The accessibility of antigenic determinants of ribosomal protein S4 in situ.

Antibodies to Escherichia coli ribosomal protein S4 react with S4 in subribosomal particles, eg, the complex of 16S RNA with S4, S7, S8, S15, S16, S17, and S19 and the RI reconstitution intermediate, but they do not react with intact 30S subunits. Antibodies were isolated by three different methods from antisera obtained during the immunization of eight rabbits. Some of these antibody preparations, which contained contaminant antibodies directed against other ribosomal proteins, reacted with subunits, but this reaction was not affected by removal of the anti-S4 antibody population. Other antibody preparations did not react with subunits. It is concluded that the antigenic determinants of S4 are accessible in some protein deficient subribosomal particles but not in intact 30S subunits.     

Selection of peptides targeting helix 31 of bacterial 16S ribosomal RNA by screening M13 phage-display libraries

Ribosomal RNA is the catalytic portion of ribosomes, and undergoes a variety of conformational changes during translation. Structural changes in ribosomal RNA can be facilitated by the presence of modified nucleotides. Helix 31 of bacterial 16S ribosomal RNA harbors two modified nucleotides, m2G966 and m?C967, that are highly conserved among bacteria, though the degree and nature of the modifications in this region are different in eukaryotes. Contacts between helix 31 and the P-site tRNA, initiation factors, and ribosomal proteins highlight the importance of this region in translation. In this work, a heptapeptide M13 phage-display library was screened for ligands that target the wild-type, naturally modified bacterial helix 31. Several peptides, including TYLPWPA, CVRPFAL, TLWDLIP, FVRPFPL, ATPLWLK, and DIRTQRE, were found to be prevalent after several rounds of screening. Several of the peptides exhibited moderate affinity (in the high nM to low μM range) to modified helix 31 in biophysical assays, including surface plasmon resonance (SPR), and were also shown to bind 30S ribosomal subunits. These peptides also inhibited protein synthesis in cell-free translation assays.     

Structure of functional A salina -- E coli hybrid ribosome by electron microscopy.

Small 40S Artemia salina and large 50S Escherichia coli ribosomal subunits can be assembled into 73S hybrid monosomes active in model assays for protein synthesis. The reciprocal combination -- small 30S E coli and large 60S A salina -- fails to form hybrids. The 73S hybrid particles strongly resemble homologous 70S E coli and 80S A salina monosomes. The morphologic differences between the corresponding eukaryotic and prokaryotic ribosomal particles, established by electron microscopy, do not significantly affect the assembly and mutual orientation of 40S A slina and 50S E coli subunits in the heterologous monosome. The fact that the structure of the interface, the supposed site of protein synthesis, is preserved in the active hybrid implies that retention or loss of biologic activity of hybrid ribosomes is determined by the extent of conformational changes in the interface.     

High-performance-liquid chromatography ofThermus aquaticus 50S and 30S ribosomal proteins

Summary The ribosomal 50S and 30S subunit proteins (r-proteins) ofThermus aquaticus have, for the first time, been characterized by size exclusion chromatography (SEC) and by reversed phase high performance liquid chromatography (RPC). To ensure that the best resolution in the RPC was obtained. the elution conditions, such as gradient time, flow rate, temperature, ionic strength of the eluent and the type of stationary phase were optimized. Correlation between experimentally found retention times and those predicted by DryLab G was better than 0.7% over 30 peaks. Protein fractions from RPC runs were desalted and processed by gel electrophoresis so that the ribosomal proteins could be identified by their position on SDS-polyacrylamide gels. The enhanced speed and quality of separation which has been achieved in this study is expected to bring advantaces in experimental work with ribosomal proteins as well as with other biopolymers. In our case the high resolution technique provides a basis for the preparation of a collection of individual ribosomal protein components for future rRNA-protein interaction studies.     

Separation of ribosomal subunits on Trisacryl GF 2000

The separation of rat liver and E. coli ribosomal subunits was attempted on Trisacryl GF 2000. Contrary to experiments with Sepharose 4B and Bio-Gel A-15 the 60S mammalian subunit did not bind to the resin at 4 degrees C but eluted within the column volume ahead of the 40S subunit. Puromycin, however, used to prepare the subunits, which on the agarose gels had eluted at the total column volume, exhibited anomalous retardation on the Trisacryl resin. Trisacryl therefore behaves as the more non-polar resin, and the binding of 60S subunits to agarose gels is a result of hydrophilic interaction.     

