Immune electron microscopic localization of dinitrophenyl-modified ribosomal protein S19 in reconstituted Escherichia coli 30 S subunits using antibodies to dinitrophenol

Escherichia coli small ribosomal subunits have been reconstituted from RNA and high performance liquid chromatography-purified proteins including protein S19 that had been modified at its amino-terminal proline residue with 1-fluoro-2,4-dinitrobenzene. As detailed in the accompanying paper (Olah, T. V., Olson, H. M., Glitz, D. G., and Cooperman, B. S. (1988) J. Biol. Chem. 263, 4795-4800), dinitrophenyl (DNP)-S19 was efficiently incorporated into the site ordinarily occupied by S19. Antibodies to DNP bound effectively to the reconstituted subunits and did not cause dissociation of the modified protein from the subunit. Electron microscopy of the immune complexes was used to localize the modified protein on the subunit surface. More than 95% of the antibody binding sites seen were consistent with a single location of protein S19 on the upper portion or head of the subunit, on the surface that faces the 50 S particle in a 70 S ribosome, and in an area relatively distant from the subunit platform. The S19 site is close to the region in which 30 S subunits are photoaffinity labeled with puromycin. Protein S19 is thus near protein S14 in the small subunit and in proximity to the peptidyl transferase center of the 70 S ribosome.     

Puromycin binding to the small subunit of Escherichia coli ribosomes. Localization of the antibiotic in subunits reconstituted with puromycin-modified components

Small (30 S) ribosomal subunits from Escherichia coli strain TPR 201 were photoaffinity-labeled with [3H]puromycin in the presence of chloramphenicol under conditions in which more than 1 mol of antibiotic was incorporated per mol of ribosomes. The subunits were than washed with 3 M NH4Cl to yield core particles and a split protein fraction; the split proteins were further fractionated with ammonium sulfate. Subunits were then reconstituted using one fraction (core, split proteins, or ammonium sulfate supernatant) from photoaffinity-modified subunits and other components from unmodified (control) subunits. The distribution of [3H]puromycin in ribosomal proteins was monitored by one-dimensional polyacrylamide gel electrophoresis, and the sites of puromycin binding were visualized by immunoelectron microscopy. Two areas of puromycin binding were identified. A high affinity puromycin site, found on the upper third of the subunit and distant from the platform, is identical to the primary site previously identified (Olson, H. M., Grant, P. G., Glitz, D. G., and Cooperman, B. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 890-894). Binding at this site is maximal in subunits reconstituted with high levels of puromycin-modified protein S14, and is decreased when unmodified S14 is incorporated. Because the percentage of antibody binding at the primary site always exceeds the percentage of puromycin label in protein S14, the primary site must include components other than S14. A secondary puromycin site of lower affinity is found on the subunit platform. This site is enriched in subunits reconstituted from puromycin-modified core particles and may include protein S7. Our results demonstrate the feasibility of localizing specifically modified components in reconstituted ribosomal subunits.     

Studies on the ability of partially iodinated 16S RNA to participate in 30S ribosome assembly.

Deproteinated 16S RNA was iodinated at pH 5.0 in an aqueous solution containing TlCl3 plus KI for 1-5 hours at 42 degrees C. Under these conditions 33 moles of iodine are incorporated per mole of RNA. As judged by sucrose gradient sedimentation, the iodinated RNA does not exhibit any large alteration in conformation as compared to unmodified 16S. The iodinated RNA was examined for its ability to reconstitute with total 30S proteins. Sedimentation velocity analysis reveals that the reconstituted subunit has a sedimentation constant of approximately 20S. In addition, protein analysis of particles reconstituted with 16S RNA iodinated for 5 hours indicates that proteins S2, S10, S13, S14, S15, S17, S18, S19, and S21 are no longer able to participate in the 30S assembly process and that proteins S6, S16 and S20 are present in reduced amounts. The ramifications of these results concerning protein-RNA and RNA-RNA interactions occurring in ribosome assembly are discussed.     

