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1.
RNA-protein interactions in the ribosome. II. Binding of ribosomal proteins to isolated fragments of the 16 S RNA 总被引:4,自引:0,他引:4
Specific fragments of the 16 S ribosomal RNA of Escherichia coli have been isolated and tested for their ability to interact with proteins of the 30 S ribosomal subunit. The 12 S RNA, a 900-nucleotide fragment derived from the 5′-terminal portion of the 16 S RNA, was shown to form specific complexes with proteins S4, S8, S15, and S20. The stoichiometry of binding at saturation was determined in each case. Interaction between the 12 S RNA and protein fraction was detected in the presence of S4, S8, S15 and S20; only these proteins were able to bind to this fragment, even when all 21 proteins of the 30 S subunit were added to the reaction mixture. Protein S4 also interacted specifically with the 9 S RNA, a fragment of 500 nucleotides that corresponds to the 5′-terminal third of the 16 S RNA, and protein S15 bound independently to the 4 S RNA, a fragment containing 140 nucleotides situated toward the middle of the RNA molecule. None of the proteins interacted with the 600-nucleotide 8 S fragment that arose from the 3′-end of the 16 S RNA.When the 16 S RNA was incubated with an unfractionated mixture of 30 S subunit proteins at 0 °C, 10 to 12 of the proteins interacted with the ribosomal RNA to form the reconstitution intermediate (RI) particle. Limited hydrolysis of this particle with T1 ribonuclease yielded 14 S and 8 S subparticles whose RNA components were indistinguishable from the 12 S and 8 S RNAs isolated from digests of free 16 S RNA. The 14 S subparticle contained proteins S6 and S18 in addition to the RNA-binding proteins S4, S8, S15, S20 and . The 8 S subparticle contained proteins S7, S9, S13 and S19. These findings serve to localize the sites at which proteins incapable of independent interaction with 16 S RNA are fixed during the early stages of 30 S subunit assembly. 相似文献
2.
Subparticles of the Escherichia coli 50 S ribosome subunit containing varying amounts of the protein L11 have been prepared. These core particles have been used to form 70 S couples containing f[3H]Met-tRNA as a substrate for the peptidyl hydrolysis reaction of in vitro termination. Studies with antibodies against L11 suggested previously that the protein was involved in this event. The peptidyl transferase of the 50 S subunit core particles containing no more than 6% of the normal complement of L11 was fully active. The 70 S couples formed from 50 S cores lacking L11 showed some decrease in their ability to bind fMet-tRNA. Ribosomes lacking the proteins retained about 50% of their activity for the peptidyl-tRNA hydrolysis event of in vitro termination. Cores lacking both and L11 were almost as active as those lacking only . L11 is, therefore, not absolutely required for peptidyl-tRNA hydrolysis at termination in vitro. The ribosome subparticles lacking L11 have been reconstituted with . Despite the absence of L11, they regained significant activity for the codon-directed in vitro termination reaction. 相似文献
3.
Hiroaki Tokimatsu William A. strycharz Albert E. Dahlberg 《Journal of molecular biology》1981,152(2):397-412
Three forms of the 50 S ribosomal subunit of Escherichia coli have been separated by agarose/acrylamide gel electrophoresis. The slowest migrating form, S-50 S, corresponded to native 50 S subunits and contained four copies of proteins . Removal of the four copies of this protein produced a more rapidly migrating form, M-50 S. The M-50 S form was then converted to the fastest migrating form, F-50 S, by removal of additional proteins, including L10 and L11. A one-step removal of a pentameric complex of four copies of plus L10 converted the S-50 S subunit directly to the F-50 S subunit. These proteins recombined specifically with the appropriate protein-deficient 50 S subunit at 3 °C to reform the S-50 S subunit, i.e. the M-50 S subunit was converted back to the S-50 S form by the addition of purified proteins ; and the F-50 S subunit bound the pentameric complex of and L10 to form S-50 S. The binding of the pentameric complex, isolated by glycerol gradient centrifugation, supports the model that all four copies of proteins are together in one part of the ribosome called the “ stalk”. Only the four copies of were removed from the 50 S subunit in low salt (0.125 m-NH4Cl) plus 50% ethanol at 0 °C. These ribosomes (in the M-50 S form) had less than 5% of the peptide-synthesizing activity of untreated control ribosomes as measured by a poly(U) translation system in vitro. Peptide-synthesizing activity was restored, upon addition of , back to the treated ribosomes to give 50 S subunits (S-50 S) with a full complement of four copies of . Antibody to proteins bound only to the S-50 S subunits, producing four new bands separated by gel electrophoresis. The bands represented complexes of one, two, three and four antibodies bound to a 50 S subunit. This result was obtained using either 50 S subunits or 70 S tight couples and indicated that all four copies of are either located at a single site in the stalk or, much less likely, are divided between two symmetrical sites. Proteins were not only accessible to their specific antibody but could also be removed from 70 S ribosomes and polyribosomes without causing their dissociation into subunits. The ribosomes and polyribosomes had an increased gel electrophoretic mobility which was reversed by addition of proteins . 相似文献
4.
