The use of cross-linking platinum reagents in the study of tRNA and mRNA interaction with ribosomes

The use of some bifunctional Pt(II)-containing cross-linking reagents for investigation of structural organization of ribosomal tRNA- and mRNA-binding centres is demonstrated for various types of [70S ribosome.mRNA-tRNA] complexes. It is shown that treatment of the complexes [70S ribosome.Ac[14C]Phe-tRNA(Phe).poly(U)], [70S ribosome.3'-32pCp-tRNA(Phe).poly(U)] and [70S ribosome.f[35S]Met-tRNA(fMet).AUGU6] with Pt(II)-derivatives results in covalent attachment of tRNA to ribosome. AcPhe-tRNA(Phe) and 3'-pCp-tRNA(Phe) bound at the P site were found to be cross-linked preferentially to 30S subunit. fMet-tRNA(fMet) within the 70S initiation complex is cross-linked to both ribosome subunits approximately in the same extent, which exceeds two-fold the level of the tRNA(Phe) cross-linking. All used tRNA species were cross-linked in the comparable degree both to rRNA and proteins of both subunits in all types of the complexes studied. 32pAUGU6 cross-links exclusively to 30S subunit (to 16S RNA only) within [70S ribosome.32pAUGU6.fMet-tRNA(fMet)] complex. In the absence of fMet-tRNAfMet the level of the cross-linking is 4-fold lower.     

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.     

Cross-linking of tobacco mosaic virus RNA and capped polyribonucleotides to 18S rRNA in wheat germ ribosome-mRNA complexes

Tobacco mosaic virus RNA, forming 40S or 80S initiation complexes with wheat germ ribosomes, was covalently bound to 18S ribosomal RNA by the photoreaction with an RNA cross-linking agent, 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT). Synthetic polyribonucleotide, poly(A, U), with the cap structure m7GpppGmC at the 5'-terminal was also cross-linked to 18S ribosomal RNA in 40S or 80S complexes with ribosomes by the AMT photoreaction. Polyuridylic acid with the same 5'-cap structure, forming 40S complexes but not 80S complexes with ribosomes, was most efficiently cross-linked to 18S ribosomal RNA by the psoralen photoreaction. These results suggest that the interactions between mRNA and 18S rRNA are not necessarily of strict complementarity but occur during formation of the complexes in eukaryotes. The 40S complexes would be then converted to 80S complexes in the presence of the AUG initiation codon or AUG-like triplets containing A and U on the polyribonucleotide chains which interact with 18S ribosomal RNA.     

The decoding region of 16S RNA; a cross-linking study of the ribosomal A, P and E sites using tRNA derivatized at position 32 in the anticodon loop.

A photo-reactive diazirine derivative was attached to the 2-thiocytidine residue at position 32 of tRNA(Arg)I from Escherichia coli. This modified tRNA was bound under suitable conditions to the A, P or E site of E.coli ribosomes. After photo-activation of the diazirine label, the sites of cross-linking to 16S rRNA were identified by our standard procedures. Each of the three tRNA binding sites showed a characteristic pattern of cross-linking. From tRNA at the A site, a major cross-link was observed to position 1378 of the 16S RNA, and a minor one to position 936. From the P site, there were major cross-links to positions 693 and to 957 and/or 966, as well as a minor cross-link to position 1338. The E site bound tRNA showed major cross-links to position 693 (identical to that from the P site) and to positions 1376/1378 (similar, but not identical, to the cross-link observed from the A site). Immunological analysis of the concomitantly cross-linked ribosomal proteins indicated that S7 was the major target of cross-linking from all three tRNA sites, with S11 as a minor product. The results are discussed in terms of the overall topography of the decoding region of the 30S ribosomal subunit.     

The ribosomal neighbourhood of the central fold of tRNA: cross-links from position 47 of tRNA located at the A, P or E site.

