Ribosomal Proteins in the Spotlight

ABSTRACT

The assignment of specific ribosomal functions to individual ribosomal proteins is difficult due to the enormous cooperativity of the ribosome; however, important roles for distinct ribosomal proteins are becoming evident. Although rRNA has a major role in certain aspects of ribosomal function, such as decoding and peptidyl-transferase activity, ribosomal proteins are nevertheless essential for the assembly and optimal functioning of the ribosome. This is particularly true in the context of interactions at the entrance pore for mRNA, for the translation-factor binding site and at the tunnel exit, where both chaperones and complexes associated with protein transport through membranes bind.     

Extraction and Isolation of Individual Ribosomal Proteins from Escherichia coli

We have described a new method for the quantitative separation of ribosomal proteins and ribosomal ribonucleic acid. A procedure for the preparation of individual ribosomal proteins by polyacrylamide gel electrophoresis is also described. By the use of gels with smaller pores, at least four of the electrophoretic components from the 30S ribosome can be split into additional protein fractions. By the methods described here, it is possible to isolate in high purity at least 15 different proteins from the 30S ribosome of Escherichia coli.     

Charge Segregation and Low Hydrophobicity Are Key Features of Ribosomal Proteins from Different Organisms

Ribosomes are large and highly charged macromolecular complexes consisting of RNA and proteins. Here, we address the electrostatic and nonpolar properties of ribosomal proteins that are important for ribosome assembly and interaction with other cellular components and may influence protein folding on the ribosome. We examined 50 S ribosomal subunits from 10 species and found a clear distinction between the net charge of ribosomal proteins from halophilic and non-halophilic organisms. We found that ∼67% ribosomal proteins from halophiles are negatively charged, whereas only up to ∼15% of ribosomal proteins from non-halophiles share this property. Conversely, hydrophobicity tends to be lower for ribosomal proteins from halophiles than for the corresponding proteins from non-halophiles. Importantly, the surface electrostatic potential of ribosomal proteins from all organisms, especially halophiles, has distinct positive and negative regions across all the examined species. Positively and negatively charged residues of ribosomal proteins tend to be clustered in buried and solvent-exposed regions, respectively. Hence, the majority of ribosomal proteins is characterized by a significant degree of intramolecular charge segregation, regardless of the organism of origin. This key property enables the ribosome to accommodate proteins within its complex scaffold regardless of their overall net charge.     

Simplification of Ribosomes in Bacteria with Tiny Genomes

The ribosome is an essential cellular machine performing protein biosynthesis. Its structure and composition are highly conserved in all species. However, some bacteria have been reported to have an incomplete set of ribosomal proteins. We have analyzed ribosomal protein composition in 214 small bacterial genomes (<1 Mb) and found that although the ribosome composition is fairly stable, some ribosomal proteins may be absent, especially in bacteria with dramatically reduced genomes. The protein composition of the large subunit is less conserved than that of the small subunit. We have identified the set of frequently lost ribosomal proteins and demonstrated that they tend to be positioned on the ribosome surface and have fewer contacts to other ribosome components. Moreover, some proteins are lost in an evolutionary correlated manner. The reduction of ribosomal RNA is also common, with deletions mostly occurring in free loops. Finally, the loss of the anti-Shine–Dalgarno sequence is associated with the loss of a higher number of ribosomal proteins.     

Coordinate control of syntheses of ribosomal ribonucleic acid and ribosomal proteins during nutritional shift-up in Saccharomyces cerevisiae.

We investigated the regulation of ribosome synthesis in Saccharomyces cerevisiae growing at different rates and in response to a growth stimulus. The ribosome content and the rates of synthesis of ribosomal ribonucleic acid and of ribosomal proteins were compared in cultures growing in minimal medium with either glucose or ethanol as a carbon source. The results demonstrated that ribosome content is proportional to growth rate. Moreover, these steady-state concentrations are regulated at the level of synthesis of ribosomal precursor ribonucleic acid and of ribosomal proteins. When cultures growing on ethanol were enriched with glucose, the rate of ribosomal ribonucleic acid synthesis, measured by pulsing cells with [methyl-3H]methionine, increased by 40% within 5 min, doubled within 15 min, and reached a steady state characteristic of the new growth medium by 30 min. Labeling with [3H]leucine reveal a coordinate increase in the rate of synthesis of 30 or more ribosomal proteins as compared with that of total cellular proteins. Their synthesis was stimulated approximately 2.5-fold within 15 min and nearly 4-fold within 60 min. The data suggest that S. cerevisiae responds to a growth stimulus by preferential stimulation of the synthesis of ribosomal ribonucleic acid and ribosomal proteins.     

