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1.
The acidic proteins B-L13 (homologous to Escherichia coli protein L7/L12) and B-L8, from the 50 S subunit of Bacillus stearothermophilus ribosomes, form a stable complex. Trypsin digestion of ribosomes generates an N-terminal fragment of B-L13 (approximately residues 1 to 47) which can associate with B-L8, displacing intact B-L13, and bind to B-L13-deficient ribosomes. Displacement of B-L13 from the B-L8 · B-L13 complex by the B-L13 N-terminal fragment causes a change in gel electrophoretic mobility of the complex, and titration of the complex with fragment indicates unambiguously that it contains four molecules of B-L13. Evidence is presented that B-L13 forms a dimer in solution, and that the dimer associates intact with B-L8. Reconstituted 50 S subunits in which B-L13 is replaced by its N-terminal fragment have the same functional properties as 50 S subunits missing B-L13 altogether: polypeptide synthesis is reduced but not abolished; ability to bind elongation factor EF-G and GTP is severely reduced; and peptidyl transferase activity and ability to associate with a 30 S subunit · Phe-tRNA · poly(U) complex are unaffected (relative to intact 50 S subunits). 相似文献
2.
A complex of ribosomal proteins B-L8 and B-L13 from Bacillus stearothermophilus has been isolated and crystallized. The cell dimensions are with the space group P41212 (or P43212). The asymmetric unit probably contains two complexes with a 4:1 stoichiometry of the proteins. 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.
Experiments are described in which the Stoichiometry of the ribosomal proteins before and after ribosome release from mRNA is compared. Polysomes labeled with 3H (or 14C) and run-off 70 S particles (Subramanian el al., 1969) labeled with 14C (or 3H) were separately isolated, mixed, and the ribosomal proteins extracted and fractionated by two-dimensional gel electrophoresis. The measurement of the isotopic ratios shows that 47 proteins out of the 53 investigated are present in the same amount in polysomes as in run-off ribosomes indicating that they remain with the ribosome during the release step. Proteins S1, S2, S6, S21, (Wittmann et al., 1971), however, show higher amounts in polysomes than in run-off ribosomes. The significance of these results is discussed. 相似文献
5.
F Sánchez-Madrid P Conde D Vázquez J P Ballesta 《Biochemical and biophysical research communications》1979,87(1):281-291
Two dimensional gel electrophoresis of ribosomal proteins from reveals the presence of three spots in the region corresponding to proteins of high acidic character. Washing the ribosomes with 0.4 M NH4Cl and 50% ethanol, followed by chromatography of the extracted proteins on DEAE-cellulose, indicated the presence of two fractions of acidic proteins; (A and Ax), having very similar molecular weights (12.000–13.000), but phosphorylated to different extents. Fractions A and Ax are immunologically distinct and their immunologic properties differ from acidic proteins found in , rat liver, and ribosomes.Protein A can be resolved into two bands by electrofocusing, and two dimensional gel electrophoresis. The two components correspond to proteins L44 and L45 according to the standard nomenclature. Proteins Ax seems to correspond to the spot that moves above and to the left of L44 and L45 and is at least three times more phosphorylated than these two proteins. 相似文献
6.
The core oligosaccharide in LPS of the Ter-15 mutant after the transformation of F'-lac episome 总被引:1,自引:0,他引:1
T Ohkawa 《Biochemical and biophysical research communications》1982,108(4):1413-1417
Ribosomes from stationary phase have a diminished activity in protein synthesis due to the presence of a bound protein (association factor I, AFI). This factor inhibits the binding of fmet-tRNA to ribosomes. The inhibition of protein synthesis can be reversed in systems directed by viral mRNA by the addition of an excess of high salt ribosomal wash obtained from mid-log cells. Under this condition AFI is released from ribosomes. 相似文献
7.
Maria Szekely Therese Loviny Richard C. Foreman 《Biochemical and biophysical research communications》1976,70(1):110-116
Binding of formylmethionyl tRNA and ribosomes to double stranded RNA has been obtained under conditions identical to those required for initiation complex formation with single stranded RNA. While natural double stranded RNAs from Penicillium virus and Penicillium virus were efficient in forming initiation complexes, the synthetic polynucleotide poly(I).poly (C) was inactive. This suggests that ribosomes can recognize initiation sequences even if these are present in base-paired form. 相似文献
8.
