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.     

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.     

Changes in subunit conformation and their reciprocal configuration in the transition from the pretranslocated to the posttranslocated state

RNA-protein contacts in pretranslocated and posttranslocated states of E. coli ribosomes have been determined by means of UV-induced cross-linking. In the two functional states as well as in free 70C ribosome, the same proteins are involved in RNA-protein intersubunit contacts, located in the region of L1 protuberance (left side of 70S ribosome). The transition from pre- to posttranslocated state is accompanied by disappearance of RNA-protein contacts in the region of L7/L12 stalk. This favours the locking-unlocking model of the translating ribosome.     

Changes of fluorescence spectra of 2'-deoxyadenosine in aqueous solution by radiation

Aqueous solution of 2'-deoxyadenosine (5 X 10(-4) M, buffered at pH 7.0) was irradiated with 60Co gamma-rays under N2, O2, N2O and t-BuOH-N2 atmospheres in order to compare with adenine radiolysis previously reported. By exposure to radiation, the fluorescence was found to increase more markedly than that from adenine under all conditions of radiolysis. This result indicates that not only base moiety but also sugar moiety participate in the formation of highly fluorescent products. In this 2'-deoxyadenosine radiolysis, both OH and e-aq take part in the formation of such products, but OH predominates over over e-aq when both active species are present, as observed in adenine radiolysis.     

Differences in 23 S rRNA-protein neighbourhood in Escherichia coli 70 S ribosomes and 70 S initiation complex. Probing by bifunctional Pt(II)-containing reagent

rRNA-protein cross-links in free E. coli 35S-labeled 70 S ribosomes and in the initiation complex 35S-labeled 70 S ribosome.AUGU6.fMet-tRNA(fMet) were studied with the aid of a new type of binuclear Pt(II) compound - dichlorotetra-ammine(1,6-hexamethylenediaminediplatinum++ +) dichloride. The use of this reagent allowed us to reveal differences in the rRNA-protein neighbourhood in free 70 S ribosomes and in the initiation complex. Proteins L3, L6, L23 and L25 were shown to cross-link to 23 S rRNA only in the initiation complex, whereas proteins L1, L13, L14, L16, L17, L18, L22, L28 and S1 did so in both free ribosomes and the complex. 16 S rRNA was found to be cross-linked preferentially to a single protein, S1, in both states of the ribosomes.     

Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P- and P/E-site tRNAPhe binding

The effects of P/P- and P/E-site tRNA(Phe) binding on the 16S rRNA structure in the Escherichia coli 70S ribosome were investigated using UV cross-linking. The identity and frequency of 16S rRNA intramolecular cross-links were determined in the presence of deacyl-tRNA(Phe) or N-acetyl-Phe-tRNA(Phe) using poly(U) or an mRNA analogue containing a single Phe codon. For N-acetyl-Phe-tRNA(Phe) with either poly(U) or the mRNA analogue, the frequency of an intramolecular cross-link C967 x C1400 in the 16S rRNA was decreased in proportion to the binding stoichiometry of the tRNA. A proportional effect was true also for deacyl-tRNA(Phe) with poly(U), but the decrease in the C967 x C1400 frequency was less than the tRNA binding stoichiometry with the mRNA analogue. The inhibition of the C967 x C1400 cross-link was similar in buffers with, or without, polyamines. The exclusive participation of C967 with C1400 in the cross-link was confirmed by RNA sequencing. One intermolecular cross-link, 16S rRNA (C1400) to tRNA(Phe)(U33), was made with either poly(U) or the mRNA analogue. These results indicate a limited structural change in the small subunit around C967 and C1400 during tRNA P-site binding sensitive to the type of mRNA that is used. The absence of the C967 x C1400 cross-link in 70S ribosome complexes with tRNA is consistent with the 30S and 70S crystal structures, which contain tRNA or tRNA analogues; the occurrence of the cross-link indicates an alternative arrangement in this region in empty ribosomes.     

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.     

A new nucleic acid-protein cross-linking reagent

Summary A new photoactivable reagent is described, which allows the formation of RNA-protein crosslinks via disulfide bridges in combination with mercaptobutyrimidate.The reconstituted L24 protein-23S RNA complex from the large subunit of E. coli ribosomes has been used as a model system for the cross-linking. The main advantages of the reagent are the absence of U.V. generated cross-links, since photoactivation is carried out at 360 nm, on one hand and the ease of cleavage of the cross-link by mild reduction (-mercaptoethanol) on the other.     