Reverse-phase high-performance liquid chromatography of Escherichia coli ribosomal small subunit proteins

Reverse-phase high-performance liquid chromatography has been explored as an approach for the separation of the proteins of the 30 S subunit of Escherichia coli ribosomes. The majority of these proteins are of similar molecular weight and isoelectric point, making separation by size exclusion or ion exchange difficult. With the use of an octadecasilyl silica column and a trifluoroacetic acid-acetonitrile solvent system, the 21 proteins of the 30 S subunit have been separated into 15 peaks. The yield of total protein recovered from the column was ≥85%. The proteins present in each peak have been identified by polyacrylamide gel electrophoretic analysis of the peaks as well as by comparison with the relative retention volumes of known purified 30 S proteins on the column. The results clearly show that this method is a powerful and rapid technique for the identification and purification of 30 S proteins. Analysis of [³H]puromycin-labeled 30 S subunit protein provides an illustrative example of its utility for affinity labeling studies.     

Reconstitution of Escherichia coli 50S ribosomal subunits containing puromycin-modified L23: functional consequences

In previous work we have shown that both puromycin [Weitzmann, C. J., & Cooperman, B. S. (1985) Biochemistry 24, 2268-2274] and p-azidopuromycin [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Coooperman, B. S. (1982) Biochemistry 21, 3809-3817] site specifically photoaffinity label protein L23 to the highest extent of any Escherichia coli ribosomal protein. In this work we demonstrate that L23 that has been photoaffinity labeled within a 70S ribosome by puromycin (puromycin-L23) can be separated from unmodified L23 by reverse-phase high-performance liquid chromatography (RP-HPLC) and further that puromycin-L23 can reconstitute into 50S subunits when added in place of unmodified L23 to a reconstitution mixture containing the other 50S components in unmodified form. We have achieved a maximum incorporation of 0.5 puromycin-L23 per reconstituted 50S subunit. As compared with reconstituted 50S subunits either containing unmodified L23 or lacking L23, reconstituted 50S subunits containing 0.4-0.5 puromycin-L23 retain virtually all (albeit low) peptidyl transferase activity but only 50-60% of mRNA-dependent tRNA binding stimulation activity. We conclude that although L23 is not directly at the peptidyl transferase center, it is sufficiently close that puromycin-L23 can interfere with tRNA binding. This conclusion is consistent with a number of other experiments placing L23 close to the peptidyl transferase center but is difficult to reconcile with immunoelectron microscopy results placing L23 near the base of the 50S subunit on the side facing away from the 30S subunit [Hackl, W., & St?ffler-Meilicke, M. (1988) Eur. J. Biochem. 174, 431-435].     

Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins.

Previous studies have shown that the 30S ribosomal subunit of Escherichia coli can be reconstituted in vitro from individually purified ribosomal proteins and 16S ribosomal RNA, which were isolated from natural 30S subunits. We have developed a 30S subunit reconstitution system that uses only recombinant ribosomal protein components. The genes encoding E. coli ribosomal proteins S2-S21 were cloned, and all twenty of the individual proteins were overexpressed and purified. Reconstitution, following standard procedures, using the complete set of recombinant proteins and purified 16S ribosomal RNA is highly inefficient. Efficient reconstitution of 30S subunits using these components requires sequential addition of proteins, following either the 30S subunit assembly map (Mizushima & Nomura, 1970, Nature 226:1214-1218; Held et al., 1974, J Biol Chem 249:3103-3111) or following the order of protein assembly predicted from in vitro assembly kinetics (Powers et al., 1993, J MoI Biol 232:362-374). In the first procedure, the proteins were divided into three groups, Group I (S4, S7, S8, S15, S17, and S20), Group II (S5, S6, S9, Sll, S12, S13, S16, S18, and S19), and Group III (S2, S3, S10, S14, and S21), which were sequentially added to 16S rRNA with a 20 min incubation at 42 degrees C following the addition of each group. In the second procedure, the proteins were divided into Group I (S4, S6, S11, S15, S16, S17, S18, and S20), Group II (S7, S8, S9, S13, and S19), Group II' (S5 and S12) and Group III (S2, S3, S10, S14, and S21). Similarly efficient reconstitution is observed whether the proteins are grouped according to the assembly map or according to the results of in vitro 30S subunit assembly kinetics. Although reconstitution of 30S subunits using the recombinant proteins is slightly less efficient than reconstitution using a mixture of total proteins isolated from 30S subunits, it is much more efficient than reconstitution using proteins that were individually isolated from ribosomes. Particles reconstituted from the recombinant proteins sediment at 30S in sucrose gradients, bind tRNA in a template-dependent manner, and associate with 50S subunits to form 70S ribosomes that are active in poly(U)-directed polyphenylalanine synthesis. Both the protein composition and the dimethyl sulfate modification pattern of 16S ribosomal RNA are similar for 30S subunits reconstituted with either recombinant proteins or proteins isolated as a mixture from ribosomal subunits as well as for natural 30S subunits.     

