High-resolution Structures of Ribosomal Subunits: Initiation, Inhibition, and Conformational Variability (original) (raw)

2001, Cold Spring Harbor Symposia on Quantitative Biology

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Abstract

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The study focuses on high-resolution crystallography of ribosomal subunits, emphasizing their dynamic behavior during protein biosynthesis. It discusses the intricate mechanisms of translation initiation, conformational variability, and the effect of inhibitors on ribosomal function, shedding light on the complex interactions between ribosomal RNA and proteins.

Functional aspects of ribosomal architecture: symmetry, chirality and regulation

Journal of Physical Organic Chemistry, 2004

High-resolution structures of both ribosomal subunits revealed that most stages of protein biosynthesis, including decoding of genetic information, are navigated and controlled by the elaborate ribosomal architectural-design. Remote interactions govern accurate substrate alignment within a flexible active-site pocket [peptidyl transferase center (PTC)], and spatial considerations, due mainly to a universal mobile nucleotide, U2585, ensure proper chirality by interfering with D-amino-acids incorporation. tRNA translocation involves two correlated motions: overall mRNA/tRNA (messenger and transfer RNA) shift, and a rotation of the tRNA single-stranded aminoacylated-3′ end around the bond connecting it with the tRNA helical-regions. This bond coincides with an axis passing through a sizable symmetry-related region, identified around the PTC in all large-subunit crystal structures. Propelled by a bulged universal nucleotide, A2602, positioned at the two-fold symmetry axis, and guided by a ribosomal-RNA scaffold along an exact pattern, the rotatory motion results in stereochemistry optimal for peptide-bond formation and in geometry ensuring nascent proteins entrance into their exit tunnel. Hence, confirming that ribosomes contribute positional rather than chemical catalysis, and that peptide bond formation is concurrent with A- to P-site tRNA passage. Connecting between the PTC, the decoding center, the tRNA entrance and exit points, the symmetry-related region can transfer intra-ribosomal signals between remote functional locations, guaranteeing smooth processivity of amino acids polymerization. Ribosomal proteins are involved in accurate substrate placement (L16), discrimination and signal transmission (L22) and protein biosynthesis regulation (CTC). Residing on the exit tunnel walls near its entrance, and stretching to its opening, protein-L22 can mediate ribosome response to cellular regulatory signals, since it can swing across the tunnel, causing gating and elongation arrest. Each of the protein CTC domains has a defined task. The N-terminal domain stabilizes the intersubunit-bridge confining the A-site-tRNA entrance. The middle domain protects the bridge conformation at elevated temperatures. The C-terminal domain can undergo substantial conformational rearrangements upon substrate binding, indicating CTC participation in biosynthesis-control under stressful conditions. Copyright © 2004 John Wiley & Sons, Ltd.

Protein Synthesis at Atomic Resolution: Mechanistics of Translation in the Light of Highly Resolved Structures for the Ribosome

Current Protein and Peptide Science, 2002

Our understanding of the process of translation has progressed rapidly since the availability of highly resolved structures for the ribosome. A wealth of information has emerged in terms of both RNA and protein structure and the interplay between them. This has prompted us to revisit the astonishing "treasure trove" of functional data regarding the ribosome that has accumulated over the past decades. Here we try a systematic synopsis of these ribosomal functions in light of the cryo-electron microscopic structures (resolution >7 Å) and the atomic x-ray structures (>2.4 Å) of the ribosome.

Decision letter: Structure of the bacterial ribosome at 2 Å resolution

2020

Using cryo-electron microscopy (cryo-EM), we determined the structure of the Escherichia coli 70S ribosome with a global resolution of 2.0 Å. The maps reveal unambiguous positioning of protein and RNA residues, their detailed chemical interactions, and chemical modifications. Notable features include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former likely conserved in all domains of life. The maps also reveal extensive solvation of the small (30S) ribosomal subunit, and interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The maps and models of the bacterial ribosome presented here now allow a deeper phylogenetic analysis of ribosomal components including structural conservation to the level of solvation. The high quality of the maps should enable future structural analyses of the chemical basis for translation and aid the development of robust tools for cryo-EM structure modeling and refinement.

Alternative conformations and motions adopted by 30S ribosomal subunits visualized by cryo-electron microscopy

RNA, 2020

It is only after recent advances in cryo-electron microscopy that it is now possible to describe at high-resolution structures of large macromolecules that do not crystalize. Purified 30S subunits interconvert between an “active” and “inactive” conformation. The active conformation was described by crystallography in the early 2000s, but the structure of the inactive form at high resolution remains unsolved. Here we used cryo-electron microscopy to obtain the structure of the inactive conformation of the 30S subunit to 3.6 Å resolution and study its motions. In the inactive conformation, an alternative base-pairing of three nucleotides causes the region of helix 44, forming the decoding center to adopt an unlatched conformation and the 3′ end of the 16S rRNA positions similarly to the mRNA during translation. Incubation of inactive 30S subunits at 42°C reverts these structural changes. The air–water interface to which ribosome subunits are exposed during sample preparation also peel...

A MULTIPLE RIBOSOMAL STRUCTURE IN PROTEIN SYNTHESIS

Proceedings of the National Academy of Sciences, 1963

It has been well established that the ribosomal particles are the site of protein synthesis, yet we have very little insight into the mechanism.1 A great deal of attention has been directed toward the question of how the ribosomes contain the information necessary to effect the alignment of amino acids in a specific sequence. This problem has been resolved recently with the discovery of a rapidly metabolizing fraction of RNA, called messenger RNA, which has the ability to attach itself to the ribosomal particle and there to determine the sequence of amino acids.2' I This view has been considerably reinforced by in vitro experiments in which naturally occurring RNA, as well as synthetic polyribonucleotides, have been shown to provide the information necessary to determine the sequence of amino acids in a polypeptide chain.4' 5 Thus, the ribosome has a passive role in transmitting information; it can apparently polymerize a variety of proteins, depending upon the particular messenger RNA which is attached to it.

