共查询到20条相似文献,搜索用时 31 毫秒
1.
2.
Paul L. Wollenzien Robert F. Murphy Charles R. Cantor Alain Expert-Bezançon Donal H. Hayes 《Journal of molecular biology》1985,184(1):67-80
Escherichia coli 16 S ribosomal RNA in reconstitution buffer has been photochemically crosslinked with aminomethyltrimethylpsoralen and chemically crosslinked with N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine. The positions of crosslinking have been detected by viewing the molecules in the electron microscope. DNA restriction fragments that contain psoralen mono-adducts were hybridized and crosslinked to the samples so that the orientations of the crosslinked molecules were seen directly. A two-dimensional histogram method has been used to classify the different types of looped crosslinked molecules. These methods allow the identification of 13 distinct types of loops in the photochemically crosslinked molecules and 31 distinct types of loops in the chemically crosslinked molecules. The psoralen experiments are a reinvestigation of some of our earlier results. Some of the crosslinks were previously reported in the incorrect orientation; with the corrected orientation, seven of the psoralen crosslinks can now be correlated with complementarities in the proposed secondary-structure models. However, there are still six other psoralen crosslinks that indicate additional contacts not found in the current models. The chemical crosslinks indicate pairs of single-stranded regions that must be close in the folded molecule. Many of these crosslinks occur between regions that are distant in the secondary structure; these crosslinks indicate part of the three-dimensional form of the folded molecule. 相似文献
3.
4.
5.
Base-pairing between distant regions of the Escherichia coli 16 S ribosomal RNA in solution. 总被引:2,自引:0,他引:2
Intramolecular crosslinks have been introduced into Escherichia coli 16 S ribosomal RNA in aqueous solution by irradiation in the presence of hydroxymethyl-trimethylpsoralen. When the crosslinked RNA is denatured and examined in the electron microscope the most striking features are a variety of large open loops. In addition, because the crosslinked molecules are shortened compared to non-crosslinked molecules, there are likely to be small hairpins not resolved by the present technique. The sizes and positions of 11 loop classes have been determined and oriented on the molecule. The frequency of occurrence of the different classes of loops depends on the crosslinking conditions. When the crosslinking is done in solutions containing Mg2+, at least four of the loop classes appear with greater frequency than they do in 3.5 mm-NaCl. The loops presumably arise because complementary sequences separated by long intervening regions are being crosslinked. These base-pairing interactions between residues distant in the primary structure appear to be prominent features of the secondary structure of rRNA in solution. 相似文献
6.
7.
Determination of the secondary structure of Drosophila melanogaster 5 S RNA by hydroxymethyltrimethylpsoralen crosslinking 总被引:7,自引:0,他引:7
The secondary structure of Drosophila melanogaster 5 S RNA was probed by 4′-hydroxymethyl-4,5′,8-trimethylpsoralen crosslinking. 5 S RNA was found to have a stable conformation in solution over a wide range of salt conditions. The structure was not affected by the intercalation of HMT. After HMT-crosslinks were formed, oligonucleotides containing the crosslinks were separated by gel electrophoresis and analyzed. Two different crosslinks were identified unambiguously. These crosslinks lead to a model very similar to that already proposed on the basis of evolutionary and enzymatic digestion data. The model proposed is in excellent agreement with all available data on eukaryotic 5 S RNA. 相似文献
8.
E. coli 30S ribosomes in the inactive conformation were irradiated at 390 nm in the presence of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT). This produces monoadducts in which AMT is attached to only one strand of an RNA duplex region. After unbound AMT was removed, some ribosomes were activated and then subjected to 360 nm irradiation; others were reirradiated without activation. Electron microscopic examination of 16S rRNA extracted from these two samples showed covalent rRNA loops indicative of rRNA crosslinks. The general pattern of loops closely matched that seen previously after direct psoralen crosslinking of 30S particles. However, the frequency of occurrence of one major class of loops formed by crosslinks between residues near position 500 and the 3' end was substantially lower for the activated samples, implying that the structure of the 16S rRNA in active and inactive 30S particles is different. 相似文献
9.
Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking. 总被引:2,自引:2,他引:0
下载免费PDF全文
![点击此处可从《RNA (New York, N.Y.)》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 A side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 A distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 A from the 4' position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit. 相似文献
10.
RNA-protein crosslinks were introduced into the 40S ribosomal subunits from Saccharomyces cerevisiae by mild UV treatment. Proteins crosslinked to the 18S rRNA molecule were separated from free proteins by repeated extraction of the treated subunits and centrifugation in glycerol gradients. After digestion with RNase to remove the RNA molecules, proteins were radio-labeled with 125I and identified by electrophoresis on two-dimensional polyacrylamide gels with carrier total 40S ribosomal proteins and autoradiography. Proteins S2, S7, S13, S14, S17/22/27, and S18 were linked to the 18S rRNA. A shorter period of irradiation resulted in crosslinking of S2 and S17/22/27 only. Several of these proteins were previously demonstrated to be present in ribosomal core particles or early assembled proteins. 相似文献
11.
Y. Gloria Chu Paul L. Wollenzien Charles R. Cantor 《Journal of biomolecular structure & dynamics》2013,31(3):647-656
Abstract E. coli 30S ribosomes in the inactive conformation were irradiated at 390 nm in the presence of 4′ -aminomethyl-4,5′,8-trimethylpsoralen (AMT). This produces monoadducts in which AMT is attached to only one strand of an RNA duplex region. After unbound AMT was removed, some ribosomes were activated and then subjected to 360 nm irradiation; others were reirradiated without activation. Electron microscopic examination of 16S rRNA extracted from these two samples showed covalent rRNA loops indicative of rRNA crosslinks. The general pattern of loops closely matched that seen previously after direct psoralen crosslinking of 30S particles. However, the frequency of occurrence of one major class of loops formed by crosslinks between residues near position 500 and the 3′ end was substantially lower for the activated samples, implying that the structure of the 16S rRNA in active and inactive 30S particles is different. 相似文献
12.
RNA-protein crosslinks were introduced into the 40S ribosomal subunits from Saccharomyces cerevisiae by mild UV treatment. Proteins crosslinked to the 18S rRNA molecule were separated from free proteins by repeated extraction of the treated subunits and centrifugation in glycerol gradients. After digestion with RNase to remove the RNA molecules, proteins were radio-labeled with 125I and identified by electrophoresis on two-dimensional polyacrylamide gels with carrier total 40S ribosomal proteins and autoradiography. Proteins S2, S7, S13, S14, S17/22/27, and S18 were linked to the 18S rRNA. A shorter period of irradiation resulted in crosslinking of S2 and S17/22/27 only. Several of these proteins were previously demonstrated to be present in ribosomal core particles or early assembled proteins. 相似文献
13.
Multiple crosslinks of proteins S7, S9, S13 to domains 3 and 4 of 16S RNA in the 30S particle.
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Functionally active 70S ribosomes containing 4-thiouracil in place of uracil (substitution level 2%) were prepared by an in vivo substitution method. RNA-protein crosslinks were introduced by 366 nm photoactivation of 4-thiouracil in the purified 30S subunits. Seven single stranded M13 probes containing rDNA inserts complementary to domains 3 and 4 of 16S RNA were constructed. These inserts approximately 100 nucleotides long starting at nucleotide 868 and ending at the 3' OH terminus were used to select contiguous RNA sections. The proteins covalently linked to each selected RNA section were identified by 2D gel electrophoresis. Proteins S7, S9, S13 were shown to be efficiently crosslinked to multiple sites belonging to both domains. 相似文献
14.
Electrophoresis in polyacrylamide gels containing both formamide and urea is a high-resolution technique for the analysis of crosslinked RNA species. Combined with a specific crosslinking agent like uv irradiation, it allows a rapid fingerprint of structural differences between RNA forms. The technique reveals significant differences in the pattern of uv crosslinking of free Escherichia coli 16 S ribosomal RNA compared with the RNA in active or inactive 30 S subunits. Ultraviolet photocrosslinks seen only in the 30 S particle are likely to be tertiary structure contacts. 相似文献
15.
16.
