Localisation of a series of intra-RNA cross-links in 16S RNA, induced by ultraviolet irradiation of Escherichia coli 30S ribosomal subunits

Mild ultraviolet irradiation of E. coli ribosomal subunits leads to the formation of a number of intra-RNA cross-links, in addition to the RNA-protein cross-links already reported (see refs. 9, 10). After partial ribonuclease digestion of the RNA from irradiated subunits, complexes containing these intra-RNA cross-links can be isolated on a two-dimensional gel electrophoresis system, and subjected to sequence analysis. A series of these cross-linked complexes is described, and the cross-linked RNA regions are compared with the secondary structures derived for 16S RNA (see refs. 6, 7).     

Investigation of the tertiary folding of Escherichia coli ribosomal RNA by intra-RNA cross-linking in vivo

"In vivo" cross-links were introduced into ribosomal RNA by direct ultraviolet irradiation of intact Escherichia coli cells, during growth in a 32P-labelled medium. Ribosomes were isolated from the irradiated cultures, dissociated into subunits and subjected to partial digestion with cobra venom nuclease. The intra-RNA cross-linked fragments were separated by two-dimensional gel electrophoresis and the sites of cross-linking determined, using our published methodology. A comparison with the data previously obtained by this procedure, after irradiation of isolated 30 S and 50 S subunits, showed that in the case of the 50 S subunit nine out of the ten previous cross-links in the 23 S RNA could be identified in the "in vivo" experiments, and correspondingly in the 30 S subunit five out of the six previous cross-links in the 16 S RNA were identified. Some new cross-links were found, as well as two cross-links in the 16 S RNA, which had hitherto only been observed after partial digestion of irradiated 30 S subunits with ribonuclease T1. The relevance of these data to the tertiary folding of the rRNA in situ is discussed, with particular reference to the work of other authors, in which "naked" RNA was used as the substrate for cross-linking and model-building studies.     

RNA-protein cross-linking in Escherichia coli 30S ribosomal subunits; determination of sites on 16S RNA that are cross-linked to proteins S3, S4, S5, S7, S8, S9, S11, S13, S19 and S21 by treatment with methyl p-azidophenyl acetimidate.

RNA-protein cross-links were introduced into E. coli 30S ribosomal subunits by treatment with methyl p-azidophenyl acetimidate. After partial nuclease digestion of the RNA moiety, a number of cross-linked RNA-protein complexes were isolated by a new three-step procedure. Protein and RNA analysis of the individual complexes gave the following results: Proteins S3, S4, S5 and S8 are cross-linked to the 5'-terminal tetranucleotide of 16S RNA. S5 is also cross-linked to the 16S RNA within an oligonucleotide encompassing positions 559-561. Proteins S11, S9, S19 and S7 are cross-linked to 16S RNA within oligonucleotides encompassing positions 702-705, 1130-1131, 1223-1231 and 1238-1240, respectively. Protein S13 is cross-linked to an oligonucleotide encompassing positions 1337-1338, and is also involved in an anomalous cross-link within positions 189-191. Protein S21 is cross-linked to the 3'-terminal dodecanucleotide of the 16S RNA.     

RNA-protein cross-linking in Escherichia coli 30S ribosomal subunits; determination of sites on 16S RNA that are cross-linked to proteins S3, S4, S7, S9, S10, S11, S17, S18 and S21 by treatment with bis-(2-chloroethyl)-methylamine.

RNA-protein cross-links were introduced into E. coli 30S ribosomal subunits by treatment with bis-(2-chloroethyl)-methylamine. After partial nuclease digestion of the RNA moiety, a number of cross-linked RNA-protein complexes were isolated by a new three-step procedure. Protein and RNA analysis of the individual complexes gave the following results: proteins S4 and S9 are cross-linked to the 16S RNA at positions 413 and 954, respectively. Proteins S11 and S21 are both cross-linked to the RNA within an oligonucleotide encompassing positions 693-697, and proteins S17, S10, S3 and S7 are cross-linked within oligonucleotides encompassing positions 278-280, 1139-1144, 1155-1158, and 1531-1542, respectively. A cross-link to protein S18 was found by a process of elimination to lie between positions 845 and 851.     

