Reverse phase high performance liquid chromatography of Escherichia coli ribosomal proteins: standardization of 70 S, 50 S, and 30 S protein chromatograms

We recently described the use of reverse phase high performance liquid chromatography for the separation of the proteins of the 30 S subunit of Escherichia coli ribosomes (Kerlavage, A. R., Kahan, L., and Cooperman, B. S. (1982) Anal. Biochem. 123, 342-348). In the present studies we report improvements in the technique and its extension to the separation of the proteins of the 50 S subunit and of 70 S ribosomes. Using an octadecasilyl silica column and a trifluoroacetic acid/acetonitrile solvent system, the 21 proteins of the 30 S subunit have been resolved into 17 peaks, the 33 proteins of the 50 S subunit into 22 peaks, and the 53 proteins of the 70 S ribosome into 31 peaks. The proteins present in each peak have been identified by polyacrylamide gel electrophoresis, by comparison with previously standardized chromatograms, and by calibration with authentic samples of purified proteins. All of the known ribosomal proteins have been identified on the chromatograms with the exception of L31 and its variant, L31'. Three protein peaks, not corresponding to known ribosomal proteins, have been observed in preparations from the total protein from 50 S subunits and 70 S ribosomes, but the significance of these peaks is unclear. The reverse phase high performance liquid chromatography technique has the potential for purifying all ribosomal proteins, as demonstrated by the increase in resolution we obtain when a peak isolated under standard gradient conditions and containing several proteins is reapplied to the column and eluted with a shallower gradient. Its utility in preparing proteins for functional studies is demonstrated by a reconstitution of active 30 S particles using 30 S proteins prepared by reverse phase high performance liquid chromatography.     

The RimP Protein Is Important for Maturation of the 30S Ribosomal Subunit

The in vivo assembly of ribosomal subunits requires assistance by auxiliary proteins that are not part of mature ribosomes. More such assembly proteins have been identified for the assembly of the 50S than for the 30S ribosomal subunit. Here, we show that the RimP protein (formerly YhbC or P15a) is important for the maturation of the 30S subunit. A rimP deletion (ΔrimP135) mutant in Escherichia coli showed a temperature-sensitive growth phenotype as demonstrated by a 1.2-, 1.5-, and 2.5-fold lower growth rate at 30, 37, and 44 °C, respectively, compared to a wild-type strain. The mutant had a reduced amount of 70S ribosomes engaged in translation and showed a corresponding increase in the amount of free ribosomal subunits. In addition, the mutant showed a lower ratio of free 30S to 50S subunits as well as an accumulation of immature 16S rRNA compared to a wild-type strain, indicating a deficiency in the maturation of the 30S subunit. All of these effects were more pronounced at higher temperatures. RimP was found to be associated with free 30S subunits but not with free 50S subunits or with 70S ribosomes. The slow growth of the rimP deletion mutant was not suppressed by increased expression of any other known 30S maturation factor.     

Preparative ion-exchange high-performance liquid chromatography of bacterial ribosomal proteins

We have developed analytical and preparative ion-exchange HPLC methods for the separation of bacterial ribosomal proteins. Proteins separated by the TSK SP-5-PW column were identified with reverse-phase HPLC and gel electrophoresis. The 21 proteins of the small ribosomal subunit were resolved into 18 peaks, and the 32 large ribosomal subunit proteins produced 25 distinct peaks. All peaks containing more than one protein were resolved using reverse-phase HPLC. Peak volumes were typically a few milliliters. Separation times were 90 min for analytical and 5 h for preparative columns. Preparative-scale sample loads ranged from 100 to 400 mg. Overall recovery efficiency for 30S and 50S subunit proteins was approximately 100%. 30S ribosomal subunit proteins purified by this method were shown to be fully capable of participating in vitro reassembly to form intact, active ribosomal subunits.     