Key intermolecular interactions in the E. coli 70S ribosome revealed by coarse-grained analysis

The ribosome is a very large complex that consists of many RNA and protein molecules and plays a central role in protein biosynthesis in all organisms. Extensive interactions between different molecules are critical to ribosomal functional dynamics. In this work, intermolecular interactions in the Escherichia coli 70S ribosome are investigated by coarse-grained (CG) analysis. CG models are defined to preserve dynamic domains in RNAs and proteins and to capture functional motions in the ribosome, and then the CG sites are connected by harmonic springs, and spring constants are obtained by matching the computed fluctuations to those of an all-atom molecular dynamics (MD) simulation. Those spring constants indicate how strong the interactions are between the ribosomal components, and they are in good agreement with various experimental data. Nearly all the bridges between the small and large ribosomal subunits are indicated by CG interactions with large spring constants. The head of the small subunit is very mobile because it has minimal CG interactions with the rest of the subunit; however, a large number of small subunit proteins bind to maintain the internal structure of the head. The results show a clear connection between the intermolecular interactions and the structural and functional properties of the ribosome because of the reduced complexity in domain-based CG models. The present approach also provides a useful strategy to map interactions between molecules within large biomolecular complexes since it is not straightforward to investigate these by either atomistic MD simulations or residue-based elastic network models.     

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

An approach that combines limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been developed to probe protease-accessible sites of ribosomal proteins from intact ribosomes. Escherichia coli and Thermus thermophilus 70S ribosomes were subjected to limited proteolysis using different proteases under strictly controlled conditions. Intact ribosomal proteins and large proteolytic peptides were recovered and directly analyzed by MALDI-MS, which allows for the determination of proteins that are resistant to proteolytic digestion by accurate measurement of molecular weights. Larger proteolytic peptides can be directly identified by the combination of measured mass, enzyme specificity, and protein database searching. Sucrose density gradient centrifugation revealed that the majority of the 70S ribosome dissociates into intact 30S and 50S subunits after 120 min of limited proteolysis. Thus, examination of ribosome populations within the first 30 to 60 min of incubation provides insight into 70S structural features. Results from E. coli and T. thermophilus revealed that a significantly larger fraction of 50S ribosomal proteins have similar limited proteolysis behavior than the 30S ribosomal proteins of these two organisms. The data obtained by this approach correlate with information available from the high-resolution crystal structures of both organisms. This new approach will be applicable to investigations of other large ribonucleoprotein complexes, is readily extendable to ribosomes from other organisms, and can facilitate additional structural studies on ribosome assembly intermediates.     

Three-dimensional electron cryomicroscopy of ribosomes

Single particle electron cryomicroscopy is nowadays routinely used to generate three-dimensional structural information of ribosomal complexes without the need of crystallization. A large number of structures of functional important ribosomal complexes have thus been determined using this technique. In E. coli 70S ribosomes all three tRNA binding sites could be localized. The ternary complex of EF-TutRNAGTP that delivers the tRNA to the ribosome was directly visualized in a ribosomal complex blocked by the antibiotic kirromycin. Three different functional states of translocation have been studied and the respective EF-G binding sites have been mapped. The level of resolution achievable with electron cryomicroscopy allows conformational changes in the domain structures of elongation factors to be modelled in terms of rigid body movements. Structural information on eukaryotic ribosomes is also available for yeast and mammalian 80S ribosomes. The structural differences between rabbit 80S and E. coli 70S ribosomes could be interpreted in terms of ribosomal RNA expansion segments in the 18S and 23S RNA. The EF-G homologue EF2 was mapped analysing the structure of an 80SEF2sodarin complex and most recently the binding of a hepatitis C virus IRES element to a yeast 40S subunit has been studied. The first electron cryomicroscopical 3D reconstructions have further been used to overcome the initial phasing problems in X-ray crystallographic studies of the ribosome facilitating structure determination of the recent atomic resolution structures of the 30S and 50S ribosomal subunits. In turn, the knowledge of the atomic structure of the ribosome makes detailed interpretations of cryo-EM maps possible at approximately 20 A resolution.     

Affinity chromatography of Drosophila melanogaster ribosomal proteins to 5S rRNA

The binding of Drosophila melanogaster ribosomal proteins to D. melanogaster 5S rRNA was studied using affinity chromatography of total ribosomal proteins (TP80) on 5S rRNA linked via adipic acid dihydrazide to Sepharose 4B. Ribosomal proteins which bound 5S rRNA at 0.3 M potassium chloride and were eluted at 1 M potassium chloride were identified as proteins 1, L4, 2/3, L14/L16, and S1, S2, S3, S4, S5, by two-dimensional polyacrylamide gel electrophoresis. Using poly A-Sepharose 4B columns as a model of non-specific binding, we found that a subset of TP80 proteins is also bound. This subset, while containing some of the proteins bound by 5S rRNA columns, was distinctly different from the latter subset, indicating that the binding to 5S rRNA was specific for that RNA species.     

Probing conformational states of modified helix 69 in 50S ribosomes

The movement of the small ribosomal subunit (30S) relative to the large ribosomal subunit (50S) during translation is widely known, but many molecular details and roles of rRNA and proteins in this process are still undefined, especially in solution models. The functional relationship of modified nucleotides to ribosome activity is one such enigma. To better understand ribosome dynamics and the influence of modified nucleotides on such processes, the focus of this work was helix 69 of 23S rRNA, which contains three pseudouridine residues in its loop region. Ribosome probing experiments with dimethylsulfate revealed that specific base accessibilities and individual nucleotide conformations in helix 69 are influenced differently by pH, temperature, magnesium, and the presence of pseudouridine modifications.     