Ribosomal protein L7/L12 cross-links to proteins in separate regions of the 50 S ribosomal subunit of Escherichia coli

The 50 S ribosomal subunits from Escherichia coli were modified by reaction with 2-iminothiolane under conditions in which 65 sulfhydryl groups, about 2/protein, were added per subunit. Earlier work showed that protein L7/L12 was modified more extensively than the average but that nearly all 50 S proteins contained sulfhydryl groups. Mild oxidation led to the formation of disulfide protein-protein cross-links. These were fractionated by urea gel electrophoresis and then analyzed by diagonal gel electrophoresis. Cross-linked complexes containing two, three, and possibly four copies of L7/L12 were evident. Cross-links between L7/L12 and other ribosomal proteins were also formed. These proteins were identified as L5, L6, L10, L11, and, in lower yield, L9, L14, and L17. The yields of cross-links to L5, L6, L10, and L11 were comparable to the most abundant cross-links formed. Similar experiments were performed with 70 S ribosomes. Protein L7/L12 in 70 S ribosomes was cross-linked to proteins L6, L10, and L11. The strong L7/L12-L5 cross-link found in 50 S subunits was absent in 70 S ribosomes. No cross-links between 30 S proteins and L7/L12 were observed.     

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.     

Yields of hydroxyl radical and reducing species in tritiated water based on the radiolysis of tetranitromethane

Yields of the nitroform anion produced in tetranitromethane aqueous solutions including tritiated water with and without isopropanol were measured. Yields of .OH and reducing species (e-aq + .OH) were calculated from the yields of the nitroform anion. The G values obtained for them were 3.8 and 2.5, respectively. The former was higher and the latter was lower than those for 60Co gamma-rays. Their beta/gamma ratio was 1.36 and 0.76, respectively. The beta/gamma ratio, 1.36, for .OH may be related to r.b.e. values higher than the one reported for 3H beta-rays.     

Uridine 5'-phosphate photohydrate formation in irradiated Escherichia coli 30S ribosomes: photochemistry and relation to ribonucleic acid-protein cross-linking

Irradiation of aqueous buffered solutions of Escherichia coli 30S ribosomes with doses of 254-nm radiation greater than 10(19) quanta causes formation of uridine 5'-phosphate (UMP) photohydrates in ribosomal 16S RNA (rRNA). The number of molecules of UMP photohydrate formed at doses less than 2 x 10(20) quanta is linearly dependent on dose of absorbed 254-nm radiation. Maximum UMP photohydrate formation is dependent on initial ribosome concentration. When solutions containing 1 A260 unit of 30S ribosomes/mL were irradiated with greater than 2 x 10(20) quanta of 254-nm radiation, maximum photohydrate formation was equal to 47 residues/ribosome. Irradiation of solutions containing 2 A260 units/mL with greater than 7 x 10(20) quanta caused formation of 102 UMP photohydrates/ribosome. These values correspond to conversion of either 15 or 33%, respectively, of the total UMP content of 30S ribosome 16S rRNA to photohydrates. Target theory analysis of UMP photohydration in 30S ribosomes showed that UMP photohydrates are formed by single-hit kinetics from two photochemically distinct precursors. Of the total 16S rRNA UMP residues, 10% was included in the most rapidly (low dose) reacting fraction. The respective photohydration cross sections are 0.014 (low dose) and 0.0095 cm2/muEinstein (high dose) for ribosome solutions containing 2 A260 units/mL. UMP photohydrate content of irradiated 30S ribosomes was compared with that of previous data for the extent of RNA-protein cross-linking at equivalent doses of absorbed 254-nm radiation. This comparison showed that at least two UMP photohydrates form per RNA-protein cross-linking event in 30S ribosomes irradiated with a dose of 254-nm radiation (1.5 x 10(19) quanta), which causes cross-linking of only three ribosomal proteins to 16S rRNA.     

rRNA-protein neighbourhood in Escherichia coli 70 S ribosomes and 70 S initiation complex. Probing by bifunctional Pt(II)-containing reagent

