The use of azidoarylimidoesters in RNA-protein cross-linking studies with Escherichia coli ribosomes

A series of related hetero-bifunctional RNA-protein cross-linking reagents has been prepared, carrying an imidoester or N-hydroxysuccinimide ester function at one end of the molecule, and a phenylazido function at the other. These compounds have been applied to RNA-protein cross-linking studies with ribosomal subunits, and one of them, p-azido-phenylacetic imidoester, has proved to be a particularly useful reagent for this purpose. The reagent first reacts specifically with protein amino groups, and subsequent photolysis of the azide group leads to cross-linking to the RNA in yields of up to 8% of the total protein. The whole reaction takes place under very mild conditions in aqueous solution.The individual proteins concerned in the cross-links have been identified by two-dimensional gel electrophoresis, and the existence of a covalent cross-link was confirmed by the isolation by two different methods of protein-oligonucleotide complexes carrying a ³²P label. Although most of the ribosomal proteins could be cross-linked to their corresponding ribosomal RNA within the individual subunits, RNA-protein cross-links at the ribosomal subunit interface were only detectable in vanishingly small amounts.The advantages of this type of genuine hetero-bifunctional reagent in RNA-protein cross-linking studies are discussed.     

Composition of the Escherichia coli 70S ribosomal interface: a cross-linking study

70S tight-couple ribosomes from Escherichia coli were cross-linked by using the bifunctional reagent phenyl-diglyoxal (PDG). The reaction was stopped after 4-h incubation while still in the linear range. In comparison with untreated ribosomes, 30% of those treated with PDG were shown, by sucrose gradient experiments, not to be separable into their subunits, but remained as 70S particles. There was no detectable change in the structure of the reacted particles when their sedimentation behavior was compared with that of native 70S controls. When the cross-linking reaction was performed in the presence of tRNAPhe and poly(U), the reacted ribosomes retained 40-50% of their tRNA binding activity. The reaction leads predominantly to the formation of RNA-protein cross-links but protein--protein as well as RNA-RNA cross-links could also be detected. Cross-linked material was extracted, and the individual RNAs were separated into 23S, 16S, and 5S RNAs. Proteins were identified electrophoretically after reversal of the RNA-protein cross-links. Proteins were found to be cross-linked to RNAs within and across the ribosomal subunits; the latter are considered to be close to or at the 70S subunit interface. The arrangement of RNA and protein at the subunit interface is discussed.     

The use of 2-iminothiolane as an RNA-protein cross-linking agent in Escherichia coli ribosomes, and the localisation on 23S RNA of sites cross-linked to proteins L4, L6, L21, L23, L27 and L29.

When E. coli ribosomal subunits are reacted with 2-iminothiolane and then subjected to a mild ultraviolet irradiation, an RNA-protein cross-linking reaction occurs. About 5% of the total protein in each subunit becomes cross-linked to the RNA, and a specific sub-set of proteins is involved in the reaction. In the case of the 50S subunit, the sites of cross-linking to the 23S RNA have been determined for six of these proteins: protein L4 is cross-linked within an oligonucleotide comprising positions 613-617 in the 23S sequence, L6 within positions 2473-2481, L21 within positions 540-548, L23 within positions 137-141, L27 within positions 2332-2337 and L29 within positions 99-107.     

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.     

The three-dimensional structure and function of Escherichia coli ribosomal RNA, as studied by cross-linking techniques

A large number of intra-RNA and RNA-protein cross-link sites have been localized within the 23S RNA from E. coli 50 S ribosomal subunits. These sites, together with other data, are sufficient to constrain the secondary structure of the 23 S molecule into a compact three-dimensional shape. Some of the features of this structure are discussed, in particular, those relating to the orientation of tRNA on the 50 S subunit as studied by site-directed cross-linking techniques. A corresponding model for the 16S RNA within the 30 S subunit has already been described, and here a site-directed cross-linking approach is being used to determine the path followed through the subunit by messenger 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 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.     

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).     

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.     

Evidence for RNA-RNA cross-link formation in Escherichia coli ribosomes.