Three-dimensional locations have been determined for Escherichia coli ribosomal proteins L1, L17 and L27 by immune electron microscopy using antibodies directed against these proteins. From the positions of immunoglobulin G attachment, observed in two characteristic projections, it was determined that these three proteins are located at single sites in different regions on the surface of the large subunit. In the quasisymmetric projection, L1 maps on the side opposite the “ stalk,” named the L1 ridge; protein L17 maps at the base of the subunit opposite the “central protuberance” (toward the side of the subunit); and protein L27 is found on the central protuberance (on the side distal to the stalk). In the asymmetric projection, proteins L1 and L27 are found on the surface of the subunit contracting the small subunit and protein L17 is on the surface of the subunit distal to the small subunit; i.e. on the cytoplasmic surface of the large subunit. Antibody binding at all three sites was eliminated when the immunoglobulin G molecules were preabsorbed with their specific proteins. 相似文献
5.
RNA-protein interactions in the ribosome. I. Characterization and ribonuclease digestion of 16 S RNA-ribosomal protein complexes 总被引:14,自引:0,他引:14
Proteins from the 30 S ribosomal subunit of Escherichia coli were fractionated by column chromatography and individually incubated with 16 S ribosomal RNA. Stable and specific complexes were formed between proteins S4, S7, S8, S15 and S20, and the 16 S RNA. Protein S13 and one or both proteins of the mixture bound more weakly to the RNA, although these interactions too were apparently specific. The binding of was found to be markedly stimulated by proteins S4, S8, S15 and S20. Limited digestion of the RNA-protein complexes with T1 or pancreatic ribonucleases yielded a variety of partially overlapping RNA fragments, which retained one or more of the proteins. Since similar fragments were recovered when 16 S RNA alone was digested under the same conditions, their stability could not be accounted for by the presence of bound protein. The integrity of the fragments was, however, strongly influenced by the magnesium ion concentration at which ribonuclease digestion was carried out. Each of the RNA fragments was characterized by fingerprinting and positioned within the sequence of the 1600-nucleotide 16 S RNA molecule. The location of ribosomal protein binding sites was delimited by the pattern of fragments to which a given protein bound. The binding sites for proteins S4, S8, S15, S20 and, possibly, S13 and as well, lie within the 5′-terminal half of the 16 S RNA molecule. In particular, the S4 binding site was localized to the first 500 nucleotides of this sequence while that for S15 lies within a 140-nucleotide sequence starting about 600 nucleotides from the 5′-terminus. The binding site for the protein S7 lies between 900 and 1500 nucleotides from the 5′-terminus of the ribosomal RNA. 相似文献
6.
Each of the 50 S ribosomal subunit proteins of Escherichia coli was tested independently in two laboratories for its ability to bind specifically to 23 S RNA. Four new RNA-binding proteins, L1, L3, L4 and L13 were identified in this way. Consistent with earlier work, proteins L2, L6, L16, L20, L23 and L24 were found to interact directly and independently with 23 S RNA as well. No binding of L17 was detected, however, contrary to previous reports, and the results for L19 were variable. The molar ratio of protein and RNA in each complex was measured at saturation. Significant differences in binding stoichiometry were noted among the various proteins. In addition, saturation levels were found to be influenced by the state of both the RNA and the proteins. 相似文献
7.