We have developed a gel electrophoresis technique for separating crosslinked RNA molecules into a series of discrete fractions. The gel used is polyacrylamide made in formamide and low salt designed to denature the RNA during electrophoresis. The mobility depends upon the position of crosslinking within each molecule, as demonstrated by electron microscopy of RNA eluted from the gel. In general, molecules with large loops electrophorese more slowly than molecules with small loops or uncrosslinked molecules. We have used this technique to re-examine the psoralen crosslinking pattern of Escherichia coli 16 S ribosomal RNA in inactivated 30 S ribosomal subunits. To determine the correct orientation of each type of crosslink, we have covalently attached DNA restriction fragments to the RNA so that the polarity of the RNA in the microscope would be known. Our previous major conclusions are confirmed: the predominant long-distance crosslink detected by gel electrophoresis involves a residue close to the 3′ end and a residue approximately 600 nucleotides away: the formamide/polyacrylamide gel is able to separate two closely spaced 1100-nucleotide interactions beginning close to the 3′ end, which were reported as one interaction before: and an interaction joining the ends is detected as before. However, one low-frequency crosslinked interaction, between positions 950 and 1400, and possibly another low-frequency interaction, between positions 550 and 870, are determined to be in the opposite polarity to that described previously. 相似文献
17.
New RNA-protein crosslinks in domains 1 and 2 of E. coli 30S ribosomal subunits obtained by means of an intrinsic photoaffinity probe. 总被引:4,自引:1,他引:3
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Functionally active 70S ribosomes containing 4-thiouridine (s4U) in place of uridine were prepared by a formerly described in vivo substitution method. Proteins were crosslinked to RNA by 366 nm photoactivation of s4U. We observe the systematic and characteristic formation of 30S dimers; they were eliminated for analysis of RNA-protein crosslinks. M13 probes containing rDNA inserts complementary to domains 1 and 2 of 16S RNA from the 5'end up to nucleotide 868 were used to select contiguous or overlapping RNA sections. The proteins covalently crosslinked to each RNA section were identified as S3, S4, S5, S7, S9, S18, S20 and S21. Several crosslinks are compatible with previously published sites for proteins S5, S18, S20 and S21; others for proteins S3, S4, S7, S9, S18 correspond necessarily to new sites. 相似文献
18.
19.
Discrete poly(A)-RNA species from rat liver mitochondria are fragments of 16S mitochondrial rRNA carrying its 5′-termini 总被引:2,自引:0,他引:2
A. M. Mazo A. G. Minchenko T. A. Avdonina G. G. Gause Jr. A. T. Pusyriov 《Molecular biology reports》1983,9(3):155-161
During electrophoresis in polyacrylamide gels containing 7M urea the major discrete components of preparations of rat liver mitochondrial poly(A)+ and poly(A)- RNA species have similar mobilities. Poly(A)- RNA components hybridize to the 16S rRNA gene of mtDNA. Analysis of 5'-terminal sequences of these components revealed their identity to the 5'-terminal sequence of 16S rRNA. These results show that poly(A)- RNA components are fragmentation products of 16S rRNA. Fragmentation occurs nonrandomly from the 3'-end of the original rRNA molecules and lead to formation of products with electrophoretic mobilities similar to those of poly(A)+ RNA components. 相似文献
20.
Identification of the modified nucleotides produced by covalent photoaddition of hydroxymethyltrimethylpsoralen to RNA. 总被引:8,自引:4,他引:4
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The reaction between RNA and 4'hydroxymethyl-4,5',8-trimethylpsoralen has been studied. Both natural RNA and synthetic RNAs were used. The base specificity of the reaction was found to be the same in natural RNA, homopolymers, and mononucleotides. Uridine was found to be the most reactive base in all cases. The kinetics of formation and reversal of monoadducts and crosslinks has been examined. Paper electrophoretic conditions are described which provide a separation of the monoaddition and crosslinked photoproducts. The relative and absolute amounts of monoadducts and crosslinks can be determined very accurately with this system. Paper electrophoresis provides good separations of the different photoproducts. The mobilities of the products are a simple function of their molecular weights and charges. 相似文献