Escherichia coli rimM and yjeQ null strains accumulate immature 30S subunits of similar structure and protein complement

Assembly of the Escherichia coli 30S ribosomal subunits proceeds through multiple parallel pathways. The protein factors RimM, YjeQ, RbfA, and Era work in conjunction to assist at the late stages of the maturation process of the small subunit. However, it is unclear how the functional interplay between these factors occurs in the context of multiple parallel pathways. To understand how these factors work together, we have characterized the immature 30S subunits that accumulate in ΔrimM cells and compared them with immature 30S subunits from a ΔyjeQ strain. The cryo-EM maps obtained from these particles showed that the densities representing helices 44 and 45 in the rRNA were partially missing, suggesting mobility of these motifs. These 30S subunits were also partially depleted in all tertiary ribosomal proteins, particularly those binding in the head domain. Using image classification, we identified four subpopulations of ΔrimM immature 30S subunits differing in the amount of missing density for helices 44 and 45, as well as the amount of density existing in these maps for the underrepresented proteins. The structural defects found in these immature subunits resembled those of the 30S subunits that accumulate in the ΔyjeQ strain. These findings are consistent with an “early convergency model” in which multiple parallel assembly pathways of the 30S subunit converge into a late assembly intermediate, as opposed to the mature state. Functionally related factors will bind to this intermediate to catalyze the last steps of maturation leading to the mature 30S subunit.     

Rapid separation of Escherichia coli 30S ribosomal proteins by fast protein liquid chromatography (FPLC)

The proteins of the 30S ribosomal subunit from Escherichia coli have been separated by reverse-phase high-performance liquid chromatography on a short alkyl chain (C1/C8)-coated phase. The reverse-phase column was connected to a fast protein liquid chromatography (FPLC) system. The 21 proteins of the 30S ribosomal subunit were resolved into 16 peaks. Eleven proteins were isolated in purified form in a single chromatographic run as shown by polyacrylamide gel electrophoresis and amino acid analysis. Interestingly, the retention times of some proteins differed from the retention times observed on other reversed-phase support materials. The results show the speed and resolution of reverse-phase FPLC for both analytical and semi-preparative separations of 30S ribosomal proteins.     

On the interpretation of small-angle x-ray solution scattering from spherical viruses.

The intermediates in the ribosome assembly in exponentially growing Escherichia coli have been identified by centrifuging a crude lysate, pulse-labeled with a radioactive RNA base, through a sucrose gradient and analyzing for precursor rRNA in the gradient fractions by gel electrophoresis. The major intermediate in the assembly of the 50 S subunit cosediments with the mature subunit, whereas two minor precursor species sediment between the 30 S and 50 S peaks. The assembly of the 30 S subunit proceeds via a minor intermediate sedimenting slightly behind the mature subunit and a major precursor particle that cosediments with the mature 30 S subunit.The fraction of the rRNA contained in these precursor particles was determined by direct determination of the amount of rRNA in the precursor particles, and from the labeling kinetics of their rRNA. The direct estimation indicated that about 2% of the total 23 S type RNA, and 3 to 5% of the total 16 S type RNA is harboured in precursor particles. In the kinetic experiments the specific activity of the nucleoside triphosphates and of the different ribosomal particles was followed after addition of a radioactive RNA precursor to the growth medium. The results were compared with a digital simulation of the flow of isotopes through the assembly pathways. This method indicated that approximately 2% of the total 23 S type RNA, as well as 2% of the total 16 S type RNA, is contained in the precursor particles.     

Molecular interactions between ribosomal proteins: a study of the S6-S18 interaction.

The proteins S6 and S18 from the 30 S ribosomal subunit of Escherichia coli were isolated to a purity of greater than 95%, characterized in solution, and investigated by sedimentation equilibrium for possible intermolecular interactions in a dilute salt reconstitution buffer. It was observed that neither protein S6 nor S18 has a tendency to self-associate in the concentration range studied. An analysis of solution mixtures containing proteins S6 and S18 revealed a species of molecular weight greater than either of the proteins. Proteins S6 and S18 were found to interact with an equilibrium constant of association of 6.6 ± 4.2 × 10⁴m^?1 at 3 °C with a Gibbs free energy of interaction, ΔG ° = ?6.1 kcal/mol. These data are part of those collected to help in building a map of the energetics in the 30 S ribosomal subunit, which provides for the stabilization of the structure.     

