Ditlev E. Brodersen

Structure of the 30S ribosomal subunit.

Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 Å resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein–RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.

Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics

The 30S ribosomal subunit has two primary functions in protein synthesis. It discriminates against aminoacyl transfer RNAs that do not match the codon of messenger RNA, thereby ensuring accuracy in translation of the genetic message in a process called decoding. Also, it works with the 50S subunit to move the tRNAs and associated mRNA by precisely one codon, in a process called translocation. Here we describe the functional implications of the high-resolution 30S crystal structure presented in the accompanying paper, and infer details of the interactions between the 30S subunit and its tRNA and mRNA ligands. We also describe the crystal structure of the 30S subunit complexed with the antibiotics paromomycin, streptomycin and spectinomycin, which interfere with decoding and translocation. This work reveals the structural basis for the action of these antibiotics, and leads to a model for the role of the universally conserved 16S RNA residues A1492 and A1493 in the decoding process.

The Structural Basis for the Action of the Antibiotics Tetracycline, Pactamycin, and Hygromycin B on the 30S Ribosomal Subunit

We have used the recently determined atomic structure of the 30S ribosomal subunit to determine the structures of its complexes with the antibiotics tetracycline, pactamycin, and hygromycin B. The antibiotics bind to discrete sites on the 30S subunit in a manner consistent with much but not all biochemical data. For each of these antibiotics, interactions with the 30S subunit suggest a mechanism for its effects on ribosome function.

Crystal Structures of the Ribosome in Complex with Release Factors RF1 and RF2 Bound to a Cognate Stop Codon

During protein synthesis, translational release factors catalyze the release of the polypeptide chain when a stop codon on the mRNA reaches the A site of the ribosome. The detailed mechanism of this process is currently unknown. We present here the crystal structures of the ribosome from Thermus thermophilus with RF1 and RF2 bound to their cognate stop codons, at resolutions of 5.9 Angstrom and 6.7 Angstrom, respectively. The structures reveal details of interactions of the factors with the ribosome and mRNA, including elements previously implicated in decoding and peptide release. They also shed light on conformational changes both in the factors and in the ribosome during termination. Differences seen in the interaction of RF1 and RF2 with the L11 region of the ribosome allow us to rationalize previous biochemical data. Finally, this work demonstrates the feasibility of crystallizing ribosomes with bound factors at a defined state along the translational pathway.

The structural basis for mRNA recognition and cleavage by the ribosome-dependent endonuclease RelE.

Summary Translational control is widely used to adjust gene expression levels. During the stringent response in bacteria, mRNA is degraded on the ribosome by the ribosome-dependent endonuclease, RelE. The molecular basis for recognition of the ribosome and mRNA by RelE and the mechanism of cleavage are unknown. Here, we present crystal structures of E. coli RelE in isolation (2.5 Å) and bound to programmed Thermus thermophilus 70S ribosomes before (3.3 Å) and after (3.6 Å) cleavage. RelE occupies the A site and causes cleavage of mRNA after the second nucleotide of the codon by reorienting and activating the mRNA for 2′-OH-induced hydrolysis. Stacking of A site codon bases with conserved residues in RelE and 16S rRNA explains the requirement for the ribosome in catalysis and the subtle sequence specificity of the reaction. These structures provide detailed insight into the translational regulation on the bacterial ribosome by mRNA cleavage.

Origins and activities of the eukaryotic exosome.

The exosome is a multi-subunit 3′-5′ exonucleolytic complex that is conserved in structure and function in all eukaryotes studied to date. The complex is present in both the nucleus and cytoplasm, where it continuously works to ensure adequate quantities and quality of RNAs by facilitating normal RNA processing and turnover, as well as by participating in more complex RNA quality-control mechanisms. Recent progress in the field has convincingly shown that the nucleolytic activity of the exosome is maintained by only two exonuclease co-factors, one of which is also an endonuclease. The additional association of the exosome with RNA-helicase and poly(A) polymerase activities results in a flexible molecular machine that is capable of dealing with the multitude of cellular RNA substrates that are found in eukaryotic cells. Interestingly, the same basic set of enzymatic activities is found in prokaryotic cells, which might therefore illustrate the evolutionary origin of the eukaryotic system. In this Commentary, we compare the structural and functional characteristics of the eukaryotic and prokaryotic RNA-degradation systems, with an emphasis on some of the functional networks in which the RNA exosome participates in eukaryotes.

