Mutations in the intersubunit bridge regions of 16S rRNA affect decoding and subunit-subunit interactions on the 70S ribosome

The small and large subunits of the ribosome are held together by a series of bridges, involving RNA–RNA, RNA–protein and protein–protein interactions. Some 12 bridges have been described for the Escherichia coli 70S ribosome. In this work, we have targeted for mutagenesis, some of the 16S rRNA residues involved in the formation of intersubunit bridges B3, B5, B6, B7b and B8. In addition to effects on subunit association, the mutant ribosomes also affect the fidelity of translation; bridges B5, B6 and B8 increase decoding errors during elongation, while disruption of bridges B3 and B7b alters the stringency of start codon selection. Moreover, mutations in the bridge B5, B6 and B8 regions of 16S rRNA also correct the growth and decoding defects associated with alterations in ribosomal protein S12. These results link bridges B5, B6 and B8 with the decoding process and are consistent with the recently described location of translation factor EF-Tu on the ribosome and the proposed involvement of h14 in activating Guanosine-5′-triphosphate (GTP) hydrolysis by aminoacyl-tRNA•EF-Tu•GTP. These observations are consistent with a model in which bridges B5, B6 and B8 contribute to the fidelity of translation by modulating GTP hydrolysis by aminoacyl-tRNA•EF-Tu•GTP ternary complexes during the elongation phase of protein synthesis.     

Structure of the mammalian 80S ribosome at 8.7 A resolution

In this paper, we present a structure of the mammalian ribosome determined at approximately 8.7 A resolution by electron cryomicroscopy and single-particle methods. A model of the ribosome was created by docking homology models of subunit rRNAs and conserved proteins into the density map. We then modeled expansion segments in the subunit rRNAs and found unclaimed density for approximately 20 proteins. In general, many conserved proteins and novel proteins interact with expansion segments to form an integrated framework that may stabilize the mature ribosome. Our structure provides a snapshot of the mammalian ribosome at the beginning of translation and lends support to current models in which large movements of the small subunit and L1 stalk occur during tRNA translocation. Finally, details are presented for intersubunit bridges that are specific to the eukaryotic ribosome. We suggest that these bridges may help reset the conformation of the ribosome to prepare for the next cycle of chain elongation.     

The activity of the acidic phosphoproteins from the 80 S rat liver ribosome

The selective removal of acidic phosphoproteins from the 80 S rat liver ribosome was accomplished by successive alcohol extractions at low salt concentration. The resulting core ribosomes lost over 90% of their translation activity and were unable to support the elongation factor 2 GTPase reaction. Both activities were partially restored when the dialyzed extracts were added back to the core ribosome. The binding of labeled adenosine diphosphoribosyl-elongation factor 2 to ribosomes was also affected by extraction and could be reconstituted, although not to the same extent as the GTPase activity associated with elongation factor 2 in the presence of the ribosome. The alcohol extracts of the 80 S ribosome contained mostly phosphoproteins P1 and P2 which could be dephosphorylated and rephosphorylated in solution by alkaline phosphatase and protein kinase, respectively. Dephosphorylation of the P1/P2 mixture in the extracts caused a decrease in the ability of these proteins to reactivate the polyphenylalanine synthesis activity of the core ribosome. However, treatment of the dephosphorylated proteins with the catalytic subunit of 3':5'-cAMP-dependent protein kinase in the presence of ATP reactivated the proteins when compared to the activity of the native extracts. Rabbit antisera raised against the alcohol-extracted proteins were capable of impairing both the polyphenylalanine synthesis reaction and the elongation factor 2-dependent GTPase reaction in the intact ribosomes.     