Proteins of the Thermus thermophilus ribosome Purification of several individual proteins and crystallization of protein TL7

The procedure of selective removal of eight proteins from the 50 S ribosomal subunit of the extreme thermophilic bacterium Thermus thermophilus has been developed based on extraction at 60°C in the presence of 0.5 M or 1 M NH₄Cl and 50% ethanol. CM-Sepharose CL column chromatography of the protein mixture under non-denaturing conditions yielded five proteins with a purity of 95% or higher. Crystals of one of these proteins, namely TL7 (probably an analog of L6 protein from the Escherichia coli ribosome) have been obtained using the ‘hanging drop’ method with ammonium sulphate as a precipitant.     

Identification of neighbor relationships among proteins in the 30 S ribosome: intermolecular cross-linkage of three proteins induced by tetranitromethane

Earlier studies have indicated that the reaction of tetranitromethane with the 30 S riboaome from Escherichia coli results in the disappearance of two protein bands from the polyacrylamide gel electrophoresis pattern (Craven et al., 1969b). As tetranitromethane is known to induce intermolecular cross-linkage in other protein systems, we studied further this reaction with the view that it might yield knowledge of protein-protein neighbor relationships within the ribosome.The use of two-dimensional polyacrylamide gel electrophoresis showed that the reaction with tetranitromethane caused the disappearance of four proteins from the pattern of 30 S ribosomal proteins. It was shown that this alteration in electrophoretic behavior was not due to simple protein modification (e.g. production of 3-nitrotyrosine), as reaction with extracted protein in 8 M-urea resulted in no observable change in the electrophoretic pattern.It was also shown that three of these proteins could be uniquely labeled with [¹⁴C]iodoacetate without changing their reactivity with tetranitromethane. Thus, ribosomes were labeled with [¹⁴C]iodoacetate, reacted with tetranitromethane and the radioactive reaction products were isolated by column chromatography and preparative gel electrophoresis. The radioactive peptide patterns of the three proteins digested by trypsin were compared with the three major reaction products. One of these products was shown to contain the radioactive tryptic peptides of all three proteins. We believe that this reaction product is an intermolecular cross-linked aggregate of these three proteins, identified as S11, S18 and S21. We suggest that these three proteins are clustered closely together in the 30 S ribosome. The fourth protein, S12, may also be involved in this aggregate.     

Characterization of different conformational forms of 30 S ribosomal subunits in isolated and associated states: possible correlations between structure and function.

The reaction pattern with N-[¹⁴C]ethylmaleimide served to follow conformational changes of 30 S ribosomal subunits that are induced by association with 50 S subunits and by the binding of aminoacyl-tRNA to 70 S ribosomes either enzymatically or non-enzymatically.The usefulness of the reaction with N-ethylmaleimide in discerning different conformational forms of the ribosome was previously demonstrated (Ginzburg et al., 1973) in an analysis of inactive and active 30 S subunits (as obtained at low Mg²⁺ and after heat reactivation, respectively). The reaction pattern of the 30 S moiety of 70 S ribosomes differs from the pattern of isolated active subunits (the only form capable of forming 70 S ribosomes) in both the nature of the labeled proteins and in being Mg²⁺-dependent. The reaction at 10 mm-Mg²⁺ reveals the following differences between isolated and reassociated 30 S subunits: (1) proteins S1, S18 and S21 that are not labeled in isolated active subunits, but are labeled in the inactive subunits, are highly reactive in 70 S ribosomes; (2) proteins S2, S4, S12 and S17 that uniquely react with N-ethylmaleimide in active subunits are all rendered inaccessible to modification after association; and (3) proteins S9, S13 and S19, that react in both active and inactive 30 S subunits, are labeled to a lesser extent in the 70 S ribosomes than in isolated subunits. This pattern is altered in two respects when the reaction with the maleimide is carried out at 20 mm-Mg²⁺; protein S18 is not modified while S17 becomes labeled.The differences in reaction pattern are considered as manifesting the existence of different conformational forms of the 30 S subunit in the dissociated and associated states as well as of different forms of 70 S ribosomes. The 30 S moiety of 70 S ribosomes at 10 mm-Mg²⁺ resembles the inactive subunit, while some of the features of the active subunit are preserved in the 70 S ribosome at 20 mmMg²⁺. The structural changes appear to be expressed in the functioning of the ribosome: non-enzymatic binding of aminoacyl-tRNA to active 30 S subunits is suppressed by 50 S subunits at 10 mm but not at 20 mm-Mg²⁺ (Kaufmann &; Zamir, 1972). The fact that elongation factor Tu-mediated binding is not suppressed by 50 S subunits raises the possibility that the function of the elongation factor might involve the facilitation of a conformational change of the ribosome. The analysis of different ribosomal binding complexes with N-ethylmaleimide showed that the binding of poly(U) alone results in a decrease in the labeling of S1 and S18. Binding of aminoacyl-tRNA, on the other hand, is closely correlated with the exposure of S17 for reaction with the maleimide. A model is outlined that accounts for this correlation as well as for the proposed role of elongation factor Tu.     