RNA-protein cross-linking in Escherichia coli ribosomal subunits: localization of sites on 16S RNA which are cross-linked to proteins S17 and S21 by treatment with 2-iminothiolane.

Treatment of E. coli ribosomal subunits with 2-iminothiolane coupled with mild ultraviolet irradiation leads to the formation of a large number of RNA-protein cross-links. In the case of the 30S subunit, a number of sites on 16S RNA that are cross-linked to proteins S7 and S8 by this procedure have already been identified (see ref. 6). Here, by using new or modified techniques for the partial digestion of the RNA and the subsequent isolation of the cross-linked RNA-protein complexes, three new iminothiolane cross-links have been localized: Protein S17 is cross-linked to the 16S RNA within an oligonucleotide encompassing positions 629-633, and protein S21 is cross-linked to two sites within oligonucleotides encompassing positions 723-724 and positions 1531-1542 (the 3'-end of the 16S RNA).     

The localization of multiple sites on 16S RNA which are cross-linked to proteins S7 and S8 in Escherichia coli 30S ribosomal subunits by treatment with 2-iminothiolane.

RNA-protein cross-links were introduced into E. coli 30S ribosomal subunits by reaction with 2-iminothiolane followed by a mild ultraviolet irradiation treatment. After removal of non-reacted protein and partial nuclease digestion of the cross-linked 16S RNA-protein moiety, a number of individual cross-linked complexes could be isolated and the sites of attachment of the proteins to the RNA determined. Protein S8 was cross-linked to the RNA at three different positions, within oligo-nucleotides encompassing positions 629-633, 651-654, and (tentatively) 593-597 in the 16S sequence. Protein S7 was cross-linked within two oligonucleotides encompassing positions 1238-1240, and 1377-1378. In addition, a site at position 723-724 was observed, cross-linked to protein S19, S20 or S21.     

The three-dimensional folding of ribosomal RNA; localization of a series of intra-RNA cross-links in 23S RNA induced by treatment of Escherichia coli 50S ribosomal subunits with bis-(2-chloroethyl)-methylamine.

Intact 50S ribosomal subunits from E.coli were cross-linked with the symmetrical bifunctional reagent bis-(2-chloroethyl)-methylamine. After deproteinization, selected regions of the 23S RNA were excised by treatment with ribonuclease H in the presence of appropriate complementary decadeoxynucleotides, and screened for the presence of intra-RNA cross-links by two-dimensional gel electrophoresis. Individual isolated cross-linked RNA fragments were analysed by our established procedures. Sixteen intra-RNA cross-links were identified, three of which corresponded to those previously published. The thirteen 'new' cross-links were localized in the 23S RNA at positions 774-78 linked to 792-94, 876-79 linked to 899-900, 979-81 or 983-84 to 2029, 1715 to 1743-46, 1911-21 to 1964, 1933 to 1966, 2032 to 2054-55, 2112 to 2169-71, 2116-17 to 2163-67, 2128-32 to 2156-59, 2392-93 to 2422-23, 2737-38 to 2763-66, and 2791 to 2890. These results are discussed in the context of three-dimensional model-building studies with the 23S RNA, with particular reference to the environment of the 'active centre' of the 50S subunit.     

Investigation of the tertiary folding of Escherichia coli 16S RNA by in situ intra-RNA cross-linking within 30S ribosomal subunits.

Intra-RNA cross-links were introduced into E. coli 30S ribosomal subunits by mild ultraviolet irradiation. The subunits were partially digested with cobra venom nuclease, followed in some cases by a second partial digestion with ribonuclease H in the presence of the hexanucleotide d-(CTTCCC). The cross-linked RNA complexes were separated by two-dimensional gel electrophoresis and the sites of cross-linking analysed by our published procedures. Tertiary structural cross-links in the 16S RNA were identified between positions 31 and 48, between oligonucleotides 1090-1094 and 1161-1164, and between oligonucleotides 1125-1127 and 1280-1281. The first of these imposes a rigid constraint on the relative orientations of helices 3 and 4 of the 16S secondary structure. A further tertiary cross-link (which could not be precisely localised) was found between regions 1-72 and 1020-1095, and secondary structural cross-links were identified between positions 497 and 545-548, and positions 1238-1240 and 1298.     