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl)uridine (acp3U) at position 47 of tRNA(Phe) from Escherichia coli was modified with a diazirine derivative and bound to ribosomes in the presence of suitable mRNA analogues under conditions specific for the ribosomal A, P or E sites. After photo-activation at 350 nm the cross-links to ribosomal proteins and RNA were identified by our standard procedures. In the 30S subunit protein S19 (and weakly S9 and S13) was the target of cross-linking from tRNA at the A site, S7, S9 and S13 from the P site and S7 from the E site. Similarly, in the 50S subunit L16 and L27 were cross-linked from the A site, L1, L5, L16, L27 and L33 from the P site and L1 and L33 from the E site. Corresponding cross-links to rRNA were localized by RNase H digestion to the following areas: in 16S rRNA between positions 687 and 727 from the P and E sites, positions 1318 and 1350 (P site) and 1350 and 1387 (E site); in the 23S rRNA between positions 865 and 910 from the A site, 1845 and 1892 (P site), 1892 and 1945 (A site), 2282 and 2358 (P site), 2242 and 2461 (P and E sites), 2461 and 2488 (A site), 2488 and 2539 (all three sites) and 2572 and 2603 (A and P sites). In most (but not all) cases, more precise localizations of the cross-link sites could be made by primer extension analysis.     

Covalent cross-linking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acid.

The covalent cross-linking of unmodified Escherichia coli N-acetylvalyl-tRNA to the 16S RNA of Escherichia coli ribosomes upon near-UV irradiation previously reported by us [Schwartz, I., & Ofengand, J. (1978) Biochemistry 17, 2524--2530] has been studied further. Up to 70% of the unmodified tRNA, nonenzymatically bound to tight-couple ribosomes at 7 mM Mg2+, could be cross-linked by 310--335-nm light. Covalent attachment was solely to the 16S RNA. It was dependent upon both irradiation and the presence of mRNA but was unaffected by the presence or absence of 4-thiouridine in the tRNA. The kinetics of cross-linking showed single-hit behavior. Twofold more cross-linking was obtained w-th tight-couple ribosomes than with salt-washed particles. Puromycin treatment after irradiation released the bound N-acetyl[3H]valine, demonstrating that the tRNA was covalently bound at the P site and that irradiation and covalent linking did not affect the peptidyl transferase reaction. Cross-linking was unaffected by the presence of O2, argon, ascorbate (1 mM), or mercaptoethanol (10 mM). Prephotolysis of a mixture of tRNA and ribosomes in the absence of puly(U2,G) did not block subsequent cross-linking in its presence nor did it generate any long-lived chemically reactive species. There was a strong tRNA specificity. E. coli tRNA1Val and tRNA1Ser and Bacillus subtilis tRNAVal and tRNAThr could be cross-linked, but E. coli tRNA2Val, 5-fluorouracil-substituted tRNA1Val, tRNAPhe, or tRNAFMet could not. By sequence comparison of the reactive and nonreactive tRNAs, the site of attachment in the tRNA was deduced to be the 5'-anticodon base, cmo5U, or ,o5U in all of the reactive tRNAs. The attachment site in 16S RNA is described in the accompanying paper [Zimmerman, R. A., Gates, S. M., Schwartz, I., & Ofengand, J. (1979) Biochemistry (following paper in this issue)]. The link between tRNA and 16S RNA is either direct or involves mRNA bases at most two nucleotides apart since use of the trinucleotide GpUpU in place of poly(U2,G) to direct the binding and cross-linking of N-acetylvalyl-tRNA to the P site did not affect either the rate or yield of cross-linking. Both B. subtilis tRNAVal (mo5U) and E. coli tRNA1Val (cmo5U) gave the same rate and yield of cross-linking when directed by the trinucleotide GpUpU. Therefore, the presence of the charged carboxyl group in the cmo5U-containing tRNA apparently does not markedly perturb the orientation of this base with respect to its reaction partner in the 16S RNA. The cross-linking of AcVal-tRNA only takes place from the P site. At 75 mM KCl and 75 mM NH4Cl, less than 0.4% cross-linking was found at the A site, while 55.5% was obtained at the P site. However, when the salt concentration was lowered to 50 mM NH4Cl, 5% cross-linking to the A site was detected, compared to 49% at the P site. Thus, a simple change in the ionic strength of the incubation mixture was able to alter the affinity labeling pattern of the ribosome.     

