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1.
Topography of 16 S RNA in 30 S subunits and 70 S ribosomes accessibility to cobra venom ribonuclease
A ribonuclease extracted from the venom of the cobra Naja oxiana, which shows an unusual specificity for double-stranded RNA regions, was used to obtain new insight on the topography of Escherichia coli ribosomal 16 S RNA in the 30 S subunit and in the 70 S couple. 32P-labeled 30 S subunits or reconstituted 70 S tight couples containing 32P-labeled 16 S RNA have been digested under progressively stronger conditions. The cleavage sites have been precisely localized and the chronology of the hydrolysis process studied.The enzyme cleaves the 16 S RNA within 30 S subunits at 21 different sites, which are not uniformly distributed along the molecule. These results provide valuable information on the 16 S RNA topography and evidence for secondary structure features.The binding of the 50 S subunit markedly reduces the rate of the 16 S RNA hydrolysis and provides protection for several cleavage sites. Four of them are clustered in the 3′-terminal 200 nucleotides of the molecule, one in the middle (at position 772) and one in the 5′ domain (at position 336). Our results provide further evidence that the 3′-terminal and central regions of the RNA chain are close to each other in the ribosome structure and lie at the interface of the two subunits. They also suggest that the 5′ domain is probably not involved exclusively in structure and assembly. 相似文献
2.
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
3.
Localization of the 3' end of Escherichia coli 16 S RNA by electron microscopy of antibody-labelled subunits. 总被引:3,自引:0,他引:3
I N Shatsky L V Mochalova M S Kojouharova A A Bogdanov V D Vasiliev 《Journal of molecular biology》1979,133(4):501-515
The 3′ end of 16 S RNA is localized on the 30 S subunit of Escherichia coli ribosomes by immune electron microscopy. It is located in the groove between the side “ledge” and the “head” of the subunit on the level of the ledge top. Thus, we have localized the 30 S subunit functional site which is believed to be responsible for binding of the specific messenger RNA sequence preceding the initiation codon. The localization of the 3′ end of 16 S RNA has been done by a new approach in immune electron microscopy. It is based on the covalent binding of low molecular weight ligands, containing the residue of phenyl-β-d-lactoside hapten, to certain points of RNA and the localization of the binding site of the antibody specific to this hapten by electron microscopy. The advantages of this approach in comparison with conventional methods of immune electron microscopy are discussed. 相似文献
4.
RNA--protein interactions in the ribosome. IV. Structure and properties of binding sites for proteins S4, S16/S17 and S20 in the 16S RNA 总被引:3,自引:0,他引:3
Proteins S4, S16/S17 and S20 of the 30 S ribosomal subunit of Escherichia coli+ associate with specific binding sites in the 16 S ribosomal RNA. A systematic investigation of the co-operative interactions that occur when two or more of these proteins simultaneously attach to the 16 S RNA indicate that their binding sites lie near to one another. The binding site for S4 has previously been located within a 550-nucleotide RNA fragment of approximately 9 S that arises from the 5′-terminal portion of the 16 S RNA upon limited hydrolysis with pancreatic ribonuclease. The 9 S RNA was unable to associate with S20 and S16/S17, however, either alone or in combination. A fragment of similar size and nucleotide sequence, termed the 9 S1 RNA, has been isolated following ribonuclease digestion of the complex of 16 S RNA with S20 and S16/S17. The 9 S1 RNA bound not only S4, but S20 and S16/S17 as well, although the fragment complex was stable only when both of the latter protein fractions were present together. Nonetheless, measurements of binding stoichiometry demonstrated the interactions to be specific under these conditions. A comparison of the 9 S and 9 S1 RNAs by electrophoresis in polyacrylamide gels containing urea revealed that the two fragments differ substantially in the number and distribution of hidden breaks. Contrary to expectation, the RNA in the ribonucleoprotein complex appeared to be more accessible to ribonuclease than the free 16 S RNA as judged by the smaller average length of the sub-fragments recovered from the 9 S1 RNA. These results suggest that the binding of S4, S16/S17 and S20 brings about a conformational alteration within the 5′ third of the 16 S RNA.To delineate further the portions of the RNA chain that interact with S4, S16/S17 and S20, specific fragments encompassing subsequences from the 5′ third of the 16 S RNA were sought. Two such fragments, designated 12 S-I and 12 S-II, were purified by polyacrylamide gel electrophoresis from partial T1 ribonuclease digests of the 16 S RNA. The two RNAs, which contain 290 and 210 nucleotides, respectively, are contiguous and together span the entire 5′-terminal 500 residues of the 16 S RNA molecule. When tested individually, neither 12 S-I nor 12 S-II bound S4, S16/S17 or S20. If heated together at 40 °C in the presence of Mg2+ ions, however, the two fragments together formed an 8 S complex which associated with S4 alone, with S16/S17 + S20 in combination, and with S4 + S16/S17 + S20 when incubated with an un fractionated mixture of 30 S subunit proteins. These results imply that each fragment contains part of the corresponding binding sites. 相似文献
5.
