Mutation from guanine to adenine in 25S rRNA at the position equivalent to E. coli A2058 does not confer erythromycin sensitivity in Sacchromyces cerevisae

The macrolide erythromycin binds to the large subunit of the prokaryotic ribosome near the peptidyltransferase center (PTC) and inhibits elongation of new peptide chains beyond a few amino acids. Nucleotides A2058 and A2059 (E. coli numbering) in 23S rRNA play a crucial role in the binding of erythromycin, and mutation of nucleotide A2058 confers erythromycin resistance in both gram-positive and gram-negative bacteria. There are high levels of sequence and structural similarity in the PTC of prokaryotic and eukaryotic ribosomes. However, eukaryotic ribosomes are resistant to erythromycin and the presence of a G at the position equivalent to E. coli nucleotide A2058 is believed to be the reason. To test this hypothesis, we introduced a G to A mutation at this position of the yeast Saccharomyces cerevisiae 25S rRNA and analyzed sensitivity toward erythromycin. Neither growth studies nor erythromycin binding assays on mutated yeast ribosomes indicated any erythromycin sensitivity in mutated yeast strains. These results suggest that the identity of nucleotide 2058 is not the only determinant responsible for the difference in erythromycin sensitivity between yeast and prokaryotes.     

Basis for prokaryotic specificity of action of aminoglycoside antibiotics.

The aminoglycosides, a group of structurally related antibiotics, bind to rRNA in the small subunit of the prokaryotic ribosome. Most aminoglycosides are inactive or weakly active against eukaryotic ribosomes. A major difference in the binding site for these antibiotics between prokaryotic and eukaryotic ribosomes is the identity of the nucleotide at position 1408 (Escherichia coli numbering), which is an adenosine in prokaryotic ribosomes and a guanosine in eukaryotic ribosomes. Expression in E.coli of plasmid-encoded 16S rRNA containing an A1408 to G substitution confers resistance to a subclass of the aminoglycoside antibiotics that contain a 6' amino group on ring I. Chemical footprinting experiments indicate that resistance arises from the lower affinity of the drug for the eukaryotic rRNA sequence. The 1408G ribosomes are resistant to the same subclass of aminoglycosides as previously observed both for eukaryotic ribosomes and bacterial ribosomes containing a methylation at the N1 position of A1408. The results indicate that the identity of the nucleotide at position 1408 is a major determinant of specificity of aminoglycoside action, and agree with prior structural studies of aminoglycoside-rRNA complexes.     

Structural origins of aminoglycoside specificity for prokaryotic ribosomes

Aminoglycoside antibiotics, including paromomycin, neomycin and gentamicin, target a region of highly conserved nucleotides in the decoding region aminoacyl-tRNA site (A site) of 16 S rRNA on the 30 S subunit. Change of a single nucleotide, A1408 to G, reduces the affinity of many aminoglycosides for the ribosome; G1408 distinguishes between prokaryotic and eukaryotic ribosomes. The structures of a prokaryotic decoding region A-site oligonucleotide free in solution and bound to the aminoglycosides paromomycin and gentamicin C1a were determined previously. Here, the structure of a eukaryotic decoding region A-site oligonucleotide bound to paromomycin has been determined using NMR spectroscopy and compared to the prokaryotic A-site-paromomycin structure. A conformational change in three adenosine residues of an internal loop, critical for high-affinity antibiotic binding, was observed in the prokaryotic RNA-paromomycin complex in comparison to its free form. This conformational change is not observed in the eukaryotic RNA-paromomycin complex, disrupting the binding pocket for ring I of the antibiotic. The lack of the conformational change supports footprinting and titration calorimetry data that demonstrate approximately 25-50-fold weaker binding of paromomycin to the eukaryotic decoding-site oligonucleotide. Neomycin, which is much less active against Escherichia coli ribosomes with an A1408G mutation, binds non-specifically to the oligonucleotide. These results suggest that eukaryotic ribosomal RNA has a shallow binding pocket for aminoglycosides, which accommodates only certain antibiotics.     

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.     