The social life of ribosomal proteins

Ribosomal proteins hold a unique position in biology because their function is so closely tied to the large rRNAs of the ribosomes in all kingdoms of life. Following the determination of the complete crystal structures of both the large and small ribosomal subunits from bacteria, the functional role of the proteins has often been overlooked when focusing on rRNAs as the catalysts of translation. In this review we highlight some of the many known and important functions of ribosomal proteins, both during translation on the ribosome and in a wider context.

Structure of the nuclear exosome component Rrp6p reveals an interplay between the active site and the HRDC domain.

The multisubunit eukaryotic exosome is an essential RNA processing and degradation machine. In its nuclear form, the exosome associates with the auxiliary factor Rrp6p, which participates in both RNA processing and degradation reactions. The crystal structure of Saccharomyces cerevisiae Rrp6p displays a conserved RNase D core with a flanking HRDC (helicase and RNase D C-terminal) domain in an unusual conformation shown to be important for the processing function of the enzyme. Complexes with AMP and UMP, the products of the RNA degradation process, reveal how the protein specifically recognizes ribonucleotides and their bases. Finally, in vivo mutational studies show the importance of the domain contacts for the processing function of Rrp6p and highlight fundamental differences between the protein and its prokaryotic RNase D counterparts.

EF-hands at atomic resolution: the structure of human psoriasin (S100A7) solved by MAD phasing

BACKGROUND The S100 family consists of small acidic proteins, belonging to the EF-hand class of calcium-binding proteins. They are primarily regulatory proteins, involved in cell growth, cell structure regulation and signal transduction. Psoriasin (S100A7) is an 11.7 kDa protein that is highly upregulated in the epidermis of patients suffering from the chronic skin disease psoriasis. Although its exact function is not known, psoriasin is believed to participate in the biochemical response which follows transient changes in the cellular Ca2+ concentration. RESULTS The three-dimensional structure of holmium-substituted psoriasin has been determined by multiple anomalous wavelength dispersion (MAD) phasing and refined to atomic resolution (1.05 A). The structure represents the most accurately determined structure of a calcium-binding protein. Although the overall structure of psoriasin is similar to those of other S100 proteins, several important differences exist, mainly in the N-terminal EF-hand motif that contains a distorted loop and lacks a crucial calcium-binding residue. It is these minor differences that may account for the different specificities among members of this family. CONCLUSIONS The structure of human psoriasin reveals that this protein, in contrast to other S100 proteins with known structure, is not likely to strongly bind more than one calcium ion per monomer. The present study contradicts the idea that calcium binding induces large changes in conformation, as suggested by previously determined structures of apo forms of S100 proteins. The substitution of Ca2+ ions in EF-hands by lanthanide ions may provide a general vehicle for structure determination of S100 proteins by means of MAD phasing.

VapC20 of Mycobacterium tuberculosis cleaves the Sarcin–Ricin loop of 23S rRNA

The highly persistent and often lethal human pathogen, Mycobacterium tuberculosis contains at least 88 toxin-antitoxin genes. More than half of these encode VapC PIN domain endoribonucleases that inhibit cell growth by unknown mechanisms. Here we show that VapC20 of M. tuberculosis inhibits translation by cleavage of the Sarcin-Ricin loop (SRL) of 23S ribosomal RNA at the same position where Sarcin and other eukaryotic ribotoxins cleave. Toxin-inhibited cells can be rescued by the expression of the antitoxin, thereby raising the possibility that vapC20 contributes to the extreme persistence exhibited by M. tuberculosis. VapC20 cleavage is inhibited by mutations in the SRL that flank the cleavage site but not by changes elsewhere in the loop. Disruption of the SRL stem abolishes cleavage; however, further mutations that restore the SRL stem structure restore cleavage, revealing that the structure rather than the exact sequence of the SRL is important for this activity.