utilize this conformational variability for optimizing their efficiency and minimizing non-productive interactions, hence disorder of functionally relevant features may be linked to less active conformations or to far from physiological conditions. Clinically relevant antibiotics bind almost exclusively to rRNA. In the small subunit they affect the decoding accuracy or limit conformational mobility and in the large subunit they either interfere with substrate binding, by interacting with components of the peptidyl transferase cavity, or hinder the progression of the growing peptide chain.

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Crystallographic, Biochemical and Genetic Studies on Halophilic Ribosomes

Cited by 12 publications

References 25 publications

The Search and Its Outcome: High-Resolution Structures of Ribosomal Particles from Mesophilic, Thermophilic, and Halophilic Bacteria at Various Functional States

The Search and Its Outcome: High-Resolution Structures of Ribosomal Particles from Mesophilic, Thermophilic, and Halophilic Bacteria at Various Functional States

Elucidating the medium-resolution structure of ribosomal particles: an interplay between electron cryo-microscopy and X-ray crystallography

High-Resolution Structures of Large Ribosomal Subunits from Mesophilic Eubacteria and Halophilic Archaea at Various Functional States

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