Fixation of colicin E3 to sensitive bacteria is followed, after a lag of 2 to 6 min, by the rapid degradation of all the RNA of the 30S ribosomal subunits, yielding a large 15.5S fragment and a smaller fragment, containing the 3′-terminal end of the 16S RNA. The small RNA fragment which was estimated to consist of about 52 nucleotides, was retained within the 30S subunit in vivo and was subsequently recovered quantitatively without apparent further degradation. Kinetic studies of the cleavage of 16S RNA indicated that this is the primary and lethal effect of colicin E3 and the primary cause of the observed inhibition of protein synthesis in vivo. Small amounts of an RNA fragment, apparently identical in size to the small E3-fragment, were also isolated from 30S particles obtained from untreated bacteria. 相似文献
6.
In Escherichia coli strains carrying null mutations in either the dnaK or dnaJ genes, the late stages of 30S and 50S ribosomal subunit biogenesis are slowed down in a temperature‐dependent manner. At high temperature (44°C), 32S and 45S particles (precursors to 50S subunits) and 21S particles (precursors to 30S subunits) accumulate. The latter are shown by 3′5′ rapid amplification of cDNA ends analysis to contain unprocessed or partially processed 16S ribosomal RNA at the 5′ end, but the 3′ end was never processed. This implies that maturation of 16S ribosomal RNA starts at the 5′‐terminus, and that the 3′‐terminus is only trimmed at a later step. At normal temperatures (30°C?37°C), ribosome assembly in both mutants is not arrested but is significantly delayed, as shown by pulse‐chase analysis. Assembly defects are partially compensated by an overexpression of other heat‐shock proteins, which occurs in the absence of their negative regulator DnaK, or by a plasmid‐driven overexpression of GroES/GroEL, suggesting the involvement of a network of chaperones in ribosome biogenesis. 相似文献
7.
The sequence of the 3′-terminal 43 nucleotides of 16S ribosomal RNA from the cyanobacterium Synechococcus AN PCC 6301 has been determined. This sequence is almost identical with the 3′-terminal sequence of 16S ribosomal RNA from maize chloroplasts. The evolutionary implications of these observations are discussed. 相似文献
8.
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10.
The location and frequency of RNA crosslinks induced by photoreaction of hydroxymethyltrimethylpsoralen with 30 S Escherichia coli ribosomal subunits have been determined by electron microscopy. At least seven distinct crosslinks between regions distant in the 16 S rRNA primary structure are seen in the inactive conformation of the 30 S particle. All correspond to crosslinked features seen when the free 16 S rRNA is treated with hydroxymethyltrimethylpsoralen. The most frequently observed crosslink occurs between residues near one end of the molecule and residues about 600 nucleotides away to generate a loop of 570 bases. The size and orientation of this feature indicate it corresponds to the crosslinked feature located at the 3′ end of free 16 S rRNA.When active 30 S particles are crosslinked in 5 mm-Mg2+, six of the seven features seen in the inactive 30 S particle can still be detected. However, the frequency of several of the features, and particularly the 570-base loop feature, is dramatically decreased. This suggests that the long-range contacts that lead to these crosslinks are either absent or inaccessible in the active conformation. Crosslinking results in some loss of functional activities of the 30 S particle. This is consistent with the notion that the presence of the crosslink that generates the 570-base loop traps the subunit in an inactive form, which cannot associate with 50 S particles.The arrangement of the interacting regions crosslinked by hydroxymethyltrimethylpsoralen suggests that the RNA may be organized into three general domains. A striking feature of the Crosslinking pattern is that three of the seven products involve regions near the 3′ end of the 16 S rRNA. These serve to tie together large sections of rRNA. Thus structural changes at the 3′ end could, in principle, be felt through the entire 30 S particle. 相似文献
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14.
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
15.
