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1.
B Poldermans C P Van Buul P H Van Knippenberg 《The Journal of biological chemistry》1979,254(18):9090-9093
The effect of the presence or absence of methyl groups on the N6 atoms of two adjacent adenosines near the 3' end of 16 S rTNA of Escherichia coli on initiation of protein biosynthesis has been studied using wild type (methylated) and kasugamycin-resistant (unmethylated) E. coli ribosomes (see preceding paper (Poldermans, B., Goosen, N., and Van Knippenberg, P. H. (1979) J. Biol. Chem. 254, 9085--9089)). Conditions of pH, temperature, and ionic strength at which binding of fMet-tRNA to ribosomes proceeds maximally are the same for wild type and mutant ribosomes. Mg2+- and factor-dependent dissociation of ribosomes as well as the association of the subunits is also the same for methylated and unmethylated ribosomes. Binding of fMet-tRNA to wild type and to mutant 70 S ribosomes requires the same amount of the three initiation factors. However, optimal fMet-tRNA binding to unmethylated 30 S ribosomes needs more of initiation factor 3 than does binding to methylated 30 S ribosomes, provided that initiation factor 1 is absent. This difference is completely abolished when mutant 30 S ribosomes are methylated using purified methylase from the wild type strain and the methyl donor S-adenosylmethionine. 相似文献
2.
A carbon-13 nuclear magnetic resonance study of the 3'-terminus of 16S ribosomal RNA of Escherichia coli specifically labeled with carbon-13 in the methylgroups of the m6(2)Am6(2)A sequence. 总被引:2,自引:2,他引:0
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R Van Charldorp J J Verhoeven P H Van Knippenberg C A Haasnoot C W Hilbers 《Nucleic acids research》1982,10(14):4237-4245
30S ribosomes were isolated from a kasugamycin resistant mutant of E. coli that lacks methylgroups on two adjacent adenines in 16S ribosomal RNA. These ribosomes were methylated in vitro with a purified methylating enzyme and 5-S-adenosyl-(13C-methyl)-L-methionine chloride ((13C-methyl)-SAM) as methyldonor. After in situ cleavage of the 16S ribosomal RNA by the bacteriocin cloacin DF13, the 49 nucleotide fragment from the 3'-end of the RNA was isolated. The carbon-13 nuclear magnetic resonance spectra of the fragment at various temperatures were compared with those of 6-N-dimethyladenosine (m6(2)A) and 6-N-dimethyladenylyl-(3' leads to 5')-6-N-dimethyladenosine (m6(2)Am6(2)A). The data show that the two methylated adenines, which are part of a four membered hairpin loop, show a strong tendency to be stacked in analogy to the dinucleotide m6(2)Am6(2). 相似文献
3.
Alterations in the ribosomes of sucrose-dependent spectinomycin-resistant (Sucd-Spcr) mutants of Escherichia coli were studied. Subunit exchange experiments showed that 30S subunits were responsible for the resistance of ribosomes to spectinomycin in all Sucd-Spcr mutants tested. Proteins of 30S ribosomes were analyzed by carboxymethyl cellulose column chromatography based on their elution positions. Mutants YM22 and YM93 had an altered 30S ribosomal protein component, S5, and mutant YM50 had an altered protein, S4. Although a shift of elution position was not detected for all the 30S ribosomal proteins from mutant YM101, the amount of protein S3 was appreciably lowered in the isolated 30S subunits. A partial reconstitution experiment with protein S3 prepared from both the wild-type strain and YM101 revealed that the mutant had altered protein S3 which is responsible for the spectinomycin resistance. These alterations in 30S subunits are discussed in relation to the interaction between ribosomes and the cytoplasmic membrane. 相似文献
4.