Proton magnetic resonance study of the influence of heme 2,4 substituents on the exchange rates of labile protons in the heme pocket of myoglobin.

Four exchangeable protons with large hyperfine shifts are assigned in the heme pocket of sperm whale met-cyano myoglobin reconstituted with heme possessing acetyl groups, ethyl groups, bromines, and hydrogens at the 2,4 position, using both relaxation and chemical-shift data. The four protons arise from the ring NH's of the proximal (F8), distal (E7), and FG2 histidines, and the peptide NH of His F8. The similarity of all chemical shifts to those of the native protein as well as the invariance of the relaxation rates of the distal histidyl ring NH dictate essentially the same structure for the heme cavity of both native and reconstituted proteins. The exchange rates with bulk water of the four labile proteins in each modified protein were determined by saturation-transfer and line width methods. All four labile protons were found to have the same exchange rate as in the native protein for acetyl and ethyl 2,4 substituents; the two resolved labile protons in the derivative with 2,4 bromine were also unchanged. The reconstituted protein with hydrogens at the 2,4 position exhibited slower exchange rates for three of the four protons, indicating an increased dynamic stability of the heme pocket in the absence of bulky 2,4 substituents.     

Protein binding sites on Escherichia coli 16S RNA; RNA regions that are protected by proteins S7, S14 and S19 in the presence or absence of protein S9.

14C-labelled proteins from E. coli 30S ribosomal subunits were isolated by HPLC, and selected groups of these proteins were reconstituted with 32P-labelled 16S RNA. The isolated reconstituted particles were partially digested with ribonuclease A, and the RNA fragments protected by the proteins were separated by gel electrophoresis and subjected to sequence analysis. Protein S7 alone gave no protected fragments, but S7 together with S14 and S19 protected an RNA region comprising the sequences 936-965, 972-1030, 1208-1262 and 1285-1379 of the 16S RNA. Addition of increasing amounts of protein S9 to the S7/S14/S19 particle resulted in a parallel increase in the protection of the hairpin loop between bases 1262 and 1285. The results are discussed in terms of the three-dimensional folding of 16S RNA in the 30S subunit.     

The protein composition of reconstituted 30S ribosomal subunits: the effects of single protein omission.

Using reverse phase HPLC, we have been able to quantify the protein compositions of reconstituted 30S ribosomal subunits, formed either with the full complement of 30S proteins in the reconstitution mix or with a single protein omitted. We denote particles formed in the latter case as SPORE (single protein omission reconstitution) particles. An important goal in 30S reconstitution studies is the formation of reconstituted subunits having uniform protein composition, preferably corresponding to one copy of each protein per reconstituted particle. Here we describe procedures involving variation of the protein:rRNA ratio that approach this goal. In SPORE particles the omission of one protein often results in the partial loss in uptake of other proteins. We also describe procedures to increase the uptake of such proteins into SPORE particles, thus enhancing the utility of the SPORE approach in defining the role of specific proteins in 30S structure and function. The losses of proteins other than the omitted protein provide a measure of protein:protein interaction within the 30S subunit. Most of these losses are predictable on the basis of other such measures. However, we do find evidence for several long-range protein:protein interactions (S6:S3, S6:S12, S10:S16, and S6:S4) that have not been described previously.     

Modification of histidine residues on proteins from the 50S subunit of the Escherichia coli ribosome. Effects on subunit assembly and peptidyl transferase centre activity.