Engineering the rRNA decoding site of eukaryotic cytosolic ribosomes in bacteria

Structural and genetic studies on prokaryotic ribosomes have provided important insights into fundamental aspects of protein synthesis and translational control and its interaction with ribosomal drugs. Comparable mechanistic studies in eukaryotes are mainly hampered by the absence of both high-resolution crystal structures and efficient genetic models. To study the interaction of aminoglycoside antibiotics with selected eukaryotic ribosomes, we replaced the bacterial drug binding site in 16S rRNA with its eukaryotic counterpart, resulting in bacterial hybrid ribosomes with a fully functional eukaryotic rRNA decoding site. Cell-free translation assays demonstrated that hybrid ribosomes carrying the rRNA decoding site of higher eukaryotes show pronounced resistance to aminoglycoside antibiotics, equivalent to that of rabbit reticulocyte ribosomes, while the decoding sites of parasitic protozoa show distinctive drug susceptibility. Our findings suggest that phylogenetically variable components of the ribosome, other than the rRNA-binding site, do not affect aminoglycoside susceptibility of the protein-synthesis machinery. The activities of the hybrid ribosomes indicate that helix 44 of the rRNA decoding site behaves as an autonomous domain, which can be exchanged between ribosomes of different phylogenetic domains for study of function.     

Defining the basis for the specificity of aminoglycoside-rRNA recognition: a comparative study of drug binding to the A sites of Escherichia coli and human rRNA

2-Deoxystreptamine (2-DOS) aminoglycoside antibiotics exert their antimicrobial activities by targeting the decoding region A site of the rRNA and inhibiting protein synthesis. A prokaryotic specificity of action is critical to therapeutic utility of 2-DOS aminoglycosides as antibiotics. Here, isothermal titration calorimetry (ITC) and fluorescence studies are presented that provide insight into the molecular basis for this prokaryotic specificity of action. Specifically, the rRNA binding properties of the 2-DOS aminoglycosides paromomycin and G418 (geneticin) are compared, using both human and Escherichia coli rRNA A site model oligonucleotides as drug targets. Paromomycin and G418 differ with respect to their specificities of action, with only paromomycin exhibiting a specificity for prokaryotic versus human ribosomes. G418 binds to both the human and E. coli rRNA A sites with a markedly lower affinity than paromomycin, with the affinities of both drugs for the human rRNA A site being lower than those they exhibit for the E. coli rRNA A site. Paromomycin induces the destacking of the base at position 1492 (by E. coli numbering) upon binding to the E. coli rRNA A site, but not the human rRNA A site. By contrast, the binding of G418 induces the destacking of base 1492 when either rRNA A site serves as the drug target. In the aggregate, these results suggest that binding-induced base destacking at the rRNA A site is a critical factor in determining the prokaryotic specificity of aminoglycoside action, with binding affinity for the A site being of secondary importance.     

Formation of the central pseudoknot in 16S rRNA is essential for initiation of translation.

The postulated central pseudoknot formed by regions 9-13/21-25 and 17-19/916-918 of 16S rRNA of Escherichia coli is phylogenetically conserved in prokaryotic as well eukaryotic species. This pseudoknot is located at the center of the secondary structure of the 16S rRNA and connects the three major domains of this molecule. We have introduced mutations into this pseudoknot by changing the base-paired residues C18 and G917, and the effect of such mutations on the ribosomal activity was studied in vivo, using a 'specialized' ribosome system. As compared with ribosomes having the wild-type pseudoknot, the translational activity of ribosomes containing an A, G or U residue at position 18 was dramatically reduced, while the activity of mutant ribosomes having complementary bases at positions 18 and 917 was at the wild-type level. The reduced translational activity of those mutants that are incapable of forming a pseudoknot was caused by their inability to form 70S ribosomal complexes. These results demonstrate that the potential formation of a central pseudoknot in 16S rRNA with any base-paired residues at positions 18 and 917 is essential to complete the initiation process.     