The association of ribosomal subparticles induced by several associating agents has been studied under different conditions. The following observations were made: 1. Spermidine was able to produce the association of subunits, and the concentration and temperature curves of this reaction were similar to those obtained with association factor. The product formed in the latter case was more stable. 2. The association at low Mg2+ concentration was higher with association factor than with polyamines. 3. The temperature-dependent binding of spermidine to 30-S subunits formed an active complex, which was converted into the 30S-50S couples by the addition of 50-S subparticles at low temperature. A similar behaviour has been previously shown for the complete association factor and its low molecular weight fraction. 4. The same unstable form of 30S-50S couples has been obtained either with spermidine or with the low molecular weight component (AFII) of the association factor. In both cases the protein fraction AFI was able to complete the reaction by stabilizing the subunit couple. 5. After glutaraldehyde fixation the products of the reactions with spermidine or association factor behaved in a similar way when they were submitted to long sucrose-gradient centrifugations. 6. The analysis of association factor preparations has shown that they contain spermidine as well as spermine. The polyamine levels in association factor could account for part of the total associating activity. 相似文献
5.
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 tRNAPhe (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. 相似文献
6.
Turbidimetric and potentiometric studies of ribosomal subunits from an erythromycin resistant mutant of Escherichia coli.
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J S Kliber G Hui Bon Hoa M Grunberg-Manago D Pardo R Rosset 《Nucleic acids research》1978,5(1):271-284
Turbidimetric and potentiometric techniques were applied to the analysis of an EryR mutant. Results show that in the mutant, the 30S subunits are drastically altered, as indicated by a higher Mg2+ requirement for subunit association and by an important difference in the titratable groups. Replacement of parental 50S by mutant 50S subunits does not decrease the association capacity with 30S parental subunits, but a structural difference is detected in the mutant 50S with potentiometric measurements. The mutation results in decreased ribosomal in vitro activities at 22 degrees C including lowered polyphenylalanine synthesis, drastic altered initiation step and the loss of erythromycin binding to the ribosomes. The results extend previous observation of a gene eryC part in the maturation of both subunits. 相似文献
7.
The behavior of E. coli ribosomes during sedimentation on sucrose gradients is predicted under a variety of conditions by computer simulations. Since numerous recent kinetic studies indicate equilibration in times short compared to the time of sedimentation, these simulations assume that the system attains local reaction equilibrium at every point in the gradient at all times. For any type of homogeneous equilibrating ribosome population, governed by a single formation constant at one atmosphere pressure for 70S couples, no more than two clearly defined zones will be resolved, although the presence of large dissociating effects due to pressure gradients in high speed experiments will spread the subunit zone. Normally the pattern will consist of a 30S zone and a so-called “70S” zone, which is in reality a mixture of 70S couples and 30S and 50S subunits in local equilibrium. The greater the dissociation into subunits, the more the “70S” zone will be slowed below the nominal rate of 70 Svedberg units. If ribosomes have been collected from the “70S” zone in several successive cycles of purification, the repeated deletion of resolved 30S subunits can result in a preparation with so large a molar excess of 50S subunits that the ensuing sucrose density gradient sedimentation pattern may exhibit a “70S” zone followed by zone of 50S subunits, insteadof a zone of 30S subunits. Our most important conclusion is that whenever a well-resolved 50S zone is present in a sucrose density gradient sedimentation experiment on E. coli ribosomes, in addition to a 30S and a “70S” zone, under conditions where ribosomes and subunits should be in reversible equilibrium, the preparation must be microheterogeneous, containing a mixture of “tight” and “loose” couples. Moreover in such cases the content of large subunits in the 50S zone must be derived entirely from “loose” couples whereas the 30S zone must contain small subunits derived from both “tight” and “loose” couples. Sedimentation patterns predicted for various mixtures of “tight” and “loose” couples display all the major characteristics of published experimental patterns for E. coli ribosomes, including the partial or complete resolution into three zones, depending on rotor velocity and level of Mg2+. 相似文献
8.
The ribosomal protein S1 was modified by reductive methylation of some of its lysyl ammonium groups (S1). With 6 out of 30 groups methylated the protein lost its capacity to form stable complexes with polyuridylate. Addition of excess polyuridylate inhibited the methylation of the lysyl groups. In equilibrium dialysis experiments it was shown that the binding constant between S1 and U15 was lowered 10-fold as compared to the native protein. The pH-dependence of the complex formation between S1 and U15 confirms a participation of the lysyl residues. When S1 depleted 30-S ribosomes were reconstituted with methylated S1 these ribosomes were inactive in the poly(U) stimulated Phe-tRNA binding. The data are discussed with respect to a grid-like interaction between the lysyl groups of the protein and the phosphodiester bonds of the polynucleotide as a molecular basis of protein nucleic acid interaction. 相似文献
9.