The cleavable homobifunctional reagent dichloro[N,N,N',N'-tetrakis(2-aminoethyl)-1,6-hexamethylenediamminedi platinum (II)] dichloride was used for studying rRNA-protein cross-links in free 35S-labelled 70 S ribosomes and within initiation complex ribosome.AUGU6.fMet-tRNA(fMet). It was shown that the sets of proteins cross-linked to 16 S and 23 S rRNA in free 70 S ribosomes and in 70 S initiation complex do not differ significantly. The authors are the first to demonstrate most of the 23 S rRNA-protein cross-links and some 16 S rRNA-protein cross-links, in particular those with L7/L12 protein.     

Chemical nature of DNA-protein cross-links produced in mammalian chromatin by hydrogen peroxide in the presence of iron or copper ions

We report on the elucidation of DNA-protein cross-links formed in isolated mammalian chromatin upon treatment with H2O2 in the presence of iron or copper ions. Analysis of chromatin samples by gas chromatography/mass spectrometry after hydrolysis and derivatization showed the presence of 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)methyl]-L-tyrosine (thymine-tyrosine cross-link) on the basis of the gas chromatographic and mass spectrometric characteristics of the trimethylsilylated authentic compound. Other DNA-protein cross-links involving thymine and the aliphatic amino acids and cytosine and tyrosine, which were known to occur in nucleohistone gamma-irradiated under anoxic conditions, were not observed. This was due to inhibition by oxygen as clearly shown by experiments that were carried out using ionizing radiation under both oxic and anoxic conditions instead of using H2O2 and metal ions. However, oxygen did not inhibit formation of the thymine-tyrosine cross-link in gamma-irradiated chromatin or in chromatin treated with H2O2 and metal ions. The yield of the thymine-tyrosine cross-link was higher upon treatment with H2O2/chelated Fe3+ ions than with H2O2/unchelated Fe3+ ions. By contrast, H2O2/unchelated Cu2+ ions produced a higher yield than H2O2/chelated Cu2+ ions. Almost complete inhibition of cross-link formation was provided by the hydroxyl radical scavengers mannitol and dimethyl sulfoxide when H2O2/chelated metal ions were used. On the other hand, scavengers only partially inhibited formation of cross-links when H2O2/unchelated metal ions were used, possibly indicating the site-specific nature of cross-linking. Superoxide dismutase afforded partial inhibition only when chelated ions were used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of a sequence region of 5S RNA from E. coli cross-linked in situ to the ribosomal protein L25.

70S ribosomes from E. coli were chemically cross-linked under conditions of in vitro protein biosynthesis. The ribosomal RNAs were extracted from reacted ribosomes and separated on sucrose gradients. The 5S RNA was shown to contain the ribosomal protein L25 covalently bound. After total RNase T1 hydrolysis of the covalent RNA-protein complex several high molecular weight RNA fragments were obtained and identified by sequencing. One fragment, sequence region U103 to U120, was shown to be directly linked to the protein first by protein specific staining of the particular fragment and second by phosphor cellulose chromatography of the covalent RNA-protein complex. The other two fragments, U89 to G106 and A34 to G51, could not be shown to be directly linked to L25 but were only formed under cross-linking conditions. While the fragment U89 to G106 may be protected from RNase T1 digestion because of a strong interaction with the covalent RNA-protein complex, the formation of the fragment A34 to G51 is very likely the result of a double monovalent modification of two neighbouring guanosines in the 5S RNA. The RNA sequences U103 to U120 established to be in direct contact to the protein L25 within the ribosome falls into the sequence region previously proposed as L25 binding site from studies with isolated 5S RNA-protein complexes.     

Gamma-radiation-induced interactions between amino acids and glucagon

The interaction of glucagon and phenylalanine mediated by the OH . radical causes formation of higher molecular weight products of glucagon and phenylalanine, loss of amino acid residues in glucagon, and formation of adducts of glucagon and phenylalanine. The relative yields of these products depend upon the molar ratio of phenylalanine to glucagon in solution. At low ratios, glucagon aggregation and loss of amino acid residues predominate; at high ratios, the formation of phenylalanine dimers (and possible trimers and tetramers) predominates. The formation of adducts reaches a maximum at a phenylalanine:glucagon molar ratio of 3-4, and then decreases gradually, as the molar ratio increases, but is still discernible even at high molar ratios. Mechanisms for the formation of adducts are suggested. The influence of the primary aqueous radical intermediates, OH., H., and e-aq, on adduct formation has been evaluated for several different amino acids by irradiating in the presence of specific radical scavengers. For the aromatic amino acids (phenylalanine, tryptophan, and tyrosine), OH. is considerably more effective than e-aq for mediating adduct formation, whereas for histidine and methionine, these primary radicals are equally effective.     