Ribosome Subunit Stapling for Orthogonal Translation in E. coli

The creation of orthogonal large and small ribosomal subunits, which interact with each other but not with endogenous ribosomal subunits, would extend our capacity to create new functions in the ribosome by making the large subunit evolvable. To this end, we rationally designed a ribosomal RNA that covalently links the ribosome subunits via an RNA staple. The stapled ribosome is directed to an orthogonal mRNA, allowing the introduction of mutations into the large subunit that reduce orthogonal translation, but have minimal effects on cell growth. Our approach provides a promising route towards orthogonal subunit association, which may enable the evolution of key functional centers in the large subunit, including the peptidyl‐transferase center, for unnatural polymer synthesis in cells.     

Sequence of the amino-terminal region of rat liver ribosomal proteins S4, S6, S8, L6, L7a, L18, L27, L30, L37, L37a, and L39.

The sequence of the amino-terminal region of eleven rat liver ribosomal proteins--S4, S6, S8, L6, L7a, L18, L27, L30, L37a, and L39--was determined. The analysis confirmed the homogeneity of the proteins and suggests that they are unique, since no extensive common sequences were found. The N-terminal regions of the rat liver proteins were compared with amino acid sequences in Saccharomyces cerevisiae and in Escherichia coli ribosomal proteins. It seems likely that the proteins L37 from rat liver and Y55 from yeast ribosomes are homologous. It is possible that rat liver L7a or L37a or both are related to S cerevisiae Y44, although the similar sequences are at the amino-terminus of the rat liver proteins and in an internal region of Y44. A number of similarities in the sequences of rat liver and E coli ribosomal proteins have been found; however, it is not yet possible to say whether they connote a common ancestry.     

Preparation of active ribosomal proteins by HPLC

Summary The proteins of the large ribosomal subunit fromEscherichia coli have been separated by size-exclusion, ion-exchange and reversed-phase high-performance-liquid chromatography (HPLC) using various buffer systems. The biological activity of the isolated proteins was tested via their ability to assemble into active 50S subunits (total reconstitution). The activity of the reconstituted subunits was measured with poly(U)-dependent poly-(Phe) synthesis. Reversed-phase HPLC techniques yielded active proteins (80–100%) by application of 2-propanol or acetonitrile. Proteins prepared by size-exclusion chromatography employing ammonium acetate as buffer also gave highly active proteins (70%). On the other hand, separation of the proteins on ion-exchange columns, using urea containing buffers, resulted in reduced activity (up to 50%).     

Electrophoretic analysis of phosphorylation of the yeast 20S proteasome

The 26S proteasome complex, consisting of two multisubunit complexes, a 20S proteasome and a pair of 19S regulatory particles, plays a major role in the nonlysosomal degradation of intracellular proteins. The 20S proteasome was purified from yeast and separated by two-dimensional gel electrophoresis (2-DE). A total of 18 spots separated by 2-DE were identified as the 20S proteasome subunits by peptide mass fingerprinting with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The alpha2-, alpha4- and alpha7-subunits gave multiple spots, which converged into one spot for each subunit when treated with alkaline phosphatase. The difference of pI between phosphorylated and dephosphorylated spots and their reaction against anti-phosphotyrosine antibody suggested that the alpha2- and alpha4-subunits are phosphorylated either at Ser or at Thr residue, and the alpha7-subunit is phosphorylated at Tyr residue(s). These phosphorylated subunits were analyzed by electrospray ionization-quadrupole time of flight-tandem MS (ESI-QTOF-MS/MS) to deduce the phosphorylation sites. The 20S proteasome has three different protease activities: chymotrypsin-like, trypsin-like and peptidylglutamyl peptide-hydrolyzing activities. The phosphatase treatment increased K(m) value for chymotrypsin-like activity of the 20S proteasome, indicating that phosphorylation may play an important role in regulating the proteasome activity.     

Interaction of the 5'-ends of 28S RNA in dimerization of hamster ribosomes.

Free ribosomes extracted from hamster cells and 28S RNA purified from these ribosomes are known to form dimers. We find that spleen phosphodiesterase inhibits ribosomal dimer formation, but only when a free 5'-hydroxyl end group, produced by the action of alkaline phosphatase, is present. Hence, formation of dimer ribosomes probably involves interaction at or near the phosphorylated 5'-ends of 28S RNA. Dimer RNA molecules show a modal length, when measured on electrom micrographs, of 2.1 mum, which is about double the length of 28S RNA. Electron micrographs of 115S dimer ribosomes often show profiles consistent with our interpretation that in dimers the 28S RNA chains are loosely linked by their 5'-ends.     

An investigation of cottonseed globulins. XXI. The 11S globulins of two varieties of the cotton plant

The 11S globulins of two varieties of the cotton plant — 108-F and Tashkent-1 — have been studied. It has been shown that the 11S globulins do not differ in quaternary structure and each consists of three subunits A, B, and C. It has been found that the subunits of these varieties differ in their amino acid compositions and the peptide maps of tryptic and chymotryptic hydrolysates.