Initiation Factor 1 (IF1) is required for the initiation of translation in Escherichia coli. However, the precise function of IF1 remains unknown. Current evidence suggests that IF1 is an RNA-binding protein that sits in the A site of the decoding region of 16 S rRNA. IF1 binding to 30 S subunits changes the reactivity of nucleotides in the A site to chemical probes. The N1 position of A1408 is enhanced, while the N1 positions of A1492 and A1493 are protected from reactivity with dimethyl sulfate (DMS). The N1-N2 positions of G530 are also protected from reactivity with kethoxal. Quantitative footprinting experiments show that the dissociation constant for IF1 binding to the 30 S subunit is 0.9 microM and that IF1 also alters the reactivity of a subset of Class III sites that are protected by tRNA, 50 S subunits, or aminoglycoside antibiotics. IF1 enhances the reactivity of the N1 position of A1413, A908, and A909 to DMS and the N1-N2 positions of G1487 to kethoxal. To characterize this RNA-protein interaction, several ribosomal mutants in the decoding region RNA were created, and IF1 binding to wild-type and mutant 30 S subunits was monitored by chemical modification and primer extension with allele-specific primers. The mutations C1407U, A1408G, A1492G, or A1493G disrupt IF1 binding to 30 S subunits, whereas the mutations G530A, U1406A, U1406G, G1491U, U1495A, U1495C, or U1495G had little effect on IF1 binding. Disruption of IF1 binding correlates with the deleterious phenotypic effects of certain mutations. IF1 binding to the A site of the 30 S subunit may modulate subunit association and the fidelity of tRNA selection in the P site through conformational changes in the 16 S rRNA. 相似文献
10.
The kinetics of association of Escherichia coli 30S and 50S ribosomal subunits have been carried out as a function of temperature after a magnesium jump from 1.5 to 3 mM. Turbidimetric recordings combined with a stopped-flow apparatus were used to follow the kinetics. The data show that the rates of formation and dissociation of the 70S particles at 3 mM Mg2+ and +25 degrees C were, respectively: k2 = 10(5) M-1 s-1, k1 = 4,5 X 10(-3) s-1; lowering the temperature decreases the rate constants with activation energies equal to E2 = 7.5 kcal/mol, E1 = 26.5 kcal/mol and enhances the association equilibrium towards the 70S species with an enthalpy change (delta H degrees assoc = -19.9 kcal/mol) dominant over the entropy change (delta S degrees assoc = -33 cal/(deg mol)). These thermodynamic parameters were compared to those obtained from studies on the interactions of codon-anticodon in yeast phenylalanine transfer RNA as well as of ribooligonucleotides. The kinetic and thermodynamic data are shown to be consistent with 16S-23S RNA interaction. 相似文献
11.
Identification of a region of Escherichia coli ribosomal protein L2 required for the assembly of L16 into the 50 S ribosomal subunit 总被引:3,自引:0,他引:3
In vitro mutagenesis of rplB was used to generate changes in a conserved region of Escherichia coli ribosomal protein L2 between Gly221 and His231. Mutants were selected by temperature sensitivity using an inducible expression system. A mutant L2 protein with the deletion of Thr222 to Asp228 was readily distinguishable from wild-type L2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ribosomes from the strain overexpressing this mutant protein were characterized by sucrose density gradient centrifugation and protein composition. In addition to 30 S and 50 S ribosomal subunits, cell lysates contained a new component that sedimented at 40 S in 1 mM Mg2+ and at 48 S in 10 mM Mg2+. These particles contained mutant L2 protein exclusively, completely lacked L16, and had reduced amounts of L28, L33, and L34. They did not reassociate with 30 S ribosomal subunits and were inactive in polyphenylalanine synthesis. Other mutants in the same conserved region, including the substitution of His229 by Gln229, produced similar aberrant 50 S particles that sedimented at 40 S and failed to associate with 30 S subunits. 相似文献
12.