Structural dynamics of bacterial ribosomes: IV. Classification of ribosomes by subunit interaction

Previous studies in this series (M. Noll et al., 1973a,b; Noll & Noll, 1974) have established that in Escherichia coli the ability of subunits to form vacant 70 S ribosome couples at 10 mm-Mg²⁺ is a stringent condition for activity in the translation of natural messenger (R17 RNA). The present study examines the structural basis of subunit interaction. It is found that vacant ribosome couples prepared by various methods fall into two classes, “tight” couples and “loose” couples, that differ in the affinity of their subunits for each other. Detection and separation of the two particle species is possible by ultracentrifugation. When analyzed on sucrose gradients at 6 mm-Mg²⁺ and moderate speed (30,000 revs/min), tight couples sediment as undissociated 70 S ribosomes, whereas loose couples are completely dissociated and sediment as 30 S and 50 S subunits. At 15 mm-Mg²⁺ in the gradient, both species sediment as a 70S peak. At 10 mm-Mg²⁺ and 60,000 revs/min, two peaks (63 S and 55 S) are seen because the high hydrostatic pressure causes more pronounced dissociation of the loose than of the tight couples.Association is dependent on the state of each subunit. Removal of Mg²⁺ produces 30 S b-particles that are unable to associate with 50 S subunits unless reconverted to the 30 S a-form by thermal activation according to Zamir et al. (1971). In the dissociated state, 50 S subunits tend to change irreversibly to a 50 S b-modification that produces loose couples upon association with 30 S a-subunits. The 50 S a → 50 S b transition could not be related to breaks in 23 S RNA detectable by sedimentation analysis. However, mild treatment of 50 S a-subunits with RNase produces particles that associate with 30 S a-subunits to couples that are less stable than the loose couples resulting from a dissociation/association step.Fresh S-30 extracts contain only tight couples (approx. 80%) and subunits (approx. 20%). Our results suggest that loose couples are artefacts derived from tight couples by a structural or conformational modification.Interaction-free subunits that previously were found to form a primitive initiation complex with poly(U) and tRNA_Phe (Schreier & Noll, 1970,1971), and to be active in phenylalanine polymerization, are shown to consist of the b-form of each subunit.It is likely that conflicting results obtained in the study of the mechanism of initiation and other aspects of ribosome function are due to the lack of structural criteria required for standardizing the ribosome preparation used by different investigators. This study provides simple methods and criteria to classify and separate physically all ribosome and ribosome subunits that have been observed into well-defined classes of predictable activity.     

Binding of protein synthesis initiation factor IF1 to 30 S ribosomal subunits: effects of other initiation factors and identification of proteins near the binding site.