Positioning of mRNA codons with respect to 18S rRNA at the P and E sites of human ribosome

Positioning of each nucleotide of the E site and the P site bound codons with respect to the 18S rRNA on the human ribosome was studied by cross-linking with mRNA analogs, derivatives of the hexaribonucleotide UUUGUU (comprising Phe and Val codons) that carried a perfluorophenylazide group on the second or the third uracil, and a derivative of the dodecaribonucleotide UUAGUAUUUAUU with a similar group on the guanine residue. The location of the modified nucleotides at any mRNA position from -3 to +3 (position +1 corresponds to the 5' nucleotide of the P site bound codon) was adjusted by the cognate tRNAs. A modified uridine at positions from -1 to +3 cross-linked to nucleotide G1207 of the 18S rRNA, and to nucleotide G961 when it was in position -2. A modified guanosine cross-linked to nucleotide G1207 if it was in position -3 of the mRNA. These data indicate that nucleotide G961 of the 18S rRNA is close only to mRNA positions -3 and -2, while G1207 is in the vicinity of positions from -3 to +3. The latter suggests that there is a sharp turn between the P and E site bound codons that brings nucleotide G1207 of the 18S rRNA close to each nucleotide of these codons. This correlates well with X-ray crystallographic data on bacterial ribosomes, indicating existence of a sharp turn between the P site and E site bound codons near a conserved nucleotide G926 of the 16S rRNA (corresponding to G1207 in 18S rRNA) close to helix 23b containing the conserved nucleotide 693 of the 16S rRNA (corresponding exactly to G961 of the 18S rRNA).     

Structural-functional topography of human ribosomes based on the data from crosslinking with mRNA analogs--oligoribonucleotide derivatives

Reviewed are data on the position of template codons with respect to 18S rRNA and certain proteins on human ribosome obtained using a set of mRNA analogs, oligoribonucleotide derivatives carrying alkylating or photoactivatable group at different positions. A comparison of data on template position on the human and Escherichia coli ribosomes has revealed both the similarity in the structure of the mRNA-binding site of bacterial and mammalian ribosomes and the peculiarities of the functioning of mammalian (in particular, human) ribosomes. The similarity manifests itself in that the template codons at the A, P, and E sites of bacterial and human ribosomes are surrounded by similar nucleotides (occupying similar positions in the conserved regions of secondary structure) of small subunit rRNA. The template forms a loop whose foot is in proximity to the 530 stem-loop conserved region of rRNA. The specific features of mammalian ribosomes appear to be associated with their lower conformational mobility as compared with bacterial ribosomes, owing to which their interaction with the template involves a lesser number of molecular contacts.     

Mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA affect the function of the decoding center of the ribosome

A dynamic structural rearrangement in the phylogenetically conserved helix 27 of Escherichia coli 16S rRNA has been proposed to directly affect the accuracy of translational decoding by switching between "accurate" and "error-prone" conformations. To examine the function of helix 27 in eukaryotes, random and site-specific mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA have been characterized. Mutations at positions of yeast 18S rRNA corresponding to E. coli 886 (rdn8), 888 (rdn6), and 912 (rdn4) increased translational accuracy in vivo and in vitro, and caused a reduction in tRNA binding to the A-site of mutant ribosomes. The double rdn4rdn6 mutation separated the killing and stop-codon readthrough effects of the aminoglycoside antibiotic, paromomycin, implicating a direct involvement of yeast helix 27 in accurate recognition of codons by tRNA or release factor eRF1. Although our data in yeast does not support a conformational switch model analogous to that proposed for helix 27 of E. coli 16S rRNA, it strongly suggests a functional conservation of this region in tRNA selection.     

Replacement of L7/L12.L10 protein complex in Escherichia coli ribosomes with the eukaryotic counterpart changes the specificity of elongation factor binding.