The central pseudoknot in 16S ribosomal RNA is needed for ribosome stability but is not essential for 30S initiation complex formation. 总被引:2,自引:1,他引:1
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To examine the function of the central pseudoknot in 16S rRNA, we have studied Escherichia coli 30S subunits with the A18 mutation in this structure element. Previously, this mutation, which changes the central base pair of helix 2, C18--G917, to an A18xG917 mismatch, was shown to inhibit translation in vivo and a defect in initiation was suggested. Here, we find that the mutant 30S particles are impaired in forming 70S tight couples and predominantly accumulate as free 30S subunits. Formation of a 30S initiation complex, as measured by toeprinting, was almost as efficient for mutant 30S subunits, derived from the tight couple fraction, as for the wild-type control. However, the A18 mutation has a profound effect on the overall stability of the subunit. The mutant ribosomes were inactivated by affinity chromatography and high salt treatment, due to easy loss of ribosomal proteins. Accordingly, the particles could be reactivated by partial in vitro reconstitution with 30S ribosomal proteins. Mutant 30S subunits from the free subunit fraction were already inactive upon isolation, but could also be reactivated by reconstitution. Apparently, the inactivity in initiation of these mutant 30S subunits is, at least in part, also due to the lack of essential ribosomal proteins. We conclude that disruption of helix 2 of the central pseudoknot by itself does not affect the formation of a 30S initiation complex. We suggest that the in vivo translational defect of the mutant ribosomes is caused by their inability to form 70S initiation complexes. 相似文献
13.
Lövgren JM Bylund GO Srivastava MK Lundberg LA Persson OP Wingsle G Wikström PM 《RNA (New York, N.Y.)》2004,10(11):1798-1812
The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes. A DeltarimM mutant is defective in 30S maturation and accumulates 17S rRNA. To study the interaction of RimM with the 30S and its involvement in 30S maturation, RimM amino acid substitution mutants were constructed. A mutant RimM (RimM-YY-->AA), containing alanine substitutions for two adjacent tyrosines within the PRC beta-barrel domain, showed a reduced binding to 30S and an accumulation of 17S rRNA compared to wild-type RimM. The (RimM-YY-->AA) and DeltarimM mutants had significantly lower amounts of polysomes and also reduced levels of 30S relative to 50S compared to a wild-type strain. A mutation in rpsS, which encodes r-protein S19, suppressed the polysome- and 16S rRNA processing deficiencies of the RimM-YY-->AA but not that of the DeltarimM mutant. A mutation in rpsM, which encodes r-protein S13, suppressed the polysome deficiency of both rimM mutants. Suppressor mutations, found in either helices 31 or 33b of 16S rRNA, improved growth of both the RimM-YY-->AA and DeltarimM mutants. However, they suppressed the 16S rRNA processing deficiency of the RimM-YY-->AA mutant more efficiently than that of the DeltarimM mutant. Helices 31 and 33b are known to interact with S13 and S19, respectively, and S13 is known to interact with S19. A GST-RimM but not a GST-RimM(YY-->AA) protein bound strongly to S19 in 30S. Thus, RimM likely facilitates maturation of the region of the head of 30S that contains S13 and S19 as well as helices 31 and 33b. 相似文献
14.