Features of 80S mammalian ribosome and its subunits

It is generally believed that basic features of ribosomal functions are universally valid, but a systematic test still stands out for higher eukaryotic 80S ribosomes. Here we report: (i) differences in tRNA and mRNA binding capabilities of eukaryotic and bacterial ribosomes and their subunits. Eukaryotic 40S subunits bind mRNA exclusively in the presence of cognate tRNA, whereas bacterial 30S do bind mRNA already in the absence of tRNA. 80S ribosomes bind mRNA efficiently in the absence of tRNA. In contrast, bacterial 70S interact with mRNA more productively in the presence rather than in the absence of tRNA. (ii) States of initiation (P_i), pre-translocation (PRE) and post-translocation (POST) of the ribosome were checked and no significant functional differences to the prokaryotic counterpart were observed including the reciprocal linkage between A and E sites. (iii) Eukaryotic ribosomes bind tetracycline with an affinity 15 times lower than that of bacterial ribosomes (K_d 30 μM and 1–2 μM, respectively). The drug does not effect enzymatic A-site occupation of 80S ribosomes in contrast to non-enzymatic tRNA binding to the A-site. Both observations explain the relative resistance of eukaryotic ribosomes to this antibiotic.     

Architecture of the protein-conducting channel associated with the translating 80S ribosome.

In vitro assembled yeast ribosome-nascent chain complexes (RNCs) containing a signal sequence in the nascent chain were immunopurified and reconstituted with the purified protein-conducting channel (PCC) of yeast endoplasmic reticulum, the Sec61 complex. A cryo-EM reconstruction of the RNC-Sec61 complex at 15.4 A resolution shows a tRNA in the P site. Distinct rRNA elements and proteins of the large ribosomal subunit form four connections with the PCC across a gap of about 10-20 A. Binding of the PCC influences the position of the highly dynamic rRNA expansion segment 27. The RNC-bound Sec61 complex has a compact appearance and was estimated to be a trimer. We propose a binary model of cotranslational translocation entailing only two basic functional states of the translating ribosome-channel complex.     

The role of ethanol extractable proteins from the 80S rat liver ribosome

80S rat liver ribosomes have been extracted with fifty percent ethanol at varying salt concentrations. The resulting 80S core ribosomes have lost almost all of their protein synthesis activity. The protein synthesis activity could be partially regained when the ethanol extracted proteins were reconstituted with the core ribosomes; however, reconstitution of the ribosome dependent EF-II GTP hydrolysis activity could not be detected. The ethanol extracts were found to contain only a few proteins, one or more of which we believe is necessary for the binding of elongation factor-II.     

Three-dimensional cryo-electron microscopy localization of EF2 in the Saccharomyces cerevisiae 80S ribosome at 17.5 A resolution

Using a sordarin derivative, an antifungal drug, it was possible to determine the structure of a eukaryotic ribosome small middle dotEF2 complex at 17.5 A resolution by three-dimensional (3D) cryo-electron microscopy. EF2 is directly visible in the 3D map and the overall arrangement of the complex from Saccharomyces cerevisiae corresponds to that previously seen in Escherichia coli. However, pronounced differences were found in two prominent regions. First, in the yeast system the interaction between the elongation factor and the stalk region of the large subunit is much more extensive. Secondly, domain IV of EF2 contains additional mass that appears to interact with the head of the 40S subunit and the region of the main bridge of the 60S subunit. The shape and position of domain IV of EF2 suggest that it might interact directly with P-site-bound tRNA.     

Structural Insights into ribosome recycling factor interactions with the 70S ribosome

At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 Å, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.     

Purification,characterization and crystallization of the human 80S ribosome

Ribosomes are key macromolecular protein synthesis machineries in the cell. Human ribosomes have so far not been studied to atomic resolution because of their particularly complex structure as compared with other eukaryotic or prokaryotic ribosomes, and they are difficult to prepare to high homogeneity, which is a key requisite for high-resolution structural work. We established a purification protocol for human 80S ribosomes isolated from HeLa cells that allows obtaining large quantities of homogenous samples as characterized by biophysical methods using analytical ultracentrifugation and multiangle laser light scattering. Samples prepared under different conditions were characterized by direct single particle imaging using cryo electron microscopy, which helped optimizing the preparation protocol. From a small data set, a 3D reconstruction at subnanometric resolution was obtained showing all prominent structural features of the human ribosome, and revealing a salt concentration dependence of the presence of the exit site tRNA, which we show is critical for obtaining crystals. With these well-characterized samples first human 80S ribosome crystals were obtained from several crystallization conditions in capillaries and sitting drops, which diffract to 26 Å resolution at cryo temperatures and for which the crystallographic parameters were determined, paving the way for future high-resolution work.     