Evidence is presented in three separate cases for the formation of RNA-RNA cross-links in intact E. coli ribosomes and ribosomal subunits. The first case is a cross-link between the 18S and 13S regions of the 23S RNA, induced by ultraviolet irradiation. The second is a cross-link at the subunit interface, generated by the bifunctional reagent bis-(2-chloroethyl)-amine. The third example is a cross-link between sections O'-D and P-A of the 16S RNA, induced as in the first case by ultraviolet irradiation. The RNA-RNA cross-links can be identified as such, despite the complications introduced by concomitant RNA-protein cross-linking reactions. The experiments represent a first attempt to introduce RNA-RNA cross-linking into studies of the topographical organization of the RNA within the ribosome.     

Chemical cross-linking of protein to RNA within intact ribosomal subunits from Escherichia coli.

Summary Bifunctional reagents, namely bis-(2-chloroethyl)-amine (nitrogen mustard) and activated esters of 3-(2-bromo-3-oxobutane-1-sulphonyl)-propionic acid (bromo-ketone reagent) are used to cross-link protein to RNA within intact ribosomal subunits. The cross-linked proteins are analysed on two different two-dimensional gel electrophoresis systems, and the existence of a stable cross-linkage is demonstrated by isolating cross-linked protein-oligonucleotide complexes from subunits containing ³²P-labelled RNA. Proteins S3, S4, S5, S9/S11 and S13 from the 30S subunit, and proteins L1 and L2 from the 50S subunit were cross-linked to RNA by the nitrogen mustard, together with a number of other so far unresolved proteins. Correspondingly S3, S4, S7, S9/S11, and L2 were cross-linked by the bromoketone reagent, although in lower yield. The reagents should prove useful for topographical studies on ribosomal subunits, and arguments are presented favouring the use of non-cleavable and relatively non-specific RNA-protein cross-linking reagents for such studies.     

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.     

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.     

Cross-linking between 16S ribosomal RNA and protein S4 in Escherichia coli ribosomal 30S subunits effected by treatment with bisulfite/hydrazine and bromopyruvate

Cytosine in nucleic acids can be modified by treatment with a mixture of bisulfite and hydrazine. The reaction is specific for single-stranded regions of nucleic acids and the product is N4-aminocytosine. Bromopyruvate has been used for alkylation of protein SH groups and through its 2-oxo group it can form a hydrazone with N4-aminocytosine. Escherichia coli ribosomal 30S subunits were treated with 1 M sodium bisulfite + 2 M hydrazine in the presence of 10 mM MgCl2 at pH 7.0 and 37 degrees C for 30 min. By this treatment, 2.4 cytosine residues/molecule 16S rRNA were derivatized into N4-aminocytosines. 35S-labeled 30S subunits were modified in this way and then treated with 10 mM bromopyruvate at pH 8.0 and 37 degrees C for 5 min. Analysis in sodium dodecyl sulfate/sucrose density gradient centrifugation showed co-sedimentation of a part of the 35S radioactivity with the RNA. The co-sedimentation was dependent on both the bisulfite/hydrazine and the bromopyruvate treatments. The RNA-protein complex was prepared from unlabeled 30S subunits. The protein portion was labeled with 125I, the RNA portion was digested with nucleases, and then the hydrazone linkage between the protein and oligonucleotides was cleaved by treatment with 0.2 M HCl. The oligonucleotides formed were removed by dialysis and the protein was identified as S4 by two-dimensional electrophoresis and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results indicate that the cysteinyl residue of protein S4 at position 31 from the N-terminus is located close to a cytosine residue which is non-base-paired and easily accessible by the externally present bisulfite/hydrazine reagent.     

Specific interaction of one subunit of eukaryotic initiation factor eIF-3 with 18S ribosomal RNA within the binary complex, eIF-3 small ribosomal subunit, as shown by cross-linking experiments.

Initiation factor eIF-3 from rat liver forms a binary complex with the small ribosomal subunit. Within this complex, 18S ribosomal RNA can be cross-linked to the 66 000 dalton subunit of eIF-3 by treating the complex with a short bifunctional reagent, diepoxybutane, with a distance of 4A between the reactive groups. In binary complexes containing eIF-3 premodified with the heterobifunctional reagent, methyl-p-azido-benzoylaminoacetimidate (10A), the 66 000 dalton subunit of eIF-3 became covalently bound to 18S rRNA after irradiation of the complex with ultraviolet light. The involvement of only one of the eight eIF-3 subunits in the formation of the covalent RNA-protein complexes indicates a highly specific interaction between 18S rRNA and eIF-3 at the attachment site of the factor on the 40S subunit.     