A cluster of methylations in the domain IV of 25S rRNA is required for ribosome stability

In all three domains of life ribosomal RNAs are extensively modified at functionally important sites of the ribosome. These modifications are believed to fine-tune the ribosome structure for optimal translation. However, the precise mechanistic effect of modifications on ribosome function remains largely unknown. Here we show that a cluster of methylated nucleotides in domain IV of 25S rRNA is critical for integrity of the large ribosomal subunit. We identified the elusive cytosine-5 methyltransferase for C2278 in yeast as Rcm1 and found that a combined loss of cytosine-5 methylation at C2278 and ribose methylation at G2288 caused dramatic ribosome instability, resulting in loss of 60S ribosomal subunits. Structural and biochemical analyses revealed that this instability was caused by changes in the structure of 25S rRNA and a consequent loss of multiple ribosomal proteins from the large ribosomal subunit. Our data demonstrate that individual RNA modifications can strongly affect structure of large ribonucleoprotein complexes.     

Crystal Structure of the Yeast Ribosomal Protein rpS3 in Complex with Its Chaperone Yar1

Eukaryotic ribosome assembly involves a plethora of factors, which ensure that a correctly folded ribosome contains all ribosomal protein components. Among these assembly factors, Yar1 has recently emerged as a molecular chaperone for ribosomal protein rpS3 of the small ribosomal subunit (40S) in yeast. In complex with its chaperone, rpS3 is imported into the nucleus and protected from aggregation. How rpS3 and other ribosomal proteins are initially sequestered and subsequently integrated into pre-ribosomal particles is currently poorly understood. Here, we present the crystal structure of yeast rpS3 in complex with its chaperone Yar1 at 2.8 Å resolution. The crystal structure rationalizes how Yar1 can protect rpS3 from aggregation while facilitating nuclear import and suggests a mechanism for a stepwise exchange of molecular partners that ribosomal proteins interact with during ribosome assembly.     

Studies on the kinetic sequence of in vitro ribosome assembly using cibacron blue F3GA as a general assembly inhibitor.

We have found that all E. coli ribosomal proteins strongly bind to an agarose affinity column derivatized with the dye Cibacron Blue F3GA. We have also shown that the capacity to bind the dye is lost when the proteins are organized within the structure of the ribosome or are members of pre-formed protein-RNA complexes. We conclude that the binding of ribosomal proteins to this dye involves specific protein-RNA recognition sites. These observations led us to discover that Cibacron Blue can be used to inhibit in vitro ribosome assembly at any stage of the assembly process. This has allowed us to determine a kinetic order of ribosome assembly.     

Identification of novel Escherichia coli ribosome-associated proteins using isobaric tags and multidimensional protein identification techniques

Biogenesis of the large ribosomal subunit requires the coordinate assembly of two rRNAs and 33 ribosomal proteins. In vivo, additional ribosome assembly factors, such as helicases, GTPases, pseudouridine synthetases, and methyltransferases, are also critical for ribosome assembly. To identify novel ribosome-associated proteins, we used a proteomic approach (isotope tagging for relative and absolute quantitation) that allows for semiquantitation of proteins from complex protein mixtures. Ribosomal subunits were separated by sucrose density centrifugation, and the relevant fractions were pooled and analyzed. The utility and reproducibility of the technique were validated via a double duplex labeling method. Next, we examined proteins from 30S, 50S, and translating ribosomes isolated at both 16 degrees C and 37 degrees C. We show that the use of isobaric tags to quantify proteins from these particles is an excellent predictor of the particles with which the proteins associate. Moreover, in addition to bona fide ribosomal proteins, additional proteins that comigrated with different ribosomal particles were detected, including both known ribosomal assembly factors and unknown proteins. The ribosome association of several of these proteins, as well as others predicted to be associated with ribosomes, was verified by immunoblotting. Curiously, deletion mutants for the majority of these ribosome-associated proteins had little effect on cell growth or on the polyribosome profiles.     