The effects of other components of the initiation complex on Escherichia coli initiation factor IFI binding to 30 S ribosomal subunits were studied. Binding of [¹⁴C]IF1 in the absence of other initiation complex components was slight. Addition of either IF2 or IF3 stimulated binding to a variable extent. Maximum binding was observed when both IF2 and IF3 were present. Addition of GTP, fMet-tRNA, and phage R17 RNA caused little or no further stimulation of [¹⁴C]IF1 binding. A maximum of 0.5 molecule of [¹⁴C]IF1 bound per 30 S subunit in the presence of an excess of each of the three factors over 30 S subunits.Complexes of 30 S subunits, [¹⁴C]IF1, IF2, and IF3 were treated with the bifunctional protein cross-linking reagent dimethyl suberimidate in order to identify the ribosomal proteins near the binding site for IF1. Non-cross-linked [¹⁴C]IF1 was removed from the complexes by sedimentation through buffer containing a high salt concentration, and total protein was extracted from the pelleted particles. Approximately 12% of the [¹⁴C]IF1 was recovered in the pellet fraction. The mixture of cross-linked products was analyzed by polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Autoradiography of the gel showed radioactive bands with molecular weights of 21,000, 25,000, and many greater than 120,000. The results indicate that [¹⁴C]IF1 was cross-linked directly to at least two ribosomal proteins. Analysis of the cross-linked mixture by radioimmunodiffusion with specific antisera prepared against each of the 30 S ribosomal proteins showed radioactivity in the precipitin bands formed with antisera against S12 and S19, and in lower yield with those against S1 and S13. Antiserum against IF2 also showed [¹⁴C]IF1 in the precipitin band. The results show that [¹⁴C]IF1 was present in covalently cross-linked complexes containing 30 S ribosomal proteins S1, S12, S13 and S19, and initiation factor IF2. The same ribosomal proteins have been implicated in the binding sites for IF2 and IF3. The results suggest that the three initiation factors bind to the 30 S subunit at the same or overlapping sites.     

Studies on proteins of animal ribosomes XXIV. Localisation of proteins in ribosomal subunits of rat liver studied by chemical substitution with p-nitrophenyl acetate and methyl acetimidate

The reaction of [³H]p-nitrophenyl acetate (NPA) or [¹⁴C]methyl acetimidate (MAI) with amino groups of ribosomal proteins from the rate has been studied.A comparison has been made between the reactivity of the proteins in situ in the ribosomal subunit with that of isolated protein mixtures.In the small subunit reactivity compared with the protein mixture was only 10–65% in the case of NPA but 45 to more than 100% in the case of MAI.In the large subunit reactivity to MAI was 10–60% that of the isolated protein mixture. This suggests that the large subunit has a denser structure than the small one.In agreement with earlier experiments with iodoacetamide the proteins S2, 5, 7, 8, 10 and 13 of the small subunit and L15, 17, 20, 24, 25, 27, 29, 33, 34, 35 and 38 in the large subunit are quite accessible while proteins S9, 14, 19, 20, 24, 25, 27, 29 and 30 of the small subunit and L1, 7, 8, 10, 11, 19, 28, 31 and 32 of the large one are relatively inaccessible.     

Localization of proteins in the inner and outer membranes of Caulobacter crescentus

Cytoplasmic and outer membranes of Caulobacter crescentus were separated by isopycnic sucrose gradient centrifugation into two peaks with buoyant densities 1.22 and 1.14 g/cm³. These peaks were identified as outer and cytoplasmic membranes by the enrichment of malate dehydrogenase and NADH oxidase in the lower density peak and the presence of flagellin, a cell surface protein, in the heavier peak. The identity of the heavier peak as outer membrane was confirmed by labeling of cells with diazotized [³⁵S]sulfanilic acid, a reagent that does not penetrate intact cells. Under these conditions only outer membrane proteins were substituted by the sulfanilic acid. The distribution of proteins between the cytoplasmic and outer membranes were examined by the analysis of [³⁵S]methionine-labeled membranes by SDS-polyacrylamide and two-dimensional gel electrophoresis. These results showed that the inner and outer membranes contain approximately equal numbers of proteins, and that the distribution of these proteins between the two layers is highly asymmetric. Although many of the proteins could be assigned to one or the other membrane fraction, a number of the outer membrane proteins in the 32 000–100 000 molecular weight range frequently contaminate the inner membrane fractions. The implications of these results for membrane isolation and separation in C. crescentus are discussed.     