Ribosomal proteins L7/L12 localized at a single region of the large subunit by immune electron microscopy. 总被引:6,自引:0,他引:6
Ribosomal proteins have been mapped by immune electron microscopy. These multiple copy proteins are located at a single region extending from the large subunit, known as the stalk. The stalk is approximately 100 Å long, about 40 Å wide and extends at an angle of approximately 50 ° from one side of the central protuberance of the large subunit. In the monomeric 70 S ribosome, the portion of the stalk proximal to the 50 S subunit is located in the vicinity of the 30 S-50 S interface.Anti- antibody binding to the stalk was shown to be solely dependent upon the presence of by the following experiments. Sucrose gradient analysis was used to demonstrate that large subunits depleted of were unable to bind anti- antibodies and that re-incorporation of restored the ability of -depleted cores to react with anti- antibodies. Anti- antibodies pre-absorbed with did not react with 50 S subunits.Anti- antibodies used in these experiments reacted only with the stalk and with no other region of the subunit. This was shown by electron microscopy and by immune electron microscopy in the following ways. Electron microscopy of 50 S subunits, -depleted 50 S cores, and reconstituted 50 S subunits was used to demonstrate that stripping removes the stalk from more than 95% of the subunits, and that re-incorporation of into depleted cores restores the stalk. Double-labelling experiments, using monomeric subunits with two or more attached anti- immunoglobulins, were used to demonstrate, independently of 50 S subunit morphology, that are located only on the stalk. 相似文献
8.
HeLa cell polysomes were oxidized with sodium periodate and reduced with sodium borohydride to induce covalent crosslinks between ribosomal RNA and nearby proteins. We proved that RNA was tryly crosslinked to protein in oxidized, and not in control, samples using denaturing cesium trichloroacetate density gradients and phenol extraction. By both one- and two-dimensional gel analysis, we found that protein S3a can be crosslinked to 18S RNA, protein L3 to 28S RNA, and proteins L7′ and L23′ to 5.8S RNA. Because of the specificity of the periodate reaction, and since we were able to crosslink protein S1 to 16S RNA in , 30S ribosomal subunits, it is likely that we have crosslinked proteins to the 3′OH ends of HeLa polysomal RNAs. 相似文献
9.
Nikolaos Robakis Miloslav Boublik 《Biochemical and biophysical research communications》1981,103(4):1401-1408
The effect of protein moiety on the conformation of 16S and 23S RNA of the ribosome has been studied by circular dichroic spectroscopy. Both rRNAs possess a comparable net content of ordered secondary structure which remains unchanged after association with ribosomal proteins into “core” particles or into complete 30S and 50S subunits, respectively. However, differences found in the stability and the cooperativity of melting of free and protein-associated rRNAs imply protein-caused variations in the distribution of the intramolecular hairpin stems and loops and/or changes in long range tertiary interactions which appear to be different for both rRNAs. While 23S RNA is maximally stabilized on the large subunit by the full set of proteins, 16S RNA on the complete small subunit shows lower stability but higher cooperativity in melting. 相似文献
10.
Structural alteration of rRNA in the L7/L12 region of 50s ribosome on removal of L7/L12 proteins 总被引:1,自引:0,他引:1
D P ByasmuniBurma 《Biochemical and biophysical research communications》1982,104(1):99-104
Core particles of 50S ribosomes depleted of proteins are degraded by RNase I at a considerably slower rate than intact 50S ribosomes. The normal rate is restored on incorporating proteins into the core particles. The capacity of the core particles to inhibit the RNase I-catalyzed hydrolysis of poly A and to bind ethidium bromide is also greater with core particles than with intact 50S ribosomes. It appears from these results that the region(s) of rRNA in the vicinity of proteins has less ordered structure which, on removal of proteins, becomes more organized. Apparently, binding of proteins to the 50S core leads to the destabilization of double-stranded regions of rRNA. 相似文献
11.
H B Stuhrmann M H Koch R Parfait J Haas K Ibel R R Crichton 《Journal of molecular biology》1978,119(2):203-212
Neutron small-angle scattering of the 70 S Escherichia coli ribosomes and of its smaller 30 S subunit has been measured in mixtures. A linear dependence of the square of the radius of gyration on the reciprocal of the contrast is found, which is qualitatively similar to the results from contrast variation with the larger 50 S subunit. The slope α in this plot is a measure of radial segregation of RNA and proteins. It is most pronounced with the 50 S subunit. The 30 S particle appears to be more homogeneous, whereas the 70 S ribosome assumes an intermediate value of α. Neither the 30 S and 50 S subunits nor the 70 S ribosome show a significant separation of the centres of mass of their RNA part and proteins. A quantitative comparison of the parameters obtained suggest that the interaction between the two subunits and the 70 S ribosomes does not involve any major change in the latter. 相似文献
12.
Characterization of ribonucleoprotein subparticles from 50 S ribosomal subunits of Escherichia coli.