Structure of the 70S ribosome from human pathogen Staphylococcus aureus

Comparative structural studies of ribosomes from various organisms keep offering exciting insights on how species-specific or environment-related structural features of ribosomes may impact translation specificity and its regulation. Although the importance of such features may be less obvious within more closely related organisms, their existence could account for vital yet species-specific mechanisms of translation regulation that would involve stalling, cell survival and antibiotic resistance. Here, we present the first full 70S ribosome structure from Staphylococcus aureus, a Gram-positive pathogenic bacterium, solved by cryo-electron microscopy. Comparative analysis with other known bacterial ribosomes pinpoints several unique features specific to S. aureus around a conserved core, at both the protein and the RNA levels. Our work provides the structural basis for the many studies aiming at understanding translation regulation in S. aureus and for designing drugs against this often multi-resistant pathogen.     

Composition and structure of the 80S ribosome from the green alga Chlamydomonas reinhardtii: 80S ribosomes are conserved in plants and animals

We have conducted a proteomic analysis of the 80S cytosolic ribosome from the eukaryotic green alga Chlamydomonas reinhardtii, and accompany this with a cryo-electron microscopy structure of the ribosome. Proteins homologous to all but one rat 40S subunit protein, including a homolog of RACK1, and all but three rat 60S subunit proteins were identified as components of the C. reinhardtii ribosome. Expressed Sequence Tag (EST) evidence and annotation of the completed C. reinhardtii genome identified genes for each of the four proteins not identified by proteomic analysis, showing that algae potentially have a complete set of orthologs to mammalian 80S ribosomal proteins. Presented at 25A, the algal 80S ribosome is very similar in structure to the yeast 80S ribosome, with only minor distinguishable differences. These data show that, although separated by billions of years of evolution, cytosolic ribosomes from photosynthetic organisms are highly conserved with their yeast and animal counterparts.     

Structure and expression of the PHO80 gene of Saccharomyces cerevisiae.

In yeast, the repression of acid phosphatase under high phosphate growth conditions requires the trans-acting factor PHO80. We have determined the DNA sequence of the PHO80 gene and found that it encodes a protein of 293 amino acids. The expression of the PHO80 gene, as measured by Northern analysis and level of a PHO80-LacZ fusion protein is independent of the level of phosphate in the growth medium. Disruption of the PHO80 gene is a non-lethal event and causes a derepressed phenotype, with acid phosphatase levels which are 3-4 fold higher than the level found in derepressed wild type cells. Furthermore, over-expression of the PHO80 gene causes a reduction in the level of acid phosphatase produced under derepressed growth conditions. Finally, we have cloned, localized and sequenced a temperature-sensitive allele of PHO80 and found the phenotype to be due to T to C transition causing a substitution of a Ser for a Leu at amino acid 163 in the protein product.     

Protein S3 in the human 80S ribosome adjoins mRNA from 3'-side of the A-site codon

The protein environment of mRNA 3' of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5'-end and a perfluoroarylazide group at one of the nucleotide residues at the 3'-end of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNAPhe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNAPhe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in triple complexes and in the absence of tRNA. Within triple complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed, it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3' of the codon in the decoding site.     

RNA--protein interactions in the ribosome. IV. Structure and properties of binding sites for proteins S4, S16/S17 and S20 in the 16S RNA