Precise localisation of three intra-RNA cross-links in 23S RNA and one in 5S RNA, induced by treatment of Escherichia coli 50S ribosomal subunits with bis-(2-chloroethyl)-methylamine.

Treatment of E. coli 50S ribosomal subunits with low doses of bis-(2-chloroethyl)-methylamine ("nitrogen mustard") leads to formation of a number of intra-RNA and RNA-protein cross-links. After partial digestion of the cross-linked subunits with cobra venom nuclease, followed by destruction of the protein moiety with proteinase K, complexes containing the intra-RNA cross-links were isolated by two-dimensional gel electrophoresis. The individual complexes were subjected to oligonucleotide analysis, either directly or after a second partial digestion procedure using ribonuclease T1, and the cross-link sites determined. In 23S RNA, the cross-links found were between bases 763 and 1567, 1210 and 1236, 1482 and 1501; in 5S RNA, base 69 was cross-linked to base 107. The significance of these cross-links in relation to the three-dimensional organization of the ribosomal RNA is discussed.     

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.     

Ribosomes bind leaderless mRNA in Escherichia coli through recognition of their 5'-terminal AUG

Leaderless mRNAs are translated in the absence of upstream signals that normally contribute to ribosome binding and translation efficiency. In order to identify ribosomal components that interact with leaderless mRNA, a fragment of leaderless cI mRNA from bacteriophage λ, with a 4-thiouridine (4^S-U) substituted at the +2 position of the AUG start codon, was used to form cross-links to Escherichia coli ribosomes during binary (mRNA+ribosome) and ternary (mRNA+ribosome+initiator tRNA) complex formation. Ribosome binding assays (i.e., toeprints) demonstrated tRNA-dependent binding of leaderless mRNA to ribosomes; however, cross-links between the start codon and 30S subunit rRNA and r-proteins formed independent of initiator tRNA. Toeprints revealed that a leaderless mRNA's 5′-AUG is required for stable binding. Furthermore, the addition of a 5′-terminal AUG triplet to a random RNA fragment can make it both competent and competitive for ribosome binding, suggesting that a leaderless mRNA's start codon is a major feature for ribosome interaction. Cross-linking assays indicate that a subset of 30S subunit r-proteins, located at either end of the mRNA tunnel, contribute to tRNA-independent contacts and/or interactions with a leaderless mRNA's start codon. The interaction of leaderless mRNA with ribosomes may reveal features of mRNA binding and AUG recognition that are distinct from known signals but are important for translation initiation of all mRNAs.     

Protein-protein cross-linking of the 50 S ribosomal subunit of Escherichia coli using 2-iminothiolane. Identification of cross-links by immunoblotting techniques

We have investigated the protein-protein cross-links formed within the 50 S subunit of the Escherichia coli ribosome using 2-iminothiolane as the cross-linking reagent. The members of the cross-links have been identified by immunoblotting from one-dimensional and two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gels using antisera specific for the individual ribosomal proteins. This method also allowed a quantitation of the yield of cross-linking for each cross-link. A total of 14 cross-links have been identified: L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L30, L17-L32, L19-L25, L20-L21, L22-L32, and L23-L34. Our results are compared with those of Traut and coworkers (Traut, R. R., Tewari, D. S., Sommer, A., Gavino, G. R., Olson, H. M., and Glitz, D. G. (1986) in Structure, Function and Genetics of Ribosomes (Hardesty, B. and Kramer, G., eds) pp. 286-308, Springer-Verlag, New York). Our cross-linking data allow us to propose the approximate locations of eight proteins of the 50 S ribosomal subunit that so far have not been localized by immunoelectron microscopy and they thus contribute considerably to our knowledge of ribosome structure.