Ribosomes Lacking Protein S20 Are Defective in mRNA Binding and Subunit Association

The functional significance of ribosomal proteins is still relatively unclear. Here, we examined the role of small subunit protein S20 in translation using both in vivo and in vitro techniques. By means of lambda red recombineering, the rpsT gene, encoding S20, was removed from the chromosome of Salmonella enterica var. Typhimurium LT2 to produce a ΔS20 strain that grew markedly slower than the wild type while maintaining a wild-type rate of peptide elongation. Removal of S20 conferred a significant reduction in growth rate that was eliminated upon expression of the rpsT gene on a high-copy-number plasmid. The in vitro phenotype of mutant ribosomes was investigated using a translation system composed of highly active, purified components from Escherichia coli. Deletion of S20 conferred two types of initiation defects to the 30S subunit: (i) a significant reduction in the rate of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in association with the 50S subunit. Both of these impairments were partially relieved by an extended incubation time with mRNA, fMet-tRNA^fMet, and initiation factors, indicating that absence of S20 disturbs the structural integrity of 30S subunits. Considering the topographical location of S20 in complete 30S subunits, the molecular mechanism by which it affects mRNA binding and subunit docking is not entirely obvious. We speculate that its interaction with helix 44 of the 16S ribosomal RNA is crucial for optimal ribosome function.     

RNA--protein interactions in the ribosome. III. Conformation and stability of ribosomal protein binding sites in the 16 S RNA

Following dialysis against distilled water, the 16 S ribosomal RNA of Escherichia coli is unable to interact with 30 S subunit protein S4 at 0 °C. The dialysed RNA recovered this capacity, however, when heated at 40 °C in the presence of 0.02m-MgCl₂ prior to addition of the protein. Furthermore, its sensitivity to ribo-nuclease markedly declined and its sedimentation rate increased as a consequence of this treatment. Although no concomitant changes in secondary structure were detected by absorbance and fluorescence techniques, the rearrangement of a small number of base-pairs was not excluded. Kinetic measurements revealed that binding site reactivation satisfies the first-order rate law and that the process is highly temperature-dependent, exhibiting an Arrhenius activation energy of 40,800 cal/mol. Together, these data suggest that dialysed RNA undergoes a unimolecular conformational transition upon pre-incubation in Mg²⁺-containing buffers and that this transition leads to renaturation of the binding site for protein S4.Similar results were obtained for several other proteins of the 30 S subunit. In particular, S7, S16/S17 and S20 all failed to interact efficiently with dialysed 16 S RNA at 0 °C. These proteins bound normally to the RNA, however, after it had been incubated at 40 °C in the presence of Mg²⁺ ions. By contrast, prior dialysis of the 16 S RNA did not affect its ability to associate with S8 and S15 at 0 °C. These two proteins interacted equally well with dialysed and pre-incubated 16 S RNA, indicating that their binding sites are not susceptible to the reversible alterations in conformation which influence the attachment of the other RNA-binding proteins to the nucleic acid molecule. The effects of dialysis and pre-incubation on the interaction of 16 S RNA with an unfractionated mixture of 30 S subunit proteins were also investigated. The dialysed RNA bound only S6, S8, S15 and S18 at 0 °C whereas, after heating at. high Mg²⁺ concentrations, the RNA associated with S4, S7, S9, S13, S16/S17, S19 and S20 as well. These results leave little doubt that the protein-binding capacities of the 16 S RNA are intimately related to its three-dimensional configuration, although individual binding sites appear to differ significantly in their stability to small changes in structure.     