Large ribonucleoprotein subparticles were recovered upon ribonuclease digestion of the 50 S ribosomal subunits of Escherichia coli, partially deproteinized by LiCl. Both their RNA and their protein compositions were analysed. The subunits, treated with LiCl at a concentration of 5.5 m, released an homogeneous subparticle containing proteins L3, L4, L13, L17, L22 and L29, about 70% of the 13 S fragment of 23 S RNA and about 50% of the 18 S one. Slightly larger species of subparticles were obtained from 50 S subunits treated with LiCl at concentrations between 3 m and 5 m; they contained in addition proteins L20, L21 and L23 or L2, L14, L20, L21 and L23 and a few small 23 S RNA fragments. No large subparticle was recovered from the 6 m-LiCl-treated 50 S subunits which contain only proteins L3, L13 and L17. These LiCl subparticles were compared with those obtained from intact, unfolded and sodium doecyl sulphatetreated 50 S subunits.These studies reveal that in the presence of 0.10 m-magnesium acetate there is a very compact area within 50 S subunits consisting of proteins L3, L4, L13, L17, L22 and L29 and of about 60% of 23 S RNA; this area probably has an essential structural role. The results also show that 23 S RNA has a more folded conformation when within the 50 S subunit than when isolated, this conformation being stabilized by some of the 50 S proteins, in particular proteins L4, L22, L20 and L21. Finally these data permit a more definite localization of the primary and/or secondary binding sites of proteins L2, L3, L4, L14, L17, L20, L21 and L22 on 23 S RNA. 相似文献
13.
Heinz-Kurt Hochkeppel Eleanor Spicer Gary R. Craven 《Journal of molecular biology》1976,101(2):155-170
A method of preparing 16 S RNA has been developed which yields RNA capable of binding specifically at least 12, and possibly 13, 30 S ribosomal proteins. This RNA, prepared by precipitation from 30 S subunits using a mixture of acetic acid and urea, is able to form stable complexes with proteins S3, S5, S9, S12, S13, S18 and possibly S11. In addition, this RNA has not been impaired in its capacity to interact with proteins S4, S7, S8, S15, S17 and S20, which are proteins that most other workers have shown to bind RNA prepared by the traditional phenol extraction procedure (Held et al., 1974; Garrett et al., 1971; Schaup et al., 1970,1971).We have applied several criteria of specificity to the binding of proteins to 16 S RNA prepared by the acetic acid-urea method. First, the new set of proteins interacts only with acetic acid-urea 16 S RNA and not with 16 S RNA prepared by the phenol method or with 23 S RNA prepared by the acetic acid-urea procedure. Second, 50 S ribosomal proteins do not interact with acetic acidurea 16 S RNA but do bind to 23 S RNA. Third, in the case of protein S9, we have shown that the bound protein co-sediments with acetic acid-urea 16 S RNA in a sucrose gradient. Additionally, a saturation binding experiment showed that approximately one mole of protein S9 binds acetic acid-urea 16 S RNA at saturation. Thus, we conclude that the method employed for the preparation of 16 S RNA greatly influences the ability of the RNA to form specific protein complexes. The significance of these results is discussed with regard to the in vitro assembly sequence. 相似文献
14.
A complex of acidic ribosomal proteins. Evidence of a four-to-one complex of proteins in the Bacillus stearothermophilus ribosome 总被引:2,自引:0,他引:2
Two acidic proteins from the 50 S subunit of Bacillus stearothermophilus ribosomes, namely B-L13 (homologous to Escherichia coli protein ) and B-L8, form a complex. Radioactive B-L13, added to ribosomes before dissociation, does not appear in the complex after electrophoresis, so the (B-L13 · B-L8) complex must exist in the ribosome before dissociation. Digestion of B. stearothermophilus ribosomes with polyacrylamide-bound trypsin causes the appearance of new B-L8 and B-L13 spots on two-dimensional polyacrylamide gel electrophoresis, in a pattern which suggests that single molecules of B-L13 are being sequentially cleaved from a four-to-one complex of B-L13 and B-L8. 相似文献
15.