L2, L3, L4, L16 and L20 are proteins of the 50S ribosomal subunit of Escherichia coli which are essential for the assembly and activity of the peptidyl transferase centre. These proteins have been modified with the histidine-specific reagent, diethylpyrocarbonate, while L17 and L18 were treated as controls. Each modified protein tested was able to participate in the reconstitution of a 50S particle when replacing its normal counterpart, although the particles assembled with modified L2 were heterogeneous. However, although they could support assembly, modified L16 and L20 were not themselves reconstituted stably, and modified L2 and L3 were found in less than stoichiometric amounts. Particles assembled in the presence of modified L16 retained significant peptidyl transferase activity (60-70% at 10 mM diethylpyrocarbonate) whereas those reconstituted with modified L2, L3, L4 or L20 had low activity (10-30% at 10 mM diethylpyrocarbonate). The particles assembled with the modified control protein, L17, retained 80% of their peptidyl transferase activity under the same conditions. The histidine residues within the essential proteins therefore contribute to ribosome structure and function in three significant ways; in the correct assembly of the ribosomal subunit (L2), for the stable assembly of the proteins within the ribosomal particle (L20 and L16 in particular), and directly or indirectly for the subsequent activity of the peptidyl transferase centre (L2, L3, L4 and L20). The essential nature of the unmodified histidines for assembly events precludes the use of the chemical-modification strategy to test the proposal that a histidine on one of the proteins might participate in the catalytic activity of the centre.     

Immunochemical accessibility of ribosomal protein S4 in the 30 S ribosome. The interaction of S4 with S5 and S12

The reactivity of protein S4-specific antibody preparations with 30 S ribosomal subunits and intermediates of in vitro subunit reconstitution has been characterized using a quantitative antibody binding assay. Anti-S4 antibody preparations did not react with native 30 S ribosomal subunits; however, they did react with various subunit assembly intermediates that lacked proteins S5 and S12. The inclusion of proteins S5 and S12 in reconstituted particles resulted in a large decrease in anti-S4 reactivity, and it was concluded that proteins S5 and S12 are primarily responsible for the masking of S4 antigenic determinants in the 30 S subunit. The effect of S5 and S12 on S4 accessibility is consistent with data from a variety of other approaches, suggesting that these proteins form a structural and functional domain in the small ribosomal subunit.     

Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6. In 90 min between 0.7 and 1.0 mol of phosphate/mol of protein S6 was incorporated by the catalytic subunit of cyclic AMP-dependent protein kinase. Of the other proteins, S3 and S7 from the small subunit and proteins L6, L18, L19 and L35 from the large subunit were predominantly phosphorylated by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates by either kinase; (3) proteins S7 and L29 were almost exclusively phosphorylated by the cyclic AMP-dependent protein kinase; (4) protein S6 and most of the other proteins were phosphorylated about two or three times faster by the cyclic AMP-dependent than by the cyclic GMP-dependent enzyme.     

The peptidyltransferase centre of the Escherichia coli ribosome. The histidine of protein L16 affects the reconstitution and control of the active centre but is not essential for release-factor-mediated peptidyl-tRNA hydrolysis and peptide bond formation

Modification of the Escherichia coli 50S ribosomal subunit with histidine-specific diethyl pyrocarbonate affects peptide bond formation and release-factor-dependent peptidyl-tRNA hydrolysis. Unmodified L16 can restore activity to a split protein fraction from the altered subunit but other proteins of the core also contain histidine residues important for the activity of the peptidyltransferase centre. When isolated and purified by centrifugation, particles reconstituted with unmodified proteins and modified L16 do not retain the altered L16. The modified protein does mediate the partial restoration of peptide bond formation and release-factor-2 activities to these particles. It must be exerting its effect during the assembly of the peptidyltransferase centre in the reconstituted particle. A particle could be reconstituted which lacks L16 and has significant activity in peptide bond formation and peptidyl-tRNA hydrolysis. L16 stimulates these activities. A tighter ribosomal binding of the release factor 2, dependent upon the absence of protein L11, can in part compensate for the loss of activity of the peptidyltransferase centre when it is assembled with either modified L16 or in the absence of L16. The protein and its histidine residue seem important, therefore, for the peptidyltransferase centre to be formed in the correct conformation but not essential for activity once the centre is assembled.     

Enzymatic methyl esterification of Escherichia coli ribosomal proteins.

Enzymatic methyl ester formation in Escherichia coli ribosomal proteins was observed. Alkali lability of the methylated proteins and derivatization of the methyl groups as methyl esters of 3,5-dinitrobenzoate indicate the presence of protein methyl esters. The esterification reaction occurs predominantly on the 30S ribosomal subunit, with protein S3 as the major esterified protein. When the purified 30S subunit was used as the methyl acceptor, protein S9 was also found to be esterified. The enzyme responsible for the esterification of free carboxyl groups in proteins, protein methylase II (S-adenosyl-L-methionine:protein carboxyl methyltransferase, EC 2.1.1.24), was identified in E. coli Q13. This enzyme is extremely unstable when compared with that from mammalian origin. By molecular sieve chromatography, E. coli protein methylase II showed multiple peaks, with a major broad peak around 120,000 daltons and several minor peaks in the lower-molecular-weight region. Rechromatography of the major enzyme peak showed activities in several fractions that are much lower in molecular weight. The substrate specificity of the E. coli enzyme is similar to that of the mammalian enzyme. The Km value for S-adenosyl-L-methionine is 1.96 X 10(-6) M, and S-adenosyl-L-homocysteine was found to be a competitive inhibitor, with a Ki value of 1.75 X 10(-6) M.     