Päivi Lehtovaara Hans Söderlund Sirkka Keränen Ralf F. Pettersson Leevi Kääriäinen 《Journal of molecular biology》1982,156(4):731-748
We have analyzed Semliki Forest virus defective interfering RNA molecules, generated by serial undiluted passaging of the virus in baby hamster kidney cells. The 42 S RNA genome (about 13 kb 2) has been greatly deleted to generate the DI RNAs, which are heterogeneous both in size (about 2 kb) and sequence content. The DI RNAs offer a system for exploring binding sites for RNA polymerase and encapsidation signals, which must have been conserved in them since they are replicated and packaged. In order to study the structural organization of DI RNAs, and to analyze which regions from the genome have been conserved, we have determined the nucleotide sequences of (1) a 2.3 kb long DI RNA molecule, DI309, (2) 3′-terminal sequences (each about 0.3 kb) of two other DI RNAs, and (3) the nucleotide sequence of 0.4 kb at the extreme 5′ end of the 42 S RNA genome.The DI309 molecule consists of a duplicated region with flanking unique terminal sequences. A 273-nucleotide sequence is present in four copies per molecule. The extreme 5′-terminal nucleotide sequence of the 42 S RNA genome is shown to contain domains that are conserved in the two DI RNAs of known structure: DI309, and the previously sequenced DI301 (Lehtovaara et al., 1981). Here we report which terminal genome sequences are conserved in the DI RNAs, and how they have been modified, rearranged or amplified. 相似文献
16.
The denaturation of ribosome and RNA by ethylene glycol (EG) has been studied in an attempt to further understand the conformation and stability of the ribosome. At high concentrations of EG, the ribosome, its subunits, and 16S RNA undergo drastic structural changes as shown by circular dichroism, ultraviolet absorption spectroscopy, and sedimentation velocity. Two separate conformational transitions were observed for the 30S subunit; one from 30 to 50% EG and another from 60 to 90% EG. This observation suggests the presence of two "domains" in the 30S subunit which differ in their stability. However, the 50S subunit undergoes a single sharp transition at 60 to 90% EG, consistent with the notion of a highly cooperative conformation. Association of the subunits stablizes part of the 30S subunit since the transition curve for the 70S ribosome does not exhibit significant change at the low EG concentration region as seen for the 30S subunit. Removal of proteins from the 30S subunit broadens the transition curve to lower EG concentrations and suggests the role of proteins in stabilizing the conformation of the 16S RNA. 相似文献
17.
Nucleotide sequence of the 3′ terminus of E. coli 16S ribosomal RNA 总被引:10,自引:0,他引:10
The 3′-terminal T1 oligonucleotide of E. coli 16S ribosomal RNA has been sequenced, using U2 and silkworm nucleases, and was found to be A-U-C-A-C-C-U-C-C-U-U-AOH. This result is discussed in view of previously reported conflicting sequences and with respect to suggested functional roles for this region of 16S RNA. 相似文献
18.
The 135-nucleotide-long “5 + S” RNA molecule found in Drosophila tissue culture cells after labelling at 37 °C has been identified as a precursor to 5 S RNA by pulse-chase experiments. The structure of the 15-nucleotide-long 3′-terminal sequence which differentiates this molecule from mature 5 S RNA has been determined. This ends in a stretch of U residues, suggestive of a polymerase termination signal. 相似文献
19.
Six 50 S ribosomal subunit proteins, each unable to interact independently with the 23 S RNA, were shown to associate specifically with ribonucleoprotein complexes consisting of intact 23 S RNA, or fragments derived from it, and one or more RNA-binding proteins. In particular, L21 and L22 depend for attachment upon L20 and L24, respectively; L5, L10 and L11 interact individually with complexes containing L2 and L16; and one or both proteins of the mixture are stimulated to bind in the presence of L1, L3, L6, L13 and L23. Moreover, L14 alone was found to interact with a fragment from the 3′ end of the 23 S RNA, even though it cannot bind to 23 S RNA. By correlating the data reported here with the findings of others, it has been possible to formulate a partial in vitro assembly map of the Escherichia coli 50 S subunit encompassing both the 5 S and 23 S RNAs as well as 21 of the 34 subunit proteins. 相似文献
20.
Colicin E3 is a ribonuclease that inactivates Escherichia coli ribosomes by cleaving the RNA of the small ribosomal subunit after nucleotide 1493. A series of oligodeoxynucleotides that complement 16 S RNA in the region of the colicin cleavage site has been synthesized, and their ability to form complexes with 30 S ribosomal subunits has been measured using a nitrocellulose filter-binding assay. The most efficiently bound probe, complementary to residues 1485-1496, was modified with antibody-recognizable derivatives at the 5'-end, the 3'-end, or both. Antibody-oligonucleotide-subunit complexes were then generated and examined by electron microscopy. Antibody binding was seen at the tip of the platform of the 30 S subunit. The complementary oligonucleotide and thus the site at which colcin E3 cleavage occurs is therefore in the same physical region as the 3'-end of the 16 S ribosomal RNA and its message-positioning "Shine-Dal-garno" sequence. 相似文献