Crystallization of 70 S ribosomes and 30 S ribosomal subunits from <i>Thermus thermophilus</i>

Trakhanov, S.; Yusupov, Marat; Agalarov, S. Ch.; Garber, M.B.; Ryazantsev, Sergey; Tischenko, Svetlana; Широков, В. Б.

doi:10.1016/0014-5793(87)80838-4

Cited by 97 publications

(52 citation statements)

References 8 publications

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“…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).…”

Section: Introductionmentioning

confidence: 99%

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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Section: Introductionmentioning

confidence: 99%

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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“…In this study, we used ribosomes from T. thermophilus, because both 70S ribosomes and small subunits from this species can be produced in a form pure enough to be crystallizable (43,44), thereby ensuring a homogeneous sample for microscopy. However, because the gene for IF3 from Thermus was still unknown, we used IF3 from another thermophile, T. maritima, whose optimal growth temperature of 80°C is close to the 72°C optimum for T. thermophilus (45).…”

Section: If3-30s Complex Formation and Preparationmentioning

confidence: 99%

Location of translational initiation factor IF3 on the small ribosomal subunit

McCutcheon

Agrawal

Philips

et al. 1999

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

134

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The location of translational initiation factor IF3 bound to the 30S subunit of the Thermus thermophilus ribosome has been determined by cryoelectron microscopy. Both the 30S⅐IF3 complex and control 30S subunit structures were determined to 27-Å resolution. The difference map calculated from the two reconstructions reveals three prominent lobes of positive density. The previously solved crystal structure of IF3 fits very well into two of these lobes, whereas the third lobe probably arises from conformational changes induced in the 30S subunit as a result of IF3 binding. Our placement of IF3 on the 30S subunit allows an understanding in structural terms of the biochemical functions of this initiation factor, namely its ability to dissociate 70S ribosomes into 30S and 50S subunits and the preferential selection of initiator tRNA by IF3 during initiation.

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“…A few years later, crystals were obtained from the large ribosomal subunits of the extreme halophilic bacterium Halobacterium marismortui, which lives in the Dead Sea (Shevack et al 1985). In 1987, 7 years after the first crystallization of ribosomal particles, parallel efforts led to the growth of crystals of the small ribosomal subunit ) and of the entire ribosome (Trakhanov et al 1987) from the extreme thermophilic bacterium Thermus thermophilus.…”

Section: From In-house X-ray Generators To Advanced Synchrotron Radiamentioning

confidence: 99%

Large facilities and the evolving ribosome, the cellular machine for genetic-code translation

Yonath

2009

J. R. Soc. Interface.

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Well-focused X-ray beams, generated by advanced synchrotron radiation facilities, yielded high-resolution diffraction data from crystals of ribosomes, the cellular nano-machines that translate the genetic code into proteins. These structures revealed the decoding mechanism, localized the mRNA path and the positions of the tRNA molecules in the ribosome and illuminated the interactions of the ribosome with initiation, release and recycling factors. They also showed that the ribosome is a ribozyme whose active site is situated within a universal symmetrical region that is embedded in the otherwise asymmetric ribosome structure. As this highly conserved region provides the machinery required for peptide bond formation and for ribosome polymerase activity, it may be the remnant of the proto-ribosome, a dimeric prebiotic machine that formed peptide bonds and non-coded polypeptide chains. Synchrotron radiation also enabled the determination of structures of complexes of ribosomes with antibiotics targeting them, which revealed the principles allowing for their clinical use, revealed resistance mechanisms and showed the bases for discriminating pathogens from hosts, hence providing valuable structural information for antibiotics improvement.

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Crystallization of 70 S ribosomes and 30 S ribosomal subunits from Thermus thermophilus

Cited by 97 publications

References 8 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

Location of translational initiation factor IF3 on the small ribosomal subunit

Large facilities and the evolving ribosome, the cellular machine for genetic-code translation

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