Fine structure of the 30 S ribosomal subunit

Elongated hollow strands were revealed on raw images and averaged by the correlation method images of the 30 S subunit of the E. coli ribosome negatively stained by uranyl acetate. The tentative three-dimensional arrangement of the 'strands' and their nature are discussed.     

Functional heterogeneity of the 30S ribosomal subunit of E. coli

Summary When 30S ribosomal subunits from E. coli are incubated with poly U, two separable components are recovered by zonal centrifugation of the incubation mixture. The faster sedimenting component is an aggregate of 30S subunits and poly U, while the slower one corresponds to the 30S ribosomal subunit. One ribosomal protein, protein 30S-1 is predominantly present in the faster sedimenting aggregate. The amount of poly U-30S subunit complex formed in the incubation mixture is limited by the amount of protein 30S-1 present. Consequently the number of ribosomal binding sites available for Phe-tRNA is limited in a similar fashion by the presence of protein 30S-1. When 30S ribosomal subunits are reconstituted in the absence of protein 30S-1, very little poly U or Phe-tRNA binding capacity is manifest under our assay conditions. We conclude that protein 30S-1 is required for maximum capacity of ribosomes to bind mRNA. Since this protein is present only on a fraction of the ribosome at any one time, it must exchange from one ribosome to another during protein synthesis.Abbreviations Poly U (polyuridylic acid) - t-RNA (transfer ribonucleic acid) - mRNA (messenger ribonucleic acid) - Phe (phenylanine) - A₂₆₀ unit (unit of material which gives an optical density of 1.0 at 260 nm in a one centimeter optical path)     

Assembly of the 30S ribosomal subunit

Ribosomes are large macromolecular complexes responsible for cellular protein synthesis. The smallest known cytoplasmic ribosome is found in prokaryotic cells; these ribosomes are about 2.5 MDa and contain more than 4000 nucleotides of RNA and greater than 50 proteins. These components are distributed into two asymmetric subunits. Recent advances in structural studies of ribosomes and ribosomal subunits have revealed intimate details of the interactions within fully assembled particles. In contrast, many details of how these massive ribonucleoprotein complexes assemble remain elusive. The goal of this review is to discuss some crucial aspects of 30S ribosomal subunit assembly.     

Assembly of the 30S ribosomal subunit

The assembly of ribosomes requires a significant fraction of the energy expenditure for rapidly growing bacteria. The ribosome is composed of three large RNA molecules and over 50 small proteins that must be rapidly and efficiently assembled into the molecular machine responsible for protein synthesis. For over 30 years, the 30S ribosome has been a key model system for understanding the process of ribosome biogenesis through in vitro assembly experiments. We have recently developed an isotope pulse-chase experiment using quantitative mass spectrometry that permits assembly kinetics to be measured in real time. Kinetic studies have revealed an assembly energy landscape that ensures efficient assembly by a flexible and robust pathway.     

Mapping structural differences between 30S ribosomal subunit assembly intermediates

Under appropriate conditions, functional Escherichia coli 30S ribosomal subunits assemble in vitro from purified components. However, at low temperatures, assembly stalls, producing an intermediate (RI) that sediments at 21S and is composed of 16S ribosomal RNA (rRNA) and a subset of ribosomal proteins (r-proteins). Incubation of RI at elevated temperatures produces a particle, RI*, of similar composition but different sedimentation coefficient (26S). Once formed, RI* rapidly associates with the remaining r-proteins to produce mature 30S subunits. To understand the nature of this transition from RI to RI*, changes in the reactivity of 16S rRNA between these two states were monitored by chemical modification and primer extension analysis. Evaluation of this data using structural and biochemical information reveals that many changes are r-protein-dependent and some are clustered in functional regions, suggesting that this transition is an important step in functional 30S subunit formation.     

