Internucleotide movements during formation of 16 S rRNA-rRNA photocrosslinks and their connection to the 30 S subunit conformational dynamics

UV light-induced RNA photocrosslinks are formed at a limited number of specific sites in the Escherichia coli and in other eubacterial 16 S rRNAs. To determine if unusually favorable internucleotide geometries could explain the restricted crosslinking patterns, parameters describing the internucleotide geometries were calculated from the Thermus thermophilus 30 S subunit X-ray structure and compared to crosslinking frequencies. Significant structural adjustments between the nucleotide pairs usually are needed for crosslinking. Correlations between the crosslinking frequencies and the geometrical parameters indicate that nucleotide pairs closer to the orientation needed for photoreaction have higher crosslinking frequencies. These data are consistent with transient conformational changes during crosslink formation in which the arrangements needed for photochemical reaction are attained during the electronic excitation times. The average structural rearrangement for UVA-4-thiouridine (s4U)-induced crosslinking is larger than that for UVB or UVC-induced crosslinking; this is associated with the longer excitation time for s4U and is also consistent with transient conformational changes. The geometrical parameters do not completely predict the crosslinking frequencies, implicating other aspects of the tertiary structure or conformational flexibility in determining the frequencies and the locations of the crosslinking sites. The majority of the UVB/C and UVA-s4U-induced crosslinks are located in four regions in the 30 S subunit, within or at the ends of RNA helix 34, in the tRNA P-site, in the distal end of helix 28 and in the helix 19/helix 27 region. These regions are implicated in different aspects of tRNA accommodation, translocation and in the termination reaction. These results show that photocrosslinking is an indicator for sites where there is internucleotide conformational flexibility and these sites are largely restricted to parts of the 30 S subunit associated with ribosome function.     

G1401: a keystone nucleotide at the decoding site of Escherichia coli 30S ribosomes.

16S ribosomal RNA contains three highly conserved single-stranded regions. Centrally located in one of these regions is the C1400 residue. Zero-length cross-linking of this residue to the anticodon of ribosome-bound tRNA showed that it was at or near the ribosomal decoding site [Ehresmann, C., Ehresmann, B., Millon, R., Ebel, J-P., Nurse, K., & Ofengand, J. (1984) Biochemistry 23, 429-437]. To assess the functional significance of sequence conservation of rRNA in the vicinity of this functionally important site, a series of site-directed mutations in this region were constructed and the effects of these mutations on the partial reactions of protein synthesis determined. Mutation of C1400 or C1402 to any other base only moderately affected a set of in vitro protein synthesis partial reactions. However, any base change from the normal G1401 residue blocked all of the tested ribosomal functions. This was also true for the deletion of G1401. Deletion of C1400 or C1402 had more complex effects. Whereas subunit association was hardly affected, 30S initiation complex formation was blocked by deletion of C1400 but much less so by deletion of C1402. Alternatively, tRNA binding to the ribosomal A site was more strongly affected by deletion of C1402 than by deletion of C1400. P site binding was inhibited by either deletion. HPLC analysis of the in vitro reconstituted mutant ribosomes showed that none of the functional effects were due to the absence or gross reduction in amount of any ribosomal protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Positioning of the mRNA stop signal with respect to polypeptide chain release factors and ribosomal proteins in 80S ribosomes

To study positioning of the mRNA stop signal with respect to polypeptide chain release factors (RFs) and ribosomal components within human 80S ribosomes, photoreactive mRNA analogs were applied. Derivatives of the UUCUAAA heptaribonucleotide containing the UUC codon for Phe and the stop signal UAAA, which bore a perfluoroaryl azido group at either the fourth nucleotide or the 3'-terminal phosphate, were synthesized. The UUC codon was directed to the ribosomal P site by the cognate tRNA(Phe), targeting the UAA stop codon to the A site. Mild UV irradiation of the ternary complexes consisting of the 80S ribosome, the mRNA analog and tRNA resulted in tRNA-dependent crosslinking of the mRNA analogs to the 40S ribosomal proteins and the 18S rRNA. mRNA analogs with the photoreactive group at the fourth uridine (the first base of the stop codon) crosslinked mainly to protein S15 (and much less to S2). For the 3'-modified mRNA analog, the major crosslinking target was protein S2, while protein S15 was much less crosslinked. Crosslinking of eukaryotic (e) RF1 was entirely dependent on the presence of a stop signal in the mRNA analog. eRF3 in the presence of eRF1 did not crosslink, but decreased the yield of eRF1 crosslinking. We conclude that (i) proteins S15 and S2 of the 40S ribosomal subunit are located near the A site-bound codon; (ii) eRF1 can induce spatial rearrangement of the 80S ribosome leading to movement of protein L4 of the 60S ribosomal subunit closer to the codon located at the A site; (iii) within the 80S ribosome, eRF3 in the presence of eRF1 does not contact the stop codon at the A site and is probably located mostly (if not entirely) on the 60S subunit.     

Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking.

Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 A side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 A distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 A from the 4' position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.     

A new RNA-RNA crosslinking reagent and its application to ribosomal 5S RNA.

The synthesis of a new RNA specific bifunctional crosslinking reagent, 1.4-phenyl-diglyoxal, is described which reacts exclusively with guanosines. The properties of the crosslinked products enabled us to develop a straightforward method for identifying the reacted nucleotides. Results obtained with ribosomal 5S RNA of Escherichia coli demonstrate the formation of an intramolecular crosslink between guanosine-2 and guanosine-112 in the stem region.     

A detailed model of the three-dimensional structure of Escherichia coli 16 S ribosomal RNA in situ in the 30 S subunit

A large body of intra-RNA and RNA-protein crosslinking data, obtained in this laboratory, was used to fold the phylogenetically and experimentally established secondary structure of Escherichia coli 16 S RNA into a three-dimensional model. All the crosslinks were induced in intact 30 S subunits (or in some cases in growing E. coli cells), and the sites of crosslinking were precisely localized on the RNA by oligonucleotide analysis. The RNA-protein crosslinking data (including 28 sites, and involving 13 of the 21 30S ribosomal were used to relate the RNA structure to the distribution of the proteins as determined by neutron scattering. The three-dimensional model of the 16 S RNA has overall dimensions of 220 A x 140 A x 90 A, in good agreement with electron microscopic estimates for the 30 S subunit. The shape of the model is also recognizably the same as that seen in electron micrographs, and the positions in the model of bases localized on the 30 S subunit by immunoelectron microscopy (the 5' and 3' termini, the m7G and m6(2)A residues, and C-1400) correspond closely to their experimentally observed positions. The distances between the RNA-protein crosslink sites in the model correlate well with the distances between protein centres of mass obtained by neutron scattering, only two out of 66 distances falling outside the expected tolerance limits. These two distances both involve protein S13, a protein noted for its anomalous behaviour. A comparison with other experimental information not specifically used in deriving the model shows that it fits well with published data on RNA-protein binding sites, mutation sites on the RNA causing resistance to antibiotics, tertiary interactions in the RNA, and a potential secondary structural "switch". Of the sites on 16 S RNA that have been found to be accessible to chemical modification in the 30 S subunit, 87% are at obviously exposed positions in the model. In contrast, 70% of the sites corresponding to positions that have ribose 2'-O-methylations in the eukaryotic 18 S RNA from Xenopus laevis are at non-exposed (i.e. internal) positions in the model. All nine of the modified bases in the E. coli 16 S RNA itself show a remarkable distribution, in that they form a "necklace" in one plane around the "throat" of the subunit. Insertions in eukaryotic 18 S RNA, and corresponding deletions in chloroplast or mammalian mitochondrial ribosomal RNA relative to E. coli 16 S RNA represent distinct sub-domains in the structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Shiga toxin, Shiga-like toxin II variant, and ricin are all single-site RNA N-glycosidases of 28 S RNA when microinjected into Xenopus oocytes

Ricin, Shiga toxin, and Shiga-like toxin II (SLT-II, Vero toxin 2) exhibit an RNA N-glycosidase activity which specifically removes a single base near the 3' end of 28 S rRNA in isolated rat liver ribosomes and deproteinized 28 S rRNA (Endo Y., Mitsui, K., Motizuki, M., & Tsurugi, K. (1987) J. Biol. Chem. 262, 5908-5912; Endo Y. & Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130, Endo, Y., Tsurugi, K., Yutsudo, T., Takeda, Y., Ogasawara, K. & Igarashi, K. (1988) Eur. J. Biochem. 171, 45-50). These workers identified the single base removed, A-4324, by examining a 28 S rRNA degradation product which was generated by contaminating ribonucleases associated with the ribosomes. To determine whether this N-glycosidase activity applies in living cells, we microinjected ricin into Xenopus oocytes. We also microinjected Shiga toxin and a variant of Shiga-like toxin II (SLT-IIv). All three toxins specifically removed A-3732, located 378 nucleotides from the 3' end of 28 S rRNA. This base is analogous to the site observed in rat 28 S rRNA for ricin, Shiga toxin, and SLT-II. Purified, glycosylated, ricin A chain contains this RNA N-glycosidase activity in oocytes. We also demonstrated that the nonglycosylated A subunit of recombinant ricin exhibits this RNA N-glycosidase activity when injected into Xenopus oocytes. Ricin, Shiga toxin, and SLT-IIv also caused a rapid decline in oocyte protein synthesis for nonsecretory proteins.     