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.     

General method of isolation and analysis of polynucleotide fragments cross-linked with proteins

A fragment of 16S RNA, cross-linked to S7 protein by UV irradiation of the 30S subunit of E. coli ribosome, was obtained by the action of T1 ribonuclease on the irradiated nucleoprotein. The digest was treated with polynucleotide kinase in the presence of [gamma-32P]ATP and the S7-cross-linked oligonucleotides were isolated. An individual oligonucleotide attached to S7 protein was obtained after proteinase treatment of the respective spot followed by electrophoresis. Sequencing of this oligonucleotide established its structure as 1233-1240 fragment of 16S RNA, the U1239 residue being the site of the S7 cross-linking. The developed general approach can be used for localizing protein - cross-linked residues in polynucleotides, whatever is the procedure employed for cross-linking.     

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.     

Codon-anticodon interaction at the ribosomal E site

The question of whether or not the tRNA at the third ribosomal binding site specific for deacylated tRNA (E site) undergoes codon-anticodon interaction was analyzed as follows. Poly(U)-programmed ribosomes each carrying two [14C]tRNAPhe molecules were subjected to a chasing experiment using various tRNA species. At 0 degree C Ac[3H]Phe-tRNAPhe did not trigger any chasing whereas deacylated cognate tRNAPhe provoked a strong effect; non-cognate tRNALys was totally ineffective. This indicates that the second [14C]tRNAPhe cannot be present at the A site but rather at the E site (confirming previous observations). In the presence of poly(U) or poly(A) ribosomes bound the cognate tRNA practically exclusively as second deacylated tRNA, i.e. [14C]tRNAPhe and [14C]tRNALys, respectively. Thus, the second deacylated tRNA binds in a codon-dependent manner. [14C]tRNALys at the P site and Ac[3H]Lys-tRNALys at the A site of poly(A)-primed ribosomes were translocated to the E and P sites, respectively, by means of elongation factor G. The E site-bound [14C]tRNALys could be significantly chased by cognate tRNALys but not by non-cognate tRNAPhe, indicating the coded nature of the E site binding. Additional evidence is presented that the ribosome accommodates two adjacent codon-anticodon interactions at either A and P or P and E sites.     

Cross-linking of streptomycin to the 16S ribosomal RNA of Escherichia coli

[3H]Dihydrostreptomycin was cross-linked to the 30S ribosomal subunit from Escherichia coli with the bifunctional reagent nitrogen mustard. The cross-linking primarily involved the 16S RNA. To localize the site of cross-linking of streptomycin to the 16S RNA, we hybridized RNA labeled with streptomycin to restriction fragments of the 16S RNA gene. Labeled RNA hybridized to DNA fragments corresponding to bases 892-917 and bases 1394-1415. These two segments of the ribosomal RNA must be juxtaposed in the ribosome, since there is a single binding site for streptomycin. This region has been implicated both in the decoding site and in the binding of initiation factor IF-3, indicating its functional importance.     