Interaction of Era with the 30S ribosomal subunit implications for 30S subunit assembly

Era (E. coliRas-like protein) is a highly conserved and essential GTPase in bacteria. It binds to the 16S ribosomal RNA (rRNA) of the small (30S) ribosomal subunit, and its depletion leads to accumulation of an unprocessed precursor of the 16S rRNA. We have obtained a three-dimensional cryo-electron microscopic map of the Thermus thermophilus 30S-Era complex. Era binds in the cleft between the head and platform of the 30S subunit and locks the subunit in a conformation that is not favorable for association with the large (50S) ribosomal subunit. The RNA binding KH motif present within the C-terminal domain of Era interacts with the conserved nucleotides in the 3' region of the 16S rRNA. Furthermore, Era makes contact with several assembly elements of the 30S subunit. These observations suggest a direct involvement of Era in the assembly and maturation of the 30S subunit.     

A role for proteins S3 and S14 in the 30 S ribosomal subunit

Small ribosomal subunits prepared by the method of Kirillov et al. (Kirillov, S. V., Makhno, V. I., Peshin, N. N., and Semenkov, Yu. P. (1986) Nucleic Acids Res. 5, 4305-4315) are active but fail to reconstitute. The inability to reconstitute is due to a deficiency in proteins S3 and S14. Supplementation of the protein component with pure S3 and S14 leads to an enhancement of the activity of the reconstituted product. Our results provide evidence that these two proteins are involved in assembly but may not be required once the 30 S subunit has been properly assembled.     

A snapshot of the 30S ribosomal subunit capturing mRNA via the Shine-Dalgarno interaction

In the initiation phase of bacterial translation, the 30S ribosomal subunit captures mRNA in preparation for binding with initiator tRNA. The purine-rich Shine-Dalgarno (SD) sequence, in the 5' untranslated region of the mRNA, anchors the 30S subunit near the start codon, via base pairing with an anti-SD (aSD) sequence at the 3' terminus of 16S rRNA. Here, we present the 3.3 A crystal structure of the Thermus thermophilus 30S subunit bound with an mRNA mimic. The duplex formed by the SD and aSD sequences is snugly docked in a "chamber" between the head and platform domains, demonstrating how the 30S subunit captures and stabilizes the otherwise labile SD helix. This location of the SD helix is suitable for the placement of the start codon AUG in the immediate vicinity of the mRNA channel, in agreement with reported crosslinks between the second position of the start codon and G1530 of 16S rRNA.     

X-ray and neutron small-angle scattering studies of the complex between protein S1 and the 30-S ribosomal subunit.

X-ray neutron solution scattering experiments have been done to investigate the influence of the binding of ribosomal protein S1 on the conformation of the 30-S ribosomal subunit of Escherichia coli. The following conclusions were made. 1. The alterations (if any) in conformation of the non-S1 parts of the 30-S subunit induced by S1 binding are too small to be detected (less than 0.1 nm change in radius of gyration). 2. The center of gravity of protein S1 bound to the 30-S subunit is quite far from the center of gravity of the particle (approximately 7.5 nm).     

An RNA secondary structure switch between the inactive and active conformations of the Escherichia coli 30 S ribosomal subunit

Psoralen cross-linking was used to produce intramolecular cross-links in the Escherichia coli 16 S ribosomal RNA in the inactive and active forms of the 30 S subunit. A number of psoralen cross-links were made in the inactive form that were not made in the active form. The most frequent of these cross-links was sequenced by a series of techniques and identified as C-924 to U-1532. In this region, a three-base complementary, (921-923).(1532-1534), forms a site where psoralen can stack and produce a cross-link between C-924 and U-1532. When psoralen monoadducts were placed on inactive subunits and the conformation was switched to the active form before cross-linking, a new cross-link involving U-1393 was detected. U-1393 is part of the complementarity, (923-925).(1391-1393), that has previously been proposed as being an element of the functional secondary structure on the basis of sequence comparison. The complementarity between (921-923).(1532-1534) occurs in most nonmitochondrial small subunit RNAs; however, there are several counter examples in which it does not occur. This suggests that this alternate secondary structure interaction is not necessary for the function of the 30 S subunit.     

