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

Abstract

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...

Functional aspects of ribosomal architecture: symmetry, chirality and regulation

“…A head-to-shoulder motion in the small subunit [Plate 1(a)] allows threading of the incoming mRNA through a dynamic pore, and the platform, located at the opposite side of the particle, is likely to perform the motions that facilitate the mRNA exit. 4,[19][20][21] The pivotal point for this motion is probably the connection between the head and the main structural junction, in the vicinity of the binding site of spectinomycin, an antibiotic found to trap the small subunit at a particular conformation, thus disabling the head motions. 22 During elongation, the mRNA advances along a curved channel on the small subunit interface, and in order to ensure cognate tRNA selection at the A-site [Plate 1(a)], a specific conformation of the small subunit is required.…”

“…22 Similarly, the two large subunit lateral protuberances, namely the L7/L12 stalk and the L1 arm [Plate 1(b)], were shown to possess dynamic properties and can undergo substantial conformational rearrangements. 21 By analyzing the available ribosomal high-resolution structures, a correlation between their structure and the functional state of the ribosome was revealed. Thus, they assume well defined, albeit dramatically different conformations in the complexed ribosome 6 compared with the unbound large ribosomal subunit, 7 They can readily become disordered [Plate 1(b)] when exposed to an environment which is very different from that leading to efficient protein biosynthesis, as seen in the crystal structure of the large subunit from Haloarcula marismortui, H50S.…”

“…2 The relative motions of these two features, which facilitate the Asite tRNA entrance (L7/L12 stalk) and the E-site tRNA exit (L1 arm), are probably associated with the motions of two features, located in the PTC vicinity, which were assigned as molecular switches. These are the multi-task inter-subunit bridge B2a [Plate 1(f)] that is likely to assist the A-to P-site tRNA acceptor-stem shift, 11,21 and the PTC nucleotide A2602 (Escherichia coli nomenclature is being used throughout) that seems to be involved in A-to P-translocation within the PTC. 8,9,11 The PTC itself possesses the dynamic properties required for facilitating the correctly bound A-site tRNA to the P-site and for assisting rearrangements of less well aligned substrates.…”

Ribosomal dynamics inferred from variations in experimental measurements

“…With the publication of the X-ray structures of the prokaryotic and archaeal ribosome, there is an opportunity to look at noncrystallographic structural measurements to assess the agreement between different types of data. The hypothesis behind this work is that data that are incompatible with published crystal structures may provide coherent patterns that suggest alternative conformations complementary or additional to those observed by crystallography (Yusupov et al 2001;Brodersen et al 2002;Ramakrishnan 2002;Yonath 2002 and references therein), and other experimental methods (see Lodmell and Dahlberg 1997;Frank and Agrawal 2000). It has been suggested that biochemical proximity measurements, such as crosslink, footprint, and cleavage experiments, may contain clues to ribosomal structural fluctuations and dynamics ribosome (see Sommer and Brimacombe 2001).…”

Electron gun for diffraction experiments off controlled molecules

“…Electron and x-ray diffraction are well established tools to investigate the structures of solid state samples [1], for example in transmission electron microscopy [2] or xray crystallography [3,4]. Furthermore, electron diffraction has found broad application for gas-phase structuredetermination in chemistry [5].…”