Identification of the nucleotide sequences involved in the interaction between Escherichia coli 5 RNA and specific 50 S subunit proteins 总被引:9,自引:0,他引:9
P.N. Gray G. Bellemare R. Monier R.A. Garrett G. Stöffler 《Journal of molecular biology》1973,77(1):133-152
Pancreatic RNase partial digests of 32P-labelled 5 S RNA-protein complexes have been fractionated by electrophoresis on polyacrylamide gels. Specific fragments of the 5 S RNA molecule have been recovered from electrophoresis bands containing polynucleotide-protein complexes. These digestion-resistant complexes are only found if RNase treatment is carried out in the presence of at least one of the two 50 S subunit proteins L18 and L25, which are able to bind to 5 S RNA individually and specifically. The sequences of the isolated fragments have been determined. From the results, it can be concluded that sequence 69 to 120 and, possibly, sequence 1 to 11, are involved in the 5 S RNA-protein interactions which are responsible for the insertion of 5 S RNA in the 50 S subunit structure. Sequence 12 to 68, on the other hand, has no strong interactions with proteins L18 and L25. Each protein certainly binds to several nucleotide residues, which are not contiguous in the primary structure. In particular, good experimental evidence has been obtained in favour of the binding of protein L25 to two distant regions of the 5 S RNA molecule, which must have a bihelical secondary structure. The importance of the 5 S RNA conformation for its proper insertion in the 50 S subunit is thus confirmed. 相似文献
16.
Analysis of kethoxal bound to ribosomal proteins from Escherichia coli 70S reacted ribosomes 总被引:1,自引:0,他引:1
To investigate ribosome topography and possible function, 70S ribosomes of were reacted with the dicarbonyl compound kethoxal. Ribosomal protein was extracted after reaction, and through two dimensional gel electrophoresis, the reactive proteins of the two subunits were identified. From the 30S subunit, the most reacted proteins were S2, S3, S4, S5 and S7 and from the 50S subunit, L1, L5, L16, L17, L18 and L27. The results with kethoxal are compared with other modifiers of ribosomal proteins. 相似文献
17.
K Igarashi Y Watanabe E Matsumoto A Kogo M Yabuki 《Biochemical and biophysical research communications》1977,76(1):18-25
From the studies on the spermidine stimulation of polyphenylalanine synthesis catalyzed by 50S and reconstituted 30S particles containing 16S RNA and 30S ribosomal proteins from and in different kinds of combinations, it is concluded that 16S RNA is mainly responsible for the stimulation of polypeptide synthesis by spermidine. 相似文献
18.
C. A. Morrison G. Tischendorf G. Stöffler R. A. Garrett 《Molecular & general genetics : MGG》1977,151(3):245-252
Summary The accessibility of each of the proteins on the 50S ribosomal subunit of Escherichia coli was investigated by establishing whether immunoglobulins (IgG), specific for each of the 34 proteins from the 50S subunit, were able to bind to the 50S subunit. The main criterion for accessibility was the formation of specific antibody-50S subunit complexes that could be detected by means of analytical ultracentrifugation.The proteins fell into two main groups. Immunoglobulins against proteins L1, L2, L3, L4, L5, L6, L7/L12, L8, L9, L10, L11, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L25, L26, L27 and L30 gave large amounts of complex (20–100%) and, therefore, these proteins were considered to be accessible sible on the surface of the 50S ribosomal subunit. The antibodies against the remaining proteins L13, L24, L28, L29 and L31 to L34 produced small amounts of complexes (10–20%). Since their effects were unequivocably stronger than those obtained with IgG's from sera of non-immunized animals, the results indicate that these proteins are probably also accessible. Nonetheless, from the ultracentrifugation studies alone definite conclusions about the exposure of the latter group of proteins could not be drawn. 相似文献
19.
G Goldberg T Zarucki-Schulz P Caldwell H Weissbach N Brot 《Biochemical and biophysical research communications》1979,91(4):1453-1461
The DNA dependent synthesis of ribosomal protein L12 and the β subunit of RNA polymerase has been investigated using DNA from a plasmid which contains the genetic information for ribosomal protein L12 and the β subunit of RNA polymerase. This DNA, however, lacks the promoter region and the genetic information for the first 26 amino acids of ribosomal protein L10. It was found that L12 and the β subunit of RNA polymerase are efficiently synthesized from this DNA. These results suggest that L12 and the β subunit of RNA polymerase can be synthesized from a promoter situated within the L10 gene. 相似文献
20.
Wen-Tah Hsu 《Biochemical and biophysical research communications》1973,52(3):974-979
The effect of T4 phage on ribosomes in terms of their ability to bind RNA viral template is examined. It is found that the 30S subunits of T4 ribosomes bind MS2 RNA as efficiently as do the subunits of uninfected ribosomes. On the other hand, analyses of the formation of 70S initiation complex, presumably from MS2 RNA-30S ribosome complex, using both labeled MS2 RNA and initiator tRNA, reveal that T4 ribosomes are only about half as active as ribosomes. The latter phenomenon has been reported previously. These results suggest that, following T4 infection, ribosomes are modified in such a way that the attachment of fMet-tRNAf to MS2 RNA-30S subunit complex is impaired. 相似文献