Localization of L11 on the Escherichia coli ribosome by singlet-singlet energy transfer

Isolated Escherichia coli ribosomal protein L11 was labeled with maleimidyl derivatives of coumarin or fluorescein at the thiol group of its single cysteine, then reconstituted singly or in pairs with other fluorescently labeled ribosomal components. The characteristics of fluorescence from the labeled protein were studied and its distance to other components was determined by non-radiative energy transfer. The distance between probes on L11 and cysteine residues on other proteins or the 3' end of the ribosomal RNAs were found to be: S1, 7.4-8.3 nm; S21, 7.6 nm; 23S RNA, 6.9 nm; 5S RNA, 7.6 nm; 16S RNA, greater than 8.5 nm. Considered together with previously published results these distances indicate that the location of L11 in the 50S subunit is below the lateral protuberance characterized by L7/L12.     

On the role of protein S4 N-terminal residues 1 through 30 in 30S ribosome function.

30S ribosomal protein S4 contains a single cysteine residue at position 31. We have selectively cleaved the peptide bond adjacent to this residue using the reagent 2-nitro-5-thiocyanobenzoic acid. The two resultant fragments were purified. The smaller S4-fragment (1-30) was found to be incapable of interacting with 16S RNA directly. This fragment also is not incorporated into a particle reconstituted from 16S RNA and 20 purified proteins with S4 missing. In contrast, the large S4-fragment (31-203) appears to be fully functional in ribosome assembly. Replacement of S4 with this fragment in the reconstitution reaction leads to a complete 30S ribosome containing all 30S proteins. This particle has a full capacity to bind poly U but has lost all activity for poly U directed phe-tRNA binding. We therefore propose that the N-terminus of protein S4 is not critical for ribosome assembly but is essential for tRNA binding.     

Chemically induced resistance to heat treatment and stress protein synthesis in cultured mammalian cells

Short exposure (1-2 h) of cultured cells, derived from a transplantable murine mammary carcinoma, to sodium arsenite, 2,4-dinitrophenol (DNP), carbonylcyanide-3-chlorophenylhydrazone (CCP) or disulfiram, induced resistance to a subsequent heat treatment, similar to heat-induced thermotolerance. Optimum resistance to a test heat treatment of 45 min at 45 degrees C after sodium arsenite exposure was obtained at a concentration of 300 microM, after DNP exposure at 3mM, after CCP at 300 microM and after disulfiram exposure in the range 1-30 microM. Exposure of cells to CCP, sodium arsenite or disulfiram led to enhanced synthesis of some proteins with the same molecular weight as 'heat shock' proteins. The pattern of enhanced synthesis of these proteins was agent specific. We could not detect significantly enhanced synthesis of the proteins after DNP using one-dimensional gel electrophoresis. These results suggest that enhanced stress protein synthesis is not a prerequisite for the development of thermal resistance.     

Topography of rna in the ribosome localization of the 16 s rna 5' end by immune electron microscopy

The location of the 5′ end of 16 S RNA on the 30 S subunit has been determined. This has been done using immune electron microscopy of the 30 S subunits reconstituted from the 16 S RNA carrying 2,4-dinitrophenyl-hapten at its 5′-terminal phosphate group. Triphenylphosphine/2,2′-dipyridyl disulfide and 2,4-dinitrophenyl-ethylenediamine have been used as condensing and nucleophilic reagents, respectively, for modification of 16 S RNA. The reaction proceeds rapidly in mild conditions and results in a high yield of the modified RNA. Thus, a new general procedure for labeling the 5′-terminal nucleotides of high molecular weight RNAs is described. The 5′ end of 16 S RNA has been found to be located on a “lower” one-third of the small ribosomal subunit, on the side opposite to the ledge (platform).