Recent results suggest that, in Neurospora crassa, one small subunit mitochondrial ribosomal protein (S-4a, Mr 52,000) is synthesized intramitochondrially (Lambowitz et al., 1976). We now find that, when wild-type cells are treated with chloramphenicol to block mitochondrial protein synthesis, the maturation of 30 S mitochondrial ribosomal subunits is rapidly inhibited and there is an accumulation of CAP-30 S particles which sediment slightly behind mature small subunits. Electrophoretic analysis suggests that the CAP-30 S particles are deficient in several proteins including S-4a and that they are enriched in a precursor RNA species that is slightly longer than 19 S RNA. Chloramphenicol also appears to inhibit the maturation of 50 S ribosomal subunits, but this effect is much less pronounced. Continued incubation in chloramphenicol leads to a decrease in the proportion of total mitochondrial ribonucleoprotein present as monomers, possibly reflecting the depletion of competent subunits. After long-term (17 h) growth in chloramphenicol, mitochondrial ribosome profiles from wild-type cells show decreased ratios of small to large subunits, a feature which is also characteristic of the poky (mi-1) mutant. Pulse-labeling experiments combined with electrophoretic analysis show that the synthesis of mitochondrial ribosomal RNAs is relatively unaffected by chloramphenicol and that, despite the deficiency of small subunits, 19 S and 25 S RNA are present in normal ratios in whole mitochondria. By contrast, 19 S RNA in poky mitochondria is rapidly degraded leading to a decreased ratio of 19 S to 25 S RNA. The significance of these results with respect to the etiology of the poky mutation is discussed and a model of mitochondrial ribosome assembly that incorporates all available data is presented. 相似文献
15.
J M Moreno L Drskj?tersen J E Kristensen K K Mortensen H U Sperling-Petersen 《FEBS letters》1999,455(1-2):130-134
We have studied the interactions between the ribosome and the domains of Escherichia coli translation initiation factor 2, using an in vitro ribosomal binding assay with wild-type forms, N- and C-terminal truncated forms of IF2 as well as isolated structural domains. A deletion mutant of the factor consisting of the two N-terminal domains of IF2, binds to both 30S and 50S ribosomal subunits as well as to 70S ribosomes. Furthermore, a truncated form of IF2, lacking the two N-terminal domains, binds to 30S ribosomal subunits in the presence of IF1. In addition, this N-terminal deletion mutant IF2 possess a low but significant affinity for the 70S ribosome which is increased by addition of IF1. The isolated C-terminal domain of IF2 has no intrinsic affinity for the ribosome nor does the deletion of this domain from IF2 affect the ribosomal binding capability of IF2. We conclude that the N-terminus of IF2 is required for optimal interaction of the factor with both 30S and 50S ribosomal subunits. A structural model for the interaction of IF2 with the ribosome is presented. 相似文献
16.
30S and 50S subunits, in the presence of either 20 mM Mg2+ or 6 mM Mg2+ and 5mM spermidine plus 25 mM putrescine, were observed to completely associate to form 70S monosomes as monitored by sucrose gradient sedimentation. Subunits maintained under the above ionic conditions were compared with 30S and 50S particles at low (6 mM) magnesium concentration with respect to the reactivity of individual ribosomal proteins to lactoperoxidase-catalyzed iodination. Altered reactivity to enzymatic iodination of ribosomal proteins S4, S9, S10, S14, S17, S19, and S20 in the small subunit of ribosomal proteins, L2, L9, L11, L27, and L30 in the large subunit following incubation with high magnesium or magnesium and polyamines suggests that a conformation change in both subunits accompanies the formation of 70S monosomes. The results further demonstrate that the effect of Mg2+ on subunit conformation is mimicked when polyamines are substituted for magnesium necessary for subunit association. 相似文献
17.
Dissociation of E. Coli 70S ribosomes in the presence of 0.1 mM Mg++ yields partially inactivated 30S and 50S subunits. This inactivation can be avoided by dissociating the 70S ribosome in a medium containing 10 mM Mg++. 400 mM Na+. Comparison of the active and inactive forms of the 30S and 50S subunits has led to the following conclusions: 1) The two forms possess identical (50S subunits) or very similar (30S subunits) hydrodynamic properties. No differences in their morphologies is detectable by electron microscopy. 2) They possess the same protein compositions except for the presence of a larger amount of protein S1 in the inactive than in the active form of the 30S subunit. 3) They differ significantly in functional properties: more efficient association of the active than of the inactive forms with the complementary subunit; extensive dimerization of inactive 30S subunits in the presence of 10 mM Mg++; no dimerization of active 30S subunits under the same conditions; six-fold higher peptidyl transferase activity of active as compared to inactive 50S subunits. 相似文献
18.