The carboxy-terminal extension of yeast ribosomal protein S14 is necessary for maturation of 43S preribosomes

Eukaryotic ribosomal proteins are required for production of stable ribosome assembly intermediates and mature ribosomes, but more specific roles for these proteins in biogenesis of ribosomes are not known. Here we demonstrate a particular function for yeast ribosomal protein rpS14 in late steps of 40S ribosomal subunit maturation and pre-rRNA processing. Extraordinary amounts of 43S preribosomes containing 20S pre-rRNA accumulate in the cytoplasm of certain rps14 mutants. These mutations not only reveal a more precise function for rpS14 in ribosome biogenesis but also uncover a role in ribosome assembly for the extended tails found in many ribosomal proteins. These studies are one of the first to relate the structure of eukaryotic ribosomes to their assembly pathway-the carboxy-terminal extension of rpS14 is located in the 40S subunit near the 3' end of 18S rRNA, consistent with a role for rpS14 in 3' end processing of 20S pre-rRNA.     

Modification of yeast ribosomal proteins. Phosphorylation.

Two-dimensional polyacrylamide-gel electrophoretic analysis of yeast ribosomal proteins labelled in vivo with 32PO43- revealed that the proteins S2 and S10 of the 40S ribosomal subunit, and the proteins L9, L30, L44 and L45 of the 60S ribosomal subunit, are phosphorylated in vivo. Most of the phosphate groups appeared to be linked to serine residues. Teh number of phosphate groups per molecule of phosphorylated protein species ranged from 0.01 to 0.79. Since most of the phosphorylated ribosomal proteins appear to associate with the pre-ribosomal particles at a very late stage of ribosome assembly, phosphorylation is more likely to play a role in the functioning of the ribosome than in its assembly.     

Depletion of yeast ribosomal proteins L16 or rp59 disrupts ribosome assembly

Two strains of Saccharomyces cerevisiae were constructed that are conditional for synthesis of the 60S ribosomal subunit protein, L16, or the 40S ribosomal subunit protein, rp59. These strains were used to determine the effects of depriving cells of either of these ribosomal proteins on ribosome assembly and on the synthesis and stability of other ribosomal proteins and ribosomal RNAs. Termination of synthesis of either protein leads to diminished accumulation of the subunit into which it normally assembles. Depletion of L16 or rp59 has no effect on synthesis of most other ribosomal proteins or ribosomal RNAs. However, most ribosomal proteins and ribosomal RNAs that are components of the same subunit as L16 or rp59 are rapidly degraded upon depletion of L16 or rp59, presumably resulting from abortive assembly of the subunit. Depletion of L16 has no effect on the stability of most components of the 40S subunit. Conversely, termination of synthesis of rp59 has no effect on the stability of most 60S subunit components. The implications of these findings for control of ribosome assembly and the order of assembly of ribosomal proteins into the ribosome are discussed.     

A decade of progress in understanding the structural basis of protein synthesis

The key reaction of protein synthesis, peptidyl transfer, is catalysed in all living organisms by the ribosome - an advanced and highly efficient molecular machine. During the last decade extensive X-ray crystallographic and NMR studies of the three-dimensional structure of ribosomal proteins, ribosomal RNA components and their complexes with ribosomal proteins, and of several translation factors in different functional states have taken us to a new level of understanding of the mechanism of function of the protein synthesis machinery. Among the new remarkable features revealed by structural studies, is the mimicry of the tRNA molecule by elongation factor G, ribosomal recycling factor and the eukaryotic release factor 1. Several other translation factors, for which three-dimensional structures are not yet known, are also expected to show some form of tRNA mimicry. The efforts of several crystallographic and biochemical groups have resulted in the determination by X-ray crystallography of the structures of the 30S and 50S subunits at moderate resolution, and of the structure of the 70S subunit both by X-ray crystallography and cryo-electron microscopy (EM). In addition, low resolution cryo-EM models of the ribosome with different translation factors and tRNA have been obtained. The new ribosomal models allowed for the first time a clear identification of the functional centres of the ribosome and of the binding sites for tRNA and ribosomal proteins with known three-dimensional structure. The new structural data have opened a way for the design of new experiments aimed at deeper understanding at an atomic level of the dynamics of the system.     