Letter: Unequal contribution to ribosomal assembly of both str alleles in Escherichia coli merodiploids and its relationship to the dominance phenomenon

Two Escherichia coli strains were constructed which are reciprocal diploids for the str locus and isogenic for this region of the chromosome (str^s/′F str^r and str^r/′Fstr³). During exponential growth the steady-state ribosomal pools of both merodiploids are comprised of about 90%, or more, of streptomycin-sensitive ribosomes. Little effect of allele position was found. A similar preponderance of sensitive 30 S subunits over those that are resistant has been found during limited subunit reconstitution when an equimolar mixture of ribosomal proteins from both phenotypes was initially present. The results indicate that the rates of 30 S subunit assembly of both phenotypes are different, and that the sensitive sub-units predominate over the resistant subunits, suggesting that the difference in biosynthetic rate may be the basis for the dominance of this phenotype in vivo. An explanation for some aspects of the physiology of str diploids have been suggested in terms of these findings.     

Initiation of ovalbumin synthesis in chicken oviduct magnum: Transient labeling of the NH2-terminus by methionine

The incorporation of [³⁵S]methionine into ovalbumin, a protein containing NH₂-terminal N-acetylglycine, has been studied in chicken oviduct magnum cells. The purification of [³⁵S]methionine-labeled ovalbumin from total oviduct proteins was accomplished by dialysis of a crude extract at pH 3.6 followed by chromatography on carboxymethyl cellulose. The radioactive ovalbumin eluted from the column in three peaks (P₀, P₁, and P₂-containing 0, 1, and 2 moles of phosphate, respectively, per mole of ovalbumin). The kinetics of labeling of peaks P₀ and P₁ showed that the ratio of radioactivity in NH₂-terminal methionine to total incorporation was greater at 2 min of labeling than at later times. The transient labeling of the NH₂-terminus of ovalbumin with methionine indicates that methionine is the initiator amino acid for the synthesis of this protein, which in its mature form contains NH₂-terminal N-acetylglycine.     

Rapid purification method for the 26S proteasome from the filamentous fungus Trichoderma reesei

We have developed a fast and simple two column chromatographic method for the purification of the 26S proteasome from the filamentous fungus Trichoderma reesei that simplifies the overall procedure and reduces the purification time from 5 to 2.5 days. The combination of only the anionic exchange POROS^® HQ column (Applied Biosystems) together with a size exclusion column has not been used previously for proteasome purification. The purified complex was analysed further by two-dimensional electrophoresis (2DE) and examined by transmission electron microscopy (TEM). A total of 102 spots separated by 2DE were identified by mass spectrometry using cross-species identification (CSI) or an in-house custom-made protein database derived from the T. reesei sequencing project. Fifty-one spots out of 102 represented unique proteins. Among them, 30 were from the 20S particle and eight were from the 19S particle. In addition, seven proteasome-interacting proteins as well as several non-proteasome related proteins were identified. Co-purification of the 19S regulatory particle was confirmed by TEM and Western blotting. The rapidity of the purification procedure and largely intact nature of the complex suggest that similar procedure may be applicable to the isolation and purification of the other protein complexes.     

Stabilization of 30 S ribosomal subunits of Bacillus subtilis W168 by spermidine and magnesium ions

An explanation for the fragility of 30 S ribosomal subunits of Bacillus subtilis has been studied. Degradation of 16 S ribosomal RNA, rather than degradation of ribosomal proteins, was found to cause the inactivation of 30 S subunits. Although RNAases were bound specifically to 30 S ribosomal subunits, the RNAases were able to function. Spermidine was found to contribute to the stabilization of 30 S ribosomal subunits by inhibiting the degradation of 16 S ribosomal RNA. A high concentration of Mg²⁺ also stabilized the 30 S ribosomal subunits of Bacillus subtilis. The polypeptide synthetic activity of 30 S ribosomal subunits prepared in the presence of spermidine was at least 4-times greater than that of 30 S ribosomal subunits prepared in the absence of spermidine; this activity was maintained without any loss for 3 months at ?70°C.