The binding of 6-demethylchlortetracycline to 70S, 50S and 30S ribosomal particles: a quantitative study by fluorescence anisotropy 总被引:3,自引:0,他引:3
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The binding of demeclocycline (6-demethylchlortetracycline) to ribosomes and ribosomal subunits from Escherichia coli was investigated by using the fluorescence anisotropy of the antibiotic to determine the extent of binding. Binding data obtained from 70S and 30S particles differed fundamentally from those obtained from 50S subunits: the first two showed a strong, specific interaction while the third did not. In addition, all three particles possessed weak, unspecific binding sites. Computer-aided least-squares analysis of the data yielded the following numbers of sites and equilibrium constants: for 30S, n1 = 1, K1 = 2.2 X 10(6) M-1, n2 K2 = 0.029 X 10(6) M-1; for 50S, n1 = 0, n2 K2 = 0.035 X 10(6) M-1; for 70S, n1 = 1, K1 = 3.2 X 10(6) M-1, n2 K2 = 0.082 X 10(6) M-1. These data resolve current disagreement in the literature and are a prerequisite for quantitative studies of the mechanism of inhibition by tetracycline of protein biosynthesis. 相似文献
19.
Wheat germ cytoplasmic ribosomes. Structure of ribosomal subunits and localization of N6,N6-dimethyladenosine by immunoelectron microscopy 总被引:1,自引:0,他引:1
Cytoplasmic ribosomes have been isolated from wheat germ, and the structure of ribosomal subunits has been examined by electron microscopy of negatively stained preparations. Small (40 S) subunits show structural features generally regarded as characteristic of eukaryotic particles, while large (60 S) subunits show shapes that are equally well described by models of prokaryotic 50 S particles. Small subunit 18 S RNA contains 2 residues of N6,N6-dimethyladenosine 19 and 20 residues from the 3'-end (Hagenbüchle, O., Santer, M., Steitz, J. A., and Mans, R. J. (1978) Cell 13, 551-563). Nucleoside analysis by high performance liquid chromatography shows no other residues of this component in the RNA. Anti-dimethyladenosine immunoglobulins were reacted with wheat germ 40 S subunits, and the resulting complexes were studied by electron microscopy in order to localize the nucleoside. In about 90% of the complexes observed, antibody-subunit contact was consistent with a single binding site. We place the dimethyladenosine residues at or near the end of the platform of the 40 S particle in a position nearly equivalent to that previously identified in prokaryotic and chloroplast subunits (Trempe, M. R., and Glitz, D. G. (1981) J. Biol. Chem. 256, 11873-11879). 相似文献
20.
Xaplanteri MA Papadopoulos G Leontiadou F Choli-Papadopoulou T Kalpaxis DL 《Journal of molecular biology》2007,369(2):489-497
In the crystal structure of the 30S ribosomal subunit from Thermus thermophilus, cysteine 24 of ribosomal protein S14 (TthS14) occupies the first position in a CXXC-X12-CXXC motif that coordinates a zinc ion. The structural and functional importance of cysteine 24, which is widely conserved from bacteria to humans, was studied by its replacement with serine and by incorporating the resulting mutant into Escherichia coli ribosomes. The capability of such modified ribosomes in binding tRNA at the P and A-sites was equal to that obtained with ribosomes incorporating wild-type TthS14. In fact, both chimeric ribosomal species exhibited 20% lower tRNA affinity compared with native E. coli ribosomes. In addition, replacement of the native E. coli S14 by wild-type, and particularly by mutant TthS14, resulted in reduced capability of the 30S subunit for association with 50S subunits. Nevertheless, ribosomes from transformed cells sedimented normally and had a full complement of proteins. Unexpectedly, the peptidyl transferase activity in the chimeric ribosomes bearing mutant TthS14 was much lower than that measured in ribosomes incorporating wild-type TthS14. The catalytic center of the ribosome is located within the 50S subunit and, therefore, it is unlikely to be directly affected by changes in the structure of S14. More probably, the perturbing effects of S14 mutation on the catalytic center seem to be propagated by adjacent intersubunit bridges or the P-site tRNA molecule, resulting in weak donor-substrate reactivity. This hypothesis was verified by molecular dynamics simulation analysis. 相似文献