Covalent cross-linking of tRNAGly1 to the ribosomal P site via the dihydrouridine loop

The dihydrouracil residue at position 20 of Escherichia coli tRNAGly1 has been replaced by the photoaffinity reagent, N-(4-azido-2-nitrophenyl)glycyl hydrazide (AGH). The location of the substituent was confirmed by the susceptibility of the modified tRNA to cleavage with aniline. When N-acetylglycyl-tRNAGly1 derivatized with AGH was bound noncovalently to the P site of E. coli 70 S ribosomes, 5-6% on average was photochemically cross-linked to the ribosomal particles in a reaction requiring poly(G,U), irradiation and the presence of the AGH label in the tRNA. Approximately two-thirds of the covalently attached tRNA was associated with 16 S RNA in the 30 S subunit. This material was judged to be in the P site by the criterion of puromycin reactivity. As partial RNAase digestion of the tRNA-16 S RNA complex produced labeled fragments from both 5' and 3' segments of the rRNA, there appeared to be more than one site of cross-linking in the 30 S subunit. The small amount of N-acetylglycyl-tRNAGly1 associated with the 50 S subunit was also linked mainly to rRNA, but it was not puromycin-reactive.     

A general approach for identification of RNA-protein cross-linking sites within native human spliceosomal small nuclear ribonucleoproteins (snRNPs). Analysis of RNA-protein contacts in native U1 and U4/U6.U5 snRNPs

We describe a novel approach to identify RNA-protein cross-linking sites within native small nuclear ribonucleoprotein (snRNP) particles from HeLa cells. It combines immunoprecipitation of the UV-irradiated particles under semi-denaturing conditions with primer extension analysis of the cross-linked RNA moiety. In a feasibility study, we initially identified the exact cross-linking sites of the U1 70-kDa (70K) protein in stem-loop I of U1 small nuclear RNA (snRNA) within purified U1 snRNPs and then confirmed the results by a large-scale preparation that allowed N-terminal sequencing and matrix-assisted laser desorption ionization mass spectrometry of purified cross-linked peptide-oligonucleotide complexes. We identified Tyr(112) and Leu(175) within the RNA-binding domain of the U1 70K protein to be cross-linked to G(28) and U(30) in stem-loop I, respectively. We further applied our immunoprecipitation approach to HeLa U5 snRNP, as part of purified 25 S U4/U6.U5 tri-snRNPs. Cross-linking sites between the U5-specific 220-kDa protein (human homologue of Prp8p) and the U5 snRNA were located at multiple nucleotides within the highly conserved loop 1 and at one site in internal loop 1 of U5 snRNA. The cross-linking of four adjacent nucleotides indicates an extended interaction surface between loop 1 and the 220-kDa protein. In summary, our approach provides a rapid method for identification of RNA-protein contact sites within native snRNP particles as well as other ribonucleoprotein particles.     

Nucleotides of 18S rRNA surrounding mRNA codons at the human ribosomal A, P, and E sites: a crosslinking study with mRNA analogs carrying an aryl azide group at either the uracil or the guanine residue

The 18S rRNA environment of the mRNA at the decoding site of human 80S ribosomes has been studied by cross-linking with derivatives of hexaribonucleotide UUUGUU (comprising Phe and Val codons) that carried a perfluorophenylazide group either at the N7 atom of the guanine or at the C5 atom of the 5'-terminal uracil residue. The location of the codons on the ribosome at A, P, or E sites has been adjusted by the cognate tRNAs. Three types of complexes have been obtained for each type derivative, namely, (1) codon UUU and Phe-tRNAPhe at the P site (codon GUU at the A site), (2) codon UUU and tRNAPhe at the P site and PheVal-tRNAVal at the A site, and (3) codon GUU and Val-tRNAVal at the P site (codon UUU at the E site). This allowed the placement of modified nucleotides of the mRNA analog at positions -3, +1, or +4 on the ribosome. Mild UV irradiation resulted in tRNA-dependent crosslinking of the mRNA analogs to the 18S rRNA. Nucleotide G961 crosslinked to mRNA position -3, nucleotide G1207 to position +1, and A1823 together with A1824 to position +4. All of these nucleotides are located in the most strongly conserved regions of the small subunit RNA structure, and correspond to nucleotides G693, G926, G1491, and A1492 of bacterial 16S rRNA. Three of them (with the exception of G1491) had been found earlier at the 70S ribosomal decoding site. The similarities and differences between the 16S and 18S rRNA decoding sites are discussed.