The L8 protein complex consisting of L7/L12 and L10 in Escherichia coli ribosomes is assembled on the conserved region of 23 S rRNA termed the GTPase-associated domain. We replaced the L8 complex in E. coli 50 S subunits with the rat counterpart P protein complex consisting of P1, P2, and P0. The L8 complex was removed from the ribosome with 50% ethanol, 10 mM MgCl(2), 0.5 M NH(4)Cl, at 30 degrees C, and the rat P complex bound to the core particle. Binding of the P complex to the core was prevented by addition of RNA fragment covering the GTPase-associated domain of E. coli 23 S rRNA to which rat P complex bound strongly, suggesting a direct role of the RNA domain in this incorporation. The resultant hybrid ribosomes showed eukaryotic translocase elongation factor (EF)-2-dependent, but not prokaryotic EF-G-dependent, GTPase activity comparable with rat 80 S ribosomes. The EF-2-dependent activity was dependent upon the P complex binding and was inhibited by the antibiotic thiostrepton, a ligand for a portion of the GTPase-associated domain of prokaryotic ribosomes. This hybrid system clearly shows significance of binding of the P complex to the GTPase-associated RNA domain for interaction of EF-2 with the ribosome. The results also suggest that E. coli 23 S rRNA participates in the eukaryotic translocase-dependent GTPase activity in the hybrid system.     

Specificities of RNA N-glycosidase activity of Mirabilis antiviral protein variants.

Mirabilis antiviral protein (MAP), a ribosome-inactivating protein, inactivates both eukaryotic and prokaryotic ribosomes by means of site-specific RNA N-glycosidase activity. In order to identify the site of this activity, some amino acid residues of MAP, conserved in homologous ribosome-inactivating proteins, were altered to other amino acids by replacing DNA fragments of the total synthetic gene of MAP. When the in vitro proteins synthesis of rabbit reticulocyte was treated with MAP variants secreted into culture media of Escherichia coli transformants, the inhibitory effect of R26L and R48L (R26L designates MAP variant with Arg-26 changed to Leu) was found to be similar to that of native MAP. Both purified Y72F and Y118F had the same effect as native MAP, and E168D had a slightly weaker effect. In contrast, on the protein synthesis of E. coli, Y118F had one-tenth the effect of native MAP, and Y72F and E168D approximately one-hundredth the effect. These three variant proteins also exhibited reduced RNA N-glycosidase activity on substrate E. coli ribosomes. These results suggest that Tyr-72 and Glu-168 are involved in RNA N-glycosidase activity. When the R171K gene was expressed in E. coli, an N-glycosidic bond of the 23 S rRNA of the host ribosome was found to be cleaved, although no product of the gene could be detected. This suggests that MAP variants can maintain their N-glycosidase activity when the conserved Glu-168 and Arg-171 are changed to similarly charged residues.     

Involvement of helix 34 of 16 S rRNA in decoding and translocation on the ribosome

Helix 34 of 16 S rRNA is located in the head of the 30 S ribosomal subunit close to the decoding center and has been invoked in a number of ribosome functions. In the present work, we have studied the effects of mutations in helix 34 both in vivo and in vitro. Several nucleotides in helix 34 that are either highly conserved or form important tertiary contacts in 16 S rRNA (U961, C1109, A1191, and A1201) were mutated, and the mutant ribosomes were expressed in the Escherichia coli MC250 Delta7 strain that lacks all seven chromosomal rRNA operons. Mutations at positions A1191 and U961 reduced the efficiency of subunit association and resulted in structural rearrangements in helix 27 (position 908) and helix 31 (position 974) of 16 S rRNA. All mutants exhibited increased levels of frameshifting and nonsense readthrough. The effects on frameshifting were specific in that -1 frameshifting was enhanced with mutant A1191G and +1 frameshifting with the other mutants. Mutations of A1191 moderately (approximately 2-fold) inhibited tRNA translocation. No significant effects were found on efficiency and rate of initiation, misreading of sense codons, or binding of tRNA to the E site. The data indicate that helix 34 is involved in controlling the maintenance of the reading frame and in tRNA translocation.     