Association of adenovirus type 2 early proteins with a soluble complex that synthesizes adenovirus DNA in vitro 总被引:3,自引:0,他引:3
H M Rho Y H Jeng W S Wold M Green 《Biochemical and biophysical research communications》1977,79(2):422-428
A soluble Ad2 DNA synthesizing complex was prepared from Ad2-infected KB cell nuclei and purified by exclusion chromatography on a BioGel A-50m column. The purified complex was able to synthesize DNA from all regions of the virus genome, as indicated by RI restriction endonuclease analysis of labeled DNA. Experiments were performed to identify Ad2-induced early polypeptides present in the complex. Ad2-infected and mock-infected cells were labeled with [35S]methionine 7–10 h postinfection, then incubated for 8 h to allow the 35S-labeled early polypeptides to become associated with the complex. The polypeptides in the purified complex and each of the cell fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The major components of the purified complex were the 73K DNA binding phosphoprotein and 11K, two adenovirus 2-induced early polypeptides. The 11K has a preferred nuclear location. Small quantities of other Ad2-induced early proteins, 21K, 15K, and possibly 8.3K were also associated with the complex. 相似文献
9.
Reelectrophoresis of the oligomer form (CP II1) of the chlorophyll light-harvesting complex (LHC) from the green alga Acetabularia yields two green bands which run at the position typical of the monomer (CP II). The upper green band (CP II1) is enriched in the 27 kDa polypeptide of the LHC, while the lower is enriched in the 26 kDa polypeptide. The fact that both bands have both chlorophyll (Chl) a and b, and in the same ratio, implies that the LHC is made up of two Chl proteins. Neither of these bands can be attributed to the Chl complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432). Resolution of CP II1 and CP II2 of spinach can be obtained if sucrose gradient fractions of an octylglucoside extract are subjected to SDS-polyacrylamide gel electrophoresis. CP II1 and CP II2 are interpreted as being fundamental subunits of the light-harvesting complex as it is defined on SDS-polyacrylamide gels. 相似文献
10.
Y D Karkhanis R L Anderson J Y Zeltner R Z Maigetter D J Carlo T H Stoudt 《Biochemical and biophysical research communications》1979,89(2):750-758
An antigenic subunit of molecular weight 66,000 daltons has been isolated from the antigenic complex of the Melvin strain of . Incubation of the complex in 8M urea at room temperature for four hours resulted in the dissociation of the subunit from the complex. It was separated from the complex by chromatography of the incubation mixture on a Sepharose 6B column in 50 mM ammonium bicarbonate pH 8.5 without 8M urea and further purified by affinity chromatography. This communication reports on a newly isolated antigenic protein devoid of LPS present in the bacteria. 相似文献
11.
The purified heavy form of elongation factor 1 (EF-1) from cysts of was found to catalyze the exchange of free GTP with a complex of EF-1L (EF-1α) and GDP. Furthermore, after heat treatment of EF-1H in the presence of GTP, the factor, while inactive by itself, stimulated aminoacyl-tRNA binding to ribosomes as well as polyphenylalanine synthesis when combined with EF-1α. These functional properties are similar to those reported recently for purified EF-1β from pig liver [ Nagata,S., Motoyoshi,K., and Iwasaki,K. (1976) Biochem. Biophys. Res. Comm. , 933–938 ]. We suggest that Artemia EF-1H consists of a EF-1α. EF-1β complex which is functionally analogous to bacterial EF-Tu · EF-Ts. 相似文献
12.
Oswald G. Baca James W. Bodley 《Biochemical and biophysical research communications》1976,70(4):1091-1096
70S ribosomes uniformly labeled with 32PO4 were subjected to varying doses of u.v. radiation and then to the combined action of the RNases A and T1. Following these treatments the ribosomal proteins were separated by trichloroacetic acid precipitation from the noncovalently attached RNA degradation fragments. Subsequent two-dimensional gel electrophoresis and autoradiography of these proteins revealed that significant 32PO4 was associated with unique ribosomal proteins, L2 was among these. 相似文献
13.
The functional role of a chlorophyll complex associated with Photosystem I (PS I) has been studied. The rate constant for P-700 photooxidation, KP-700, which under light-limiting conditions is directly proportional to the size of the functional light-harvesting antenna, has been measured in two PS I preparations, one of which contains the chlorophyll complex and the other lacking the complex. KP-700 for the former preparation is half of that of the preparation which has the chlorophyll complex present. This difference reflects a decrease in the functional light-harvesting antenna in the PS I complex devoid of the chlorophyll complex. Experiments involving reconstitution of the chlorophyll complex with the antenna-depleted PS I preparation indicate a substantial recovery of the KP-700 rate. These results demonstrate that the chlorophyll complex functions as a light-harvesting antenna in PS I. 相似文献
14.