Identification of the collar-like structure of the 30S ribosomal subunit from E. coli by dark field electron microscopy

We have used dark field electron microscopy to study a fragment of the small (30S) subunit of the E. coli ribosome. This fragment is almost the same size as the parent particle but RNA sequencing studies have shown it to lack, as a major constituent, a 150-nucleotide stretch at the 3' end of the rRNA, and two minor sections constituting 20 nucleotides from the 5' end and the 15 nucleotides of the sequence 687-701. The protein composition of the fragment was essentially unchanged. Samples of this material, and controls, were examined in the electron microscope after treatment with a buffered uranyl acetate solution for positive staining. Careful comparison revealed the following differences. The structural feature that we call the "collar" was missing in the fragment. Of the three parallel uranyl-staining bands that we have observed in micrographs of whole 30S subunits, the fragment consistently lacked the uppermost band. These observations identify the top uranyl-adsorbing band as being the 3' end of the ribosomal RNA and show that it can be equated with the collar-like structure.     

Interaction of translation initiation factor 3 with the 30S ribosomal subunit.

Hydroxyl radical footprinting and directed probing from Fe(II)-derivatized IF3 have been used to map the interaction of IF3 relative to 16S rRNA and tRNA(Met)(f) in the 30S ribosomal subunit. Our results place the two domains of IF3 on opposite sides of the initiator tRNA, with the C domain at the platform interface and the N domain at the E site. The C domain coincides with the location of helix 69 of 23S rRNA, explaining the ability of IF3 to block subunit association. The N domain neighbors proteins S7 and S11 and may interfere with E site tRNA binding. Our model suggests that IF3 influences initiator tRNA selection indirectly.     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.     

Long range RNA-RNA interactions in the 30 S ribosomal subunit of E. coli.

We have attempted to identify long-range interactions in the tertiary structure of RNA in the E. coli 30 S ribosome. Native subunits were cleaved with ribonuclease and separated into nucleoprotein fragments which were deproteinized and fractionated into multi-oligonucleotide complexes under conditions intended to preserve RNA-RNA interactions. The final products were denatured by urea and heat and their constituent oligonucleotides resolved and sequenced. Many complexes contained complementary sequences known to be bound together in the RNA secondary structure, attesting to the validity of the technique. Other co-migrating oligonucleotides, not joined in the secondary structure, contained mutually complementary sequences in locations that allow base-pairing interaction without disrupting pre-existing secondary structure. In seven instances the complementary relationship was found to have been preserved during phylogenetic diversification.     

Solution NMR structure of the 30S ribosomal protein S28E from Pyrococcus horikoshii

We report NMR assignments and solution structure of the 71-residue 30S ribosomal protein S28E from the archaean Pyrococcus horikoshii, target JR19 of the Northeast Structural Genomics Consortium. The structure, determined rapidly with the aid of automated backbone resonance assignment (AutoAssign) and automated structure determination (AutoStructure) software, is characterized by a four-stranded beta-sheet with a classic Greek-key topology and an oligonucleotide/oligosaccharide beta-barrel (OB) fold. The electrostatic surface of S28E exhibits positive and negative patches on opposite sides, the former constituting a putative binding site for RNA. The 13 C-terminal residues of the protein contain a consensus sequence motif constituting the signature of the S28E protein family. Surprisingly, this C-terminal segment is unstructured in solution.     

The spatial arrangement of the complex between eukaryotic initiation factor eIF-3 and 40 S ribosomal subunit. Cross-linking between factor and ribosomal proteins

The binding site for eIF-3 on the small ribosomal subunit was studied (a) by use of a complex of eIF-3 and derived 40 S ribosomal subunit from rat liver, and (b) by use of native small ribosomal subunits from rabbit reticulocytes. After treatment of both complexes with dimethyl 4,7-dioxo-5,6-dihydroxy-3,8-diazadecanbisimidate ribosomal proteins S3a, S4, S6, S7, S8, S9, S10, S23/24 and S27 became covalently linked to eIF-3 and were isolated together with the factor by gradient centrifugation. The ribosomal proteins were identified by two-dimensional polyacrylamide gel electrophoresis after periodate cleavage of the link(s).