RNA-protein cross-linking in Escherichia coli 50S ribosomal subunits; determination of sites on 23S RNA that are cross-linked to proteins L2, L4, L24 and L27 by treatment with 2-iminothiolane.

RNA-protein cross-links were introduced into E. coli 50S ribosomal subunits by treatment with 2-iminothiolane followed by mild ultraviolet irradiation. After partial digestion of the RNA, the cross-linked RNA-protein complexes were separated by our recently published three-step procedure. In cases where this separation was inadequate, a further purification step was introduced, involving affinity chromatography with antibodies to the ribosomal 50S proteins. Analysis of the isolated complexes enabled four new cross-link sites on the 23S RNA to be identified, as well as re-confirming several previously established sites. The new sites are as follows: Protein L2 is cross-linked within an oligonucleotide at positions 1818-1823 in the 23S RNA, protein L4 within positions 320-325, protein L24 within positions 99-107, and protein L27 within positions 2320-2323.     

Localisation of a series of intra-RNA cross-links in the secondary and tertiary structure of 23S RNA, induced by ultraviolet irradiation of Escherichia coli 50S ribosomal subunits.

Intra-RNA cross-links were introduced into E. coli 50S ribosomal subunits by mild ultraviolet irradiation. The subunits were partially digested with cobra venom nuclease, and the cross-linked RNA complexes were isolated by two-dimensional electrophoresis. Many of the complexes were submitted to a second partial digestion procedure. Oligonucleotide analysis of the RNA fragments obtained in this manner enabled cross-links between the following ribonuclease T1 oligonucleotides in the 23S RNA to be established: positions 292-296 and 339-350; 601-604 and 652-656; 1018-1022 and 1140-1149; 1433-1435 and 1556-1560; 1836-1839 and 1898-1903; 2832-2834 (tentative) and 2878-2885; 2849-2852 and 2865-2867 (tentative); 739-748 and 2609-2618; 571-577 and 2030-2032; 1777-1792 (tentative) and 2584-2588. The first seven of these cross-links lie within the secondary structure of the 23S RNA, whereas the last three are tertiary structural cross-links. The degree of precision of the individual determinations was variable, depending on the nucleotide sequence in the vicinity of the cross-link site concerned.     

Intra-RNA cross-linking in Escherichia coli 30S ribosomal subunits: selective isolation of cross-linked products by hybridization to specific cDNA fragments.

M13 clones were constructed with cDNA inserts corresponding to specific regions of E. coli ribosomal RNA. The DNA from the clones was immobilized by coupling to diazobenzyloxymethyl cellulose, and was used for the selective isolation by hybridization of cross-linked RNA complexes containing the complementary sequences. Immobilized DNA samples with inserts complementary to four different regions covering bases 735-1384 of the 16S RNA were hybridized with a mixture of 16S RNA fragments generated by partial digestion of 30S subunits that had been cross-linked by ultraviolet irradiation in vivo. After dehybridization, the individual RNA fragments and cross-linked complexes were separated by gel electrophoresis and analysed by our usual procedures. Nine cross-links are described; four of these are hitherto unobserved "secondary structural" cross-links, and one is a new "tertiary structural" cross-link between positions 243-247 and 891-894 of the 16S RNA.     

The topography of the 3''-terminal region of Escherichia coli 16S ribosomal RNA; an intra-RNA cross-linking study.

30S ribosomal subunits, 70S ribosomes or polysomes from E. coli were subjected to mild ultraviolet irradiation, and the 3'-terminal region of the 16S RNA was excised by 'addressed cleavage' using ribonuclease H in the presence of suitable complementary oligodeoxynucleotides. RNA fragments from this region containing intra-RNA cross-links were separated by two-dimensional gel electrophoresis and the cross-link sites identified by our standard procedures. Five new cross-links were found in the 30S subunit, which were localized at positions 1393-1401 linked to 1531-1532, 1393-1401 linked to 1506, 1393-1401 to 1502-1504, 1402-1403 to 1498-1501, and 1432 to 1465-69, respectively. In 70S ribosomes or polysomes the first four of these were absent, but instead two cross-links between the 1400-region and tRNA were observed. These results are discussed in the context of the tertiary folding of the 3'-terminal region of the 16S RNA and its known functional significance as part of the ribosomal decoding centre.     