Ribosomal proteins: Structure,function, and evolution

The question concerning reasons for the variety of ribosomal proteins that arose for more than 40 years ago is still open. Ribosomes of modern organisms contain 50–80 individual proteins. Some are characteristic for all domains of life (universal ribosomal proteins), whereas others are specific for bacteria, archaea, or eucaryotes. Extensive information about ribosomal proteins has been obtained since that time. However, the role of the majority of ribosomal proteins in the formation and functioning of the ribosome is still not so clear. Based on recent data of experiments and bioinformatics, this review presents a comprehensive evaluation of structural conservatism of ribosomal proteins from evolutionarily distant organisms. Considering the current knowledge about features of the structural organization of the universal proteins and their intermolecular contacts, a possible role of individual proteins and their structural elements in the formation and functioning of ribosomes is discussed. The structural and functional conservatism of the majority of proteins of this group suggests that they should be present in the ribosome already in the early stages of its evolution.     

Janus chaperones: Assistance of both RNA- and protein-folding by ribosomal proteins

Ribosomal proteins assist the assembly and increase the stability of ribosomal RNA, without requiring ATP for their action. Some ribosomal proteins are also known to have essential functions outside the ribosome, i.e. promiscuity of functions that appears to correlate with their structural disorder. Here we addressed if certain ribosomal proteins with RNA chaperone activity and with a significant level of disorder also have protein-chaperone activity in vitro. Four proteins of the large subunit of Escherichia coli ribosome, L15, L16, L18 and L19 have been tested in three chaperone assays, in which all of them exhibited potent chaperone activity, commensurable with that of heat shock protein 90 kDa. These observations highlight possible novel aspects of the promiscuous functions of ribosomal proteins outside of the ribosome.     

Structure of the 80S ribosome from Saccharomyces cerevisiae--tRNA-ribosome and subunit-subunit interactions.

A cryo-EM reconstruction of the translating yeast 80S ribosome was analyzed. Computationally separated rRNA and protein densities were used for docking of appropriately modified rRNA models and homology models of yeast ribosomal proteins. The core of the ribosome shows a remarkable degree of conservation. However, some significant differences in functionally important regions and dramatic changes in the periphery due to expansion segments and additional ribosomal proteins are evident. As in the bacterial ribosome, bridges between the subunits are mainly formed by RNA contacts. Four new bridges are present at the periphery. The position of the P site tRNA coincides precisely with its prokaryotic counterpart, with mainly rRNA contributing to its molecular environment. This analysis presents an exhaustive inventory of an eukaryotic ribosome at the molecular level.     

Mechanisms of In Vivo Ribosome Maintenance Change in Response to Nutrient Signals

Control of protein homeostasis is fundamental to the health and longevity of all organisms. Because the rate of protein synthesis by ribosomes is a central control point in this process, regulation, and maintenance of ribosome function could have amplified importance in the overall regulatory circuit. Indeed, ribosomal defects are commonly associated with loss of protein homeostasis, aging, and disease (1, 2, 3, 4), whereas improved protein homeostasis, implying optimal ribosomal function, is associated with disease resistance and increased lifespan (5, 6, 7). To maintain a high-quality ribosome population within the cell, dysfunctional ribosomes are targeted for autophagic degradation. It is not known if complete degradation is the only mechanism for eukaryotic ribosome maintenance or if they might also be repaired by replacement of defective components. We used stable-isotope feeding and protein mass spectrometry to measure the kinetics of turnover of ribosomal RNA (rRNA) and 71 ribosomal proteins (r-proteins) in mice. The results indicate that exchange of individual proteins and whole ribosome degradation both contribute to ribosome maintenance in vivo. In general, peripheral r-proteins and those with more direct roles in peptide-bond formation are replaced multiple times during the lifespan of the assembled structure, presumably by exchange with a free cytoplasmic pool, whereas the majority of r-proteins are stably incorporated for the lifetime of the ribosome. Dietary signals impact the rates of both new ribosome assembly and component exchange. Signal-specific modulation of ribosomal repair and degradation could provide a mechanistic link in the frequently observed associations among diminished rates of protein synthesis, increased autophagy, and greater longevity (5, 6, 8, 9).