Genetic interaction between yeast Saccharomyces cerevisiae release factors and the decoding region of 18 S rRNA

Functional and structural similarities between tRNA and eukaryotic class 1 release factors (eRF1) described previously, provide evidence for the molecular mimicry concept. This concept is supported here by the demonstration of a genetic interaction between eRF1 and the decoding region of the ribosomal RNA, the site of tRNA-mRNA interaction. We show that the conditional lethality caused by a mutation in domain 1 of yeast eRF1 (P86A), that mimics the tRNA anticodon stem-loop, is rescued by compensatory mutations A1491G (rdn15) and U1495C (hyg1) in helix 44 of the decoding region and by U912C (rdn4) and G886A (rdn8) mutations in helix 27 of the 18 S rRNA. The rdn15 mutation creates a C1409-G1491 base-pair in yeast rRNA that is analogous to that in prokaryotic rRNA known to be important for high-affinity paromomycin binding to the ribosome. Indeed, rdn15 makes yeast cells extremely sensitive to paromomycin, indicating that the natural high resistance of the yeast ribosome to paromomycin is, in large part, due to the absence of the 1409-1491 base-pair. The rdn15 and hyg1 mutations also partially compensate for inactivation of the eukaryotic release factor 3 (eRF3) resulting from the formation of the [PSI+] prion, a self-reproducible termination-deficient conformation of eRF3. However, rdn15, but not hyg1, rescues the conditional cell lethality caused by a GTPase domain mutation (R419G) in eRF3. Other antisuppressor rRNA mutations, rdn2(G517A), rdn1T(C1054T) and rdn12A(C526A), strongly inhibit [PSI+]-mediated stop codon read-through but do not cure cells of the [PSI+] prion. Interestingly, cells bearing hyg1 seem to enable [PSI+] strains to accumulate larger Sup35p aggregates upon Sup35p overproduction, suggesting a lower toxicity of overproduced Sup35p when the termination defect, caused by [PSI+], is partly relieved.     

A comparison of the yeast and rabbit 80 S ribosome reveals the topology of the nascent chain exit tunnel, inter-subunit bridges and mammalian rRNA expansion segments

Protein synthesis in eukaryotes is mediated by both cytoplasmic and membrane-bound ribosomes. During the co-translational translocation of secretory and membrane proteins, eukaryotic ribosomes dock with the protein conducting channel of the endoplasmic reticulum. An understanding of these processes will require the detailed structure of a eukaryotic ribosome. To this end, we have compared the three-dimensional structures of yeast and rabbit ribosomes at 24 A resolution. In general, we find that the active sites for protein synthesis and translocation have been highly conserved. It is interesting that a channel was visualized in the neck of the small subunit whose entrance is formed by a deep groove. By analogy with the prokaryotic small subunit, this channel may provide a conserved portal through which mRNA is threaded into the decoding center. In addition, both the small and large subunits are built around a dense tubular network. Our analysis further suggests that the nascent chain exit tunnel and the docking surface for the endoplasmic reticulum channel are formed by this network. We surmise that many of these features correspond to rRNA, based on biochemical and structural data.Ribosomal function is critically dependent on the specific association of small and large subunits. Our analysis of eukaryotic ribosomes reveals four conserved inter-subunit bridges with a geometry similar to that found in prokaryotes. In particular, a double-bridge connects the small subunit platform with the interface canyon on the large subunit. Moreover, a novel bridge is formed between the platform and the base of the L1 domain. Finally, size differences between mammalian and yeast large subunit rRNAs have been correlated with five expansion segments that form two large spines and three extended fingers. Overall, we find that expansion segments within the large subunit rRNA have been incorporated at positions distinct from the active sites for protein synthesis and translocation.     

Variable and conserved elements of human ribosomes surrounding the mRNA at the decoding and upstream sites

This study is centred upon an important biological problem concerning the structural organization of mammalian ribosomes that cannot be studied by X-ray analysis because 80S ribosome crystals are still unavailable. Here, positioning of the mRNA on 80S ribosomes was studied using mRNA analogues containing the perfluorophenylazide cross-linker on either the guanosine or an uridine residue. The modi-fied nucleotides were directed to positions from −9 to +6 with respect to the first nucleotide of the P site bound codon by a tRNA cognate to the triplet targeted to the P site. Upon mild UV-irradiation, the modified nucleotides at positions +4 to +6 cross-linked to protein S15 and 18S rRNA nucleotides A1823–A1825. In addition, modified guanosines in positions +5 and +6 also cross-linked to G626, and in position +1 to G1702. Cross-linking from the upstream positions was mainly to protein S26 that has no prokaryotic homologues. These findings indicate that the tail of mammalian S15 comes closer to the decoding site than that of its prokaryotic homologue S19, and that the environments of the upstream part of mRNA on 80S and 70S ribosomes differ. On the other hand, the results confirm the widely accepted idea regarding the conserved nature of the decoding site of the small subunit rRNA.     

Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Escherichia coli ribosomes

The cytotoxin colicin E3 targets the 30S subunit of bacterial ribosomes and specifically cleaves 16S rRNA at the decoding centre, thereby inhibiting translation. Although the cleavage site is well known, it is not clear which step of translation is inhibited. We studied the effects of colicin E3 cleavage on ribosome functions by analysing individual steps of protein synthesis. We find that the cleavage affects predominantly the elongation step. The inhibitory effect of colicin E3 cleavage originates from the accumulation of sequential impaired decoding events, each of which results in low occupancy of the A site and, consequently, decreasing yield of elongating peptide. The accumulation leads to an almost complete halt of translation after reading of a few codons. The cleavage of 16S rRNA does not impair monitoring of codon-anticodon complexes or GTPase activation during elongation-factor Tu-dependent binding of aminoacyl-tRNA, but decreases the stability of the codon-recognition complex and slows down aminoacyl-tRNA accommodation in the A site. The tRNA-mRNA translocation is faster on colicin E3-cleaved than on intact ribosomes and is less sensitive to inhibition by the antibiotic viomycin.     

A ribosomal protein is specifically recognized by saporin, a plant toxin which inhibits protein synthesis.

Many plants express enzymes which specifically remove an adenine residue from the skeleton of the 28 S RNA in the major subunit of the eukaryotic ribosome (ribosome inactivating proteins, RIPs). The site of action of RIPs (A4324 in the rRNA from rat liver) is in a loop structure whose nucleotide sequence all around the target adenine is also conserved in those species which are completely or partially insensitive to RIPs. In this paper we identify a covalent complex between saporin (the RIP extracted from Saponaria officinalis) and ribosomal proteins from yeast (Saccharomyces cerevisiae), by means of chemical crosslinking and immunological or avidin-biotin detection. The main complex (mol. wt. congruent to 60 kDa) is formed only with a protein from the 60 S subunit of yeast ribosomes, and is not detected with ribosomes from E. coli, a resistant species. This observation supports the hypothesis for a molecular recognition mechanism involving one or more ribosomal proteins, which could provide a 'receptor' site for the toxin and favour optimal binding of the target adenine A4324 to the active site of the RIP.     

Cross-linking of the anticodon of Escherichia coli and Bacillus subtilis acetylvalyl-tRNA to the ribosomal P site. Characterization of a unique site in both E. coli 16S and yeast 18S ribosomal RNA

The nucleotide residues involved in the cross-link between P site bound acetylvalyl-tRNA (AcVal-tRNA) and 16-18S rRNA have been identified. This cross-link was formed by irradiation of Escherichia coli or Bacillus subtilis AcVal-tRNA bound to the P site of E. coli ribosomes or by irradiation of E. coli AcVal-tRNA bound to the P site of yeast ribosomes. The three cross-linked RNA heterodimers were obtained in 10-35% purity by disruption of the irradiated ribosome-tRNA complex with sodium dodecyl sulfate followed by sucrose gradient centrifugation. After total digestion with RNase T1, and labeling at either the 5'- or the 3'-end, the cross-linked oligomers could be identified and isolated before and after photolytic splitting of the cross-link. One of the oligomers was shown to be UACACACCG, a unique rRNA nonamer present in an evolutionarily conserved region. This oligomer was found in all three heterodimers. The other oligomer of the dimer had the sequence expected for the RNase T1 product encompassing the anticodon of the tRNA used. The precise site of cross-linking was determined by two novel methods. Bisulfite modification of the oligonucleotide dimer converted all C residues to U, except for any cross-linked C which would be resistant by being part of a cyclobutane dimer. Sequencing gel analysis of the UACACACCG oligomer showed that the C residue protected was the 3'-penultimate C residue, C1400 in E. coli rRNA or C1626 in yeast rRNA.(ABSTRACT TRUNCATED AT 250 WORDS)