Secondary structure of the large subunit ribosomal RNA from Escherichia coli, Zea mays chloroplast, and human and mouse mitochondrial ribosomes.

Short base-paired RNA fragments, and fragments containing intra-RNA cross-links, were isolated from E. coli 23S rRNA or 50S ribosomal subunits by two-dimensional gel electrophoresis. The interactions thus found were used as a first basis for constructing a secondary structure model of the 23S rRNA. Sequence comparison with the 23S rDNA from Z. mays chloroplasts, as well as with the 16S (large subunit) rDNA from human and mouse mitochondria, enabled the experimental model to be improved and extrapolated to give complete secondary structures of all four species. The structures are organized in well-defined domains, with over 450 compensating base changes between the two 23S species. Some ribosomal structural "'switches" were found, one involving 5S rRNA.     

The tertiary folding of Escherichia coli 16S RNA, as studied by in situ intra-RNA cross-linking of 30S ribosomal subunits with bis-(2-chloroethyl)-methylamine.

Intra-RNA cross-links were introduced into E. coli 30S ribosomal subunits by treatment with bis-(2-chloroethyl)methylamine. The subunits were partially digested with cobra venom nuclease, and the cross-linked complexes were separated by two-dimensional electrophoresis and analysed according to our published procedures. Tertiary structural cross-links in the 16S RNA were identified between nucleotides 31 and 306, and between the tetranucleotide 693-696 and nucleotides 794 or 799. Secondary structural cross-links, lying at the ends of double-helical regions, were found between nucleotides 46 and the trinucleotide 362-364, and between the dinucleotide 148-149 and nucleotide 174. Cross-links within double-helical elements were identified between the tetranucleotide 128-131 and nucleotide 232, between nucleotide 250 and the dinucleotide 274-275, and between nucleotides 1413 and 1486. Adenine as well as guanine residues were involved in the cross-links.     

Multiple crosslinks of proteins S7 and S9 to domains 3 and 4 of 16S ribosomal RNA in the Escherichia coli 30S particle

RNA-protein cross-links were introduced into Escherichia coli 30S subunits by treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. 16S rRNA, cross-linked to 30S ribosomal proteins, was isolated and hybridized with seven single-stranded bacteriophage M13-DNA probes. These probes, each carrying an inserted rDNA fragment, were used to select contiguous RNA sections covering domains 3 and 4 (starting at nucleotide 868 and ending at the 3'OH terminus) of the 16S rRNA. The proteins covalently linked to each selected RNA section were identified by two-dimensional polyacrylamide gel electrophoresis. Proteins S7 and S9 were shown to be efficiently cross-linked to multiple sites belonging to both domains.     

RNA-protein cross-linking in Eschericia coli 30S ribosomal subunits: a method for the direct analysis of the RNA regions involved in the cross-links.

A prerequisite for topographical studies on ribosomal subunits involving RNA-protein cross-linking is that the cross-linking sites on the RNA should be determined. Methodology is presented which offers a solution to this problem, using as a test system 30S subunits in which protein S7 has been cross-linked to the 16S RNA by ultraviolet irradiation. The method is based on a gel separation system in the presence of a non-ionic detergent. When a ribonucleoprotein fragment containing RNA-protein cross-links is applied to this system, non-cross-linked protein is removed, and simultaneously the cross-linked RNA-protein complex is separated from non-cross-linked RNA. Oligonucleotide analysis of the S7-RNA complex isolated in this manner showed it to consist of a region of RNA from sections P-A of the 16S RNA. A single characteristic oligonucleotide was absent from this region, and it was tentatively concluded that this missing oligonucleotide contains the actual site of cross-linking.     

Covalent cross-linking of poly(A) to Escherichia coli ribosomes, and localization of the cross-link site within the 16S RNA.