Photo-induced protein cross-linking to 5S RNA and 28-5.8S RNA within rat-liver 60S ribosomal subunits

Rat liver 60S ribosomal subunits were irradiated with 254-nm ultraviolet light (1.26 X 10(4) quanta/subunit), under conditions which preserved their functional activity. Cross-linked RNA-protein complexes were recovered after unreacted proteins had been removed by repeated acetic acid extractions. Proteins linked to the whole rRNA, to 5S RNA and to 28-5.8 S RNAs were identified by two-dimensional gel electrophoresis after RNA hydrolysis by ribonucleases T1 and A. Our results showed that numerous proteins interact with rRNAs (at least ten with 28-5.8 S RNA, eight with 5S RNA and among these three are common to both) and have been discussed in the light of all the available data.     

Radiochemical cross-linking between proteins and RNA within 70 S ribosomal particles from E. coli MRE600

In the absence of oxygen, gamma-irradiation produces covalent links between some ribosomal proteins and 16 S RNA to 23 S RNA, within 70 S ribosomes from E. coli MRE600. Under optimal conditions minimizing the structural modifications induced by radiations, in situ formed cross-links appear specific and reflect close RNA-protein contacts. In view of these results, the spatial organization of the 30 S, 50 S subunit interfaces is discussed. In addition, the gamma-irradiation technique reveals that subunit association induces modifications of some protein--RNA interactions.     

The alpha and gamma subunits of initiation factor eIF-2 can be cross-linked to 18S ribosomal RNA within the quaternary initiation complex, eIF-2.Met-tRNAf.GDPCP.small ribosomal subunit.

Initiation factor eIF-2 from rat liver was reacted with the hetero-bifunctional cross-linking reagents ABAI or APTPI without diminishing its ability to form the quaternary initiation complex with Met-tRNAf, GDPCP and the small ribosomal subunit. Upon irradiation with UV light, subunits alpha and gamma of eIF-2 became covalently linked to 18S ribosomal RNA. The subunits were identified electrophoretically after isolation of the covalent protein-rRNA complexes and subsequent degradation of the rRNA by nuclease and alkali treatments. The close proximity of the two factor subunits to sequences of ribosomal RNA within the quaternary complex could be confirmed in a second set of experiments using unmodified, 125I-labeled factor and diepoxybutane as cross-linking reagent.     

Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies.

We have investigated protein-rRNA cross-links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2-iminothiolane as cross-linking agents. We identified amino acids cross-linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross-linked to rRNA have been sequenced in which no direct cross-linked amino acid could be detected. The cross-linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross-linked peptides in ribosomal protein S3 shows a common primary sequence motif--the KH motif--directly involved in interaction with rRNA, and the cross-linked amino acid in ribosomal protein L36 lies within the zinc finger-like motif of this protein. The cross-linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three-dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein-antibiotic cross-linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.     

Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli

We have carried out an extensive protein-protein cross-linking study on the 50S ribosomal subunit of Escherichia coli using four different cross-linking reagents of varying length and specificity. For the unambiguous identification of the members of the cross-linked protein complexes, immunoblotting techniques using antisera specific for each individual ribosomal protein have been used, and for each cross-link, the cross-linking yield has been determined. With the smallest cross-linking reagent diepoxybutane (4 A), four cross-links have been identified, namely, L3-L19, L10-L11, L13-L21, and L14-L19. With the sulfhydryl-specific cross-linking reagent o-phenylenedimaleimide (5.2 A) and p-phenylenedimaleimide (12 A), the cross-links L2-L9, L3-L13, L3-L19, L9-L28, L13-L20, L14-L19, L16-L27, L17-L32, and L20-L21 were formed; in addition, the cross-link L23-L29 was exclusively found with the shorter o-phenylenedimaleimide. The cross-links obtained with dithiobis(succinimidyl propionate) (12 A) were L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L32, L19-L25, L20-L21, and L23-L34. The good agreement of the cross-links obtained with the different cross-linking reagents used in this study demonstrates the reliability of our cross-linking approach. Incorporation of our cross-linking results into the three-dimensional model of the 50S ribosomal subunit derived from immunoelectron microscopy yields the locations for 29 of the 33 proteins within the larger ribosomal subunit.