Proteins S4, S16/S17 and S20 of the 30 S ribosomal subunit of Escherichia coli+ associate with specific binding sites in the 16 S ribosomal RNA. A systematic investigation of the co-operative interactions that occur when two or more of these proteins simultaneously attach to the 16 S RNA indicate that their binding sites lie near to one another. The binding site for S4 has previously been located within a 550-nucleotide RNA fragment of approximately 9 S that arises from the 5′-terminal portion of the 16 S RNA upon limited hydrolysis with pancreatic ribonuclease. The 9 S RNA was unable to associate with S20 and S16/S17, however, either alone or in combination. A fragment of similar size and nucleotide sequence, termed the 9 S¹ RNA, has been isolated following ribonuclease digestion of the complex of 16 S RNA with S20 and S16/S17. The 9 S¹ RNA bound not only S4, but S20 and S16/S17 as well, although the fragment complex was stable only when both of the latter protein fractions were present together. Nonetheless, measurements of binding stoichiometry demonstrated the interactions to be specific under these conditions. A comparison of the 9 S and 9 S¹ RNAs by electrophoresis in polyacrylamide gels containing urea revealed that the two fragments differ substantially in the number and distribution of hidden breaks. Contrary to expectation, the RNA in the ribonucleoprotein complex appeared to be more accessible to ribonuclease than the free 16 S RNA as judged by the smaller average length of the sub-fragments recovered from the 9 S¹ RNA. These results suggest that the binding of S4, S16/S17 and S20 brings about a conformational alteration within the 5′ third of the 16 S RNA.To delineate further the portions of the RNA chain that interact with S4, S16/S17 and S20, specific fragments encompassing subsequences from the 5′ third of the 16 S RNA were sought. Two such fragments, designated 12 S-I and 12 S-II, were purified by polyacrylamide gel electrophoresis from partial T₁ ribonuclease digests of the 16 S RNA. The two RNAs, which contain 290 and 210 nucleotides, respectively, are contiguous and together span the entire 5′-terminal 500 residues of the 16 S RNA molecule. When tested individually, neither 12 S-I nor 12 S-II bound S4, S16/S17 or S20. If heated together at 40 °C in the presence of Mg²⁺ ions, however, the two fragments together formed an 8 S complex which associated with S4 alone, with S16/S17 + S20 in combination, and with S4 + S16/S17 + S20 when incubated with an un fractionated mixture of 30 S subunit proteins. These results imply that each fragment contains part of the corresponding binding sites.     

Unbalanced ribosome assembly in Saccharomyces cerevisiae expressing mutant 5 S rRNAs.

Mutant 5 S rRNA genes were expressed in Saccharomyces cerevisiae to further define the function of the ribosomal 5 S RNA. RNA synthesis and utilization were assayed using previously constructed markers which have been shown to be functionally neutral and easily detected by gel electrophoresis. Most mutations were found not to affect the growth rate because they were poorly expressed or could be accommodated effectively in the ribosomal structure. Two of the mutants, Y5A99U56U57 and Y5U90i5 adversely affected cell growth as well as protein synthesis in vitro. Polyribosome profiles in both of these mutants were substantially shorter, and an analysis of the ribosomal subunit composition revealed a significant imbalance with a 25-35% excess in 40 S subunits. Kinetic analyses of RNA labeling indicated very low cellular levels of mutant RNA either because it was poorly expressed (Y5U90i5) or rapidly degraded before being incorporated into mature 60 subunits (Y5A99U56U57). The results suggest that the 5 S RNA is required for the assembly of stable ribosomal 60 S subunits and raise the possibility that this RNA or, more likely, its corresponding ribonucleoprotein complex is critical for subunit assembly or even RNA processing.     

RNA-protein interactions in the ribosome: VIII. Co-operative interactions in the 50 S subunit of Escherichia coli

Six 50 S ribosomal subunit proteins, each unable to interact independently with the 23 S RNA, were shown to associate specifically with ribonucleoprotein complexes consisting of intact 23 S RNA, or fragments derived from it, and one or more RNA-binding proteins. In particular, L21 and L22 depend for attachment upon L20 and L24, respectively; L5, L10 and L11 interact individually with complexes containing L2 and L16; and one or both proteins of the $\text{L}\text{17}\text{L}\text{27}$ mixture are stimulated to bind in the presence of L1, L3, L6, L13 and L23. Moreover, L14 alone was found to interact with a fragment from the 3′ end of the 23 S RNA, even though it cannot bind to 23 S RNA. By correlating the data reported here with the findings of others, it has been possible to formulate a partial in vitro assembly map of the Escherichia coli 50 S subunit encompassing both the 5 S and 23 S RNAs as well as 21 of the 34 subunit proteins.