Structure of the E. coli signal recognition particle bound to a translating ribosome

Nature, 2007

The prokaryotic signal recognition particle (SRP) targets membrane proteins into the inner membrane 1-4 . It binds translating ribosomes and screens the emerging nascent chain for a hydrophobic signal sequence, such as the transmembrane helix of inner membrane proteins. If such a sequence emerges, the SRP binds tightly, allowing the SRP receptor to lock on. This assembly delivers the ribosome-nascent chain complex to the protein translocation machinery in the membrane. Using cryo-electron microscopy and single-particle reconstruction, we obtained a 16 Å structure of the Escherichia coli SRP in complex with a translating E. coli ribosome containing a nascent chain with a transmembrane helix anchor. We also obtained structural information on the SRP bound to an empty E. coli ribosome. The latter might share characteristics with a scanning SRP complex, whereas the former represents the next step: the targeting complex ready for receptor binding. High-resolution structures of the bacterial ribosome and of the bacterial SRP components are available, and their fitting explains our electron microscopic density. The structures reveal the regions that are involved in complex formation, provide insight into the conformation of the SRP on the ribosome and indicate the conformational changes that accompany high-affinity SRP binding to ribosome nascent chain complexes upon recognition of the signal sequence.

New Insights into Ribosome Structure and Function

Cold Spring Harbor perspectives in biology, 2018

In the past 4 years, because of the advent of new cameras, many ribosome structures have been solved by cryoelectron microscopy (cryo-EM) at high, often near-atomic resolution, bringing new mechanistic insights into the processes of translation initiation, peptide elongation, termination, and recycling. Thus, cryo-EM has joined X-ray crystallography as a powerful technique in structural studies of translation. The significance of this new development is that structures of ribosomes in complex with their functional binding partners can now be determined to high resolution in multiple states as they perform their work. The aim of this article is to provide an overview of these new studies and assess the contributions they have made toward an understanding of translation and translational control.

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Structural basis of the ribosomal machinery for peptide bond formation, translocation, and nascent chain progression

Molecular Cell, 2003

Noller et al., 1992; Garrett and Rodriguez-Fon-Weizmann Institute seca, 1995; Samaha et al., 1995). Crystal structures of 76100 Rehovot complexes of Thermus thermophilus ribosomes (T70S) Israel with tRNA (Yusupov et al., 2001) as well as of the large 2 Max-Planck-Research Unit for Ribosomal Structure ribosomal subunits from the archaeon Haloarcula maris-Notkestrasse 85 mortui (H50S) and the mesophilic eubacterium Deino-22603 Hamburg coccus radiodurans (D50S) with various substrate pepti-Germany dyl-transferase analogs or inhibitors (Nissen et al., 2000; 3 Max-Planck-Institute for Molecular Genetics Schluenzen et al., 2001; Schmeing et al., 2002; Hansen Ihnestrasse 73 et al., 2002) show that the PTC can be described as a 4 FB Biologie, Chemie, Pharmazie pocket with a tunnel emerging from it. It is located at Frei University Berlin the bottom of a cavity containing all of the nucleotides Takustrasse 3

Ribosomal crystallography: Peptide bond formation and its inhibition

Biopolymers, 2003

Ribosomes, the universal cellular organelles catalyzing the translation of genetic code into proteins, are protein/RNA assemblies, of a molecular weight 2.5 mega Daltons or higher. They are built of two subunits that associate for performing protein biosynthesis. The large subunit creates the peptide bond and provides the path for emerging proteins. The small has key roles in initiating the process and controlling its fidelity.

Structure and Dynamics of the Mammalian Ribosomal Pretranslocation Complex

Molecular Cell, 2011

Although the structural core of the ribosome is conserved in all kingdoms of life, eukaryotic ribosomes are significantly larger and more complex than their bacterial counterparts. The extent to which these differences influence the molecular mechanism of translation remains elusive. Multiparticle cryo-electron microscopy and single-molecule FRET investigations of the mammalian pretranslocation complex reveal spontaneous, large-scale conformational changes, including an intersubunit rotation of the ribosomal subunits. Through structurally related processes, tRNA substrates oscillate between classical and at least two distinct hybrid configurations facilitated by localized changes in their L-shaped fold. Hybrid states are favored within the mammalian complex. However, classical tRNA positions can be restored by tRNA binding to the E site or by the eukaryotic-specific antibiotic and translocation inhibitor cycloheximide. These findings reveal critical distinctions in the structural and energetic features of bacterial and mammalian ribosomes, providing a mechanistic basis for divergent translation regulation strategies and species-specific antibiotic action.

The Escherichia coli large ribosomal subunit at 7.5 Å resolution

Structure, 1999

In recent years, the three-dimensional structure of the ribosome has been visualised in different functional states by single-particle cryo-electron microscopy (cryo-EM) at 13-25 Å resolution. Even more recently, X-ray crystallography has achieved resolution levels better than 10 Å for the ribosomal structures of thermophilic and halophilic organisms. We present here the 7.5 Å solution structure of the 50S large subunit of the Escherichia coli ribosome, as determined by cryo-EM and angular reconstitution.