Poly(A) can be cross-linked to E. coli 70S ribosomes in the presence of tRNALys by mild ultraviolet irradiation. The cross-linking reaction is exclusively with the 30S subunit, and involves primarily the RNA moiety. Following a partial nuclease digestion, cross-linked complexes containing poly(A) and fragments of the 16S RNA were isolated by affinity chromatography on oligo(dT)-cellulose. The complexes were purified by gel electrophoresis and subjected to oligonucleotide analysis, which revealed a single cross-link site within positions 1394-1399 of the 16S RNA. The same pattern of cross-linking, at about one-fifth of the intensity, was observed in the absence of tRNALys. The cross-link site to poly(A), together with other sites in the 16S RNA that have been implicated in ribosomal function, is discussed in the framework of our recent model for the three-dimensional structure of 16S RNA; all of the functional sites are clustered together in two distinct groups in the model.     

Conformation of 4.5S RNA in the signal recognition particle and on the 30S ribosomal subunit

The signal recognition particle (SRP) from Escherichia coli consists of 4.5S RNA and protein Ffh. It is essential for targeting ribosomes that are translating integral membrane proteins to the translocation pore in the plasma membrane. Independently of Ffh, 4.5S RNA also interacts with elongation factor G (EF-G) and the 30S ribosomal subunit. Here we use a cross-linking approach to probe the conformation of 4.5S RNA in SRP and in the complex with the 30S ribosomal subunit and to map the binding site. The UV-activatable cross-linker p-azidophenacyl bromide (AzP) was attached to positions 1, 21, and 54 of wild-type or modified 4.5S RNA. In SRP, cross-links to Ffh were formed from AzP in all three positions in 4.5S RNA, indicating a strongly bent conformation in which the 5' end (position 1) and the tetraloop region (including position 54) of the molecule are close to one another and to Ffh. In ribosomal complexes of 4.5S RNA, AzP in both positions 1 and 54 formed cross-links to the 30S ribosomal subunit, independently of the presence of Ffh. The major cross-linking target on the ribosome was protein S7; minor cross-links were formed to S2, S18, and S21. There were no cross-links from 4.5S RNA to the 50S subunit, where the primary binding site of SRP is located close to the peptide exit. The functional role of 4.5S RNA binding to the 30S subunit is unclear, as the RNA had no effect on translation or tRNA translocation on the ribosome.     

Cross-links between ribosomal proteins of 30S subunits in 70S tight couples and in 30S subunits

Ribosome 70S tight couples and 30S subunits derived from them were modified with 2-iminothiolane under conditions where about two sulfhydryl groups per protein were added to the ribosomal particles. The 70S and 30S particles were not treated with elevated concentrations of NH4Cl, in contrast to those used in earlier studies. The modified particles were oxidized to promote disulfide bond formation. Proteins were extracted from the cross-linked particles by using conditions to preclude disulfide interchange. Disulfide-linked protein complexes were fractionated on the basis of charge by electrophoresis in polyacrylamide/urea gels at pH 5.5. The proteins from sequential slices of the urea gels were analyzed by two-dimensional diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Final identification of proteins in cross-linked complexes was made by radioiodination of the proteins, followed by two-dimensional polyacrylamide/urea gel electrophoresis. Attention was focused on cross-links between 30S proteins. We report the identification of 27 cross-linked dimers and 2 trimers of 30S proteins, all but one of which were found in both 70S ribosomes and free 30S subunits in similar yield. Seven of the cross-links, S3-S13, S13-S21, S14-S19, S7-S12, S9-S13, S11-S21, and S6-S18-S21, have not been reported previously when 2-iminothiolane was used. Cross-links S3-S13, S13-S21, S7-S12, S11-S21, and S6-S18-S21 are reported for the first time. The identification of the seven new cross-links is illustrated and discussed in detail. Ten of the dimers reported in the earlier studies of Sommer & Traut (1976) [Sommer, A., & Traut, R. R. (1976) J. Mol. Biol. 106, 995-1015], using 30S subunits treated with high salt concentrations, were not found in the experiments reported here.