Hans-Peter Schär Herbert Zuber Michael G. Rossmann 《Journal of molecular biology》1982,154(2):349-353
Crystals of lactate dehydrogenase from Bacillus stearothermophilus have been obtained in five different crystal morphologies belonging to at least two different space groups. Apo-lactate dehydrogenase can crystallize in space group P6122 or P6522 (). A complex of lactate dehydrogenase with NADH and the effector fructose 1,6-diphosphate can crystallize in the same space group as the apoenzyme and in P6322 (). Both forms are suitable for high resolution X-ray diffraction studies. 相似文献
15.
Deborah A. Eppstein Charles E. Samuel 《Biochemical and biophysical research communications》1977,79(1):145-153
A low-molecular-weight interferon-mediated ribosome-associated inhibitor of reovirus mRNA translation was purified from the 0.5 KCl ribosomal salt-wash fraction of mouse L929 cells. The inhibitor possessed nucleolytic activity with reovirus [3H]mRNA as a substrate. Loss of translational inhibitory activity correlated with the thermal inactivation of the nuclease. A low-molecular-weight (10K) component present in the Bio-Gel P150 chromatography fractions which contained the interferon-mediated nucleolytic activity was labeled in vivo with [14C]valine; a smaller component present in the same fractions was phosphorylated in vitro with [γ-32P]ATP. The 10K components were resolved from ~50K, ~30K and ~20K phosphorylatable proteins associated with ribosomes that possess the interferon-mediated inhibitor(s) of viral mRNA translation. 相似文献
16.
The aminoacyl-tRNA synthetase-tRNA complex: detection by differential labelling of lysine residues involved in complex formation 总被引:2,自引:0,他引:2
The interaction of tRNATyr with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by differential acetylation of lysine residues. The synthetase was trace-labelled in the free form and as the synthetase-tRNATyr complex with [3H]acetic anhydride. In a second step the two 3H-labelled enzyme preparations were fully acetylated with cold reagent under denaturing conditions and were mixed with synthetase that had been homogeneously labelled with excess [14C]acetic anhydride. Peptides containing labelled lysine residues were isolated after chymotryptic digestion and their ratios were determined. These ratios reflect the reactivity of primary amino groups towards acetic anhydride.Involvement of lysine side-chains in complex formation with tRNATyr was suggested from altered ratios. Out of the 22 primary amino groups of tyrosyl-tRNA synthetase at least three showed reduced reactivities towards acetic anhydride in the synthetase-tRNATyr complex by factors of 1.6, 1.9 and 6.8, respectively. The sequences around these lysine residues have been determined enabling their placement when the primary and tertiary structure of the enzyme are available (G. L. E. Koch, to be published). No lysine residue of increased reactivity in the synthetase-tRNATyr complex has been detected.Only one molecule of tRNATyr binds to the dimeric synthetase molecule under the conditions of the differential labelling. If the binding site for the tRNA is on one of the two identical subunits, any observed decrease in chemical reactivity of a particular lysine residue should not exceed a factor of two. The detection of a lysine residue which reacts about seven times more slowly in the synthetase-tRNA complex could therefore indicate that the single binding site is formed by both enzyme subunits. 相似文献
17.
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
18.
A possible complex containing RNA processing enzymes 总被引:5,自引:0,他引:5
S K Jain B Pragai D Apirion 《Biochemical and biophysical research communications》1982,106(3):768-778
The three enzymes, RNAase III, RNAase E and RNAase P participate in the processing of RNA precursors in . In extracts which contain a mutated RNAase III or RNAase E under certain conditions RNAase P activity is not expressed while in the wild-type extract it is. Upon high-speed centrifugation of a cell extract from a strain of ,, which contains all these three enzymes, the majority of RNAase P, RNAase III and RNAase E activities sediment as particles heavier than their known sizes. In a sucrose density gradient of the cell extract, part of RNAase E and RNAase P activities co-sediment while most of the RNAase III activity is found toward the top of the gradient. This behavior is distinct from other ribonucleases such as RNAase II and RNAase H, which do not sediment as complexes. This complex does not seem to be caused merely by the association of the enzymes with ribosomes. 相似文献
19.
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
20.
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