Immunoelectron microscopic localization of the S19 site on the 30 S ribosomal subunit which is crosslinked to A site bound transfer RNA

Phe-tRNA of Escherichia coli, specifically derivatized at the S4U8 position with the 9 A long p-azidophenacyl photoaffinity probe, was crosslinked exclusively to protein S19 of the 30 S ribosomal subunit when the transfer RNA occupied the ribosomal A site (Lin et al., 1983). Two antigenic sites for S19 are known, on opposite sides of the head of the subunit. In this work, discrimination between these two sites was accomplished by affinity immunoelectron microscopy. A dinitrophenyl group was placed on the acp3U47 residue of the same tRNA molecules bearing the photoprobe on S4U8. Addition of this group affected neither aminoacylation, A site binding, nor crosslinking. It also made possible specific affinity purification of crosslinked tRNA-30 S complexes from unreactive 30 S. Reaction of the 2,4-dinitrophenyl-labeled tRNA-30 S complex with antibody was followed by immunoelectron microscopy to reveal the sites of attachment. All of the bound antibody was associated with the ribosome region corresponding to only one of the two known antigenic sites for S19, namely the one closer to the large side projection of the 30 S subunit. A site within this region must be within 10 A of the S4U8 residue of tRNA when it is bound in the ribosomal A site.     

Analysis of the conformation of the 3' major domain of Escherichia coli16S ribosomal RNA using site-directed photoaffinity crosslinking.

The 3' major domain of Escherichia coli 16S rRNA, which occupies the head of the small ribosomal subunit, is involved in several functions of the ribosome. We have used a site-specific crosslinking procedure to gain further insights into the higher-order structure of this domain. Circularly permuted RNAs were used to introduce an azidophenacyl group at specific positions within the 3' major domain. Crosslinks were generated in a high-ionic strength buffer that has been used for ribosome reconstitution studies and so enables the RNA to adopt a structure recognized by ribosomal proteins. The crosslinking sites were identified by primer extension and confirmed by assessing the mobility of the crosslinked RNA lariats in denaturing polyacrylamide gels. Eight crosslinks were characterized. Among them, one crosslink demonstrates that helix 28 is proximal to the top of helix 34, and two others show that the 1337 region, located in an internal loop at the junction of helices 29, 30, 41, and 42, is proximal to the center of helix 30 and to a segment connecting helix 28 to helix 29. These relationships of vicinity have previously been observed in native 30S subunits, which suggests that the free domain adopts a conformation similar to that within the 30S subunit. Furthermore, crosslinks were obtained in helix 34, which suggest that the upper and lower portions of this helix are in close proximity.     

Protein S3 in the human 80S ribosome adjoins mRNA from 3'-side of the A-site codon

The protein environment of mRNA 3' of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5'-end and a perfluoroarylazide group at one of the nucleotide residues at the 3'-end of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNAPhe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNAPhe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in triple complexes and in the absence of tRNA. Within triple complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed, it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3' of the codon in the decoding site.     

The effects of stem I and loop A on the processing of 5 S rRNA from Drosophila melanogaster.

The 135-nucleotide Drosophila melanogaster 5 S RNA precursor is processed by removal of 15 nucleotides from its 3' end before incorporation into the large ribosomal subunit. Mature 5 S RNA consists of five helical stem-loops; stem IV and part of V are dispensable, whereas stem III and the 1/118 G-C base pair closest to the processing site at nucleotide 120 are required for processing (Preiser, P., and Levinger, L. (1991) J. Biol. Chem. 266, 7509-7516; Preiser, P., and Levinger, L. (1991) J. Biol. Chem. 266, 23602-23605). We have investigated the effects of stem I and loop A transversions, transitions, selected additions and deletions on 5 S RNA processing. Stem I single substitutions generally prevent processing, whereas compensatory double substitutions restore a range of processing rates. Proximal to the processing site, stem I double substitutions inhibit processing. In the distal portion of stem I and loop A, the processing effect of paired sequence changes varies widely in an irregular pattern. The 7/112 GU pair and nucleotide 13A least tolerate sequence changes; several mutations clustered close to the stem I-loop A boundary stimulate processing. We interpret these results in terms of the RNA helix path and possible RNA-protein contacts.     

Structure and functions of 5S rRNA.

The ribosome is a macromolecular assembly that is responsible for protein biosynthesis in all organisms. It is composed of two-subunit, ribonucleoprotein particles that translate the genetic material into an encoded polypeptides. The small subunit is the site of codon-anticodon interaction between the messenger RNA (mRNA) and transfer RNA (tRNA) substrates, and the large subunit catalyses peptide bond formation. The peptidyltransferase activity is fulfilled by 23S rRNA, which means that ribosome is a ribozyme. 5S rRNA is a conserved component of the large ribosomal subunit that is thought to enhance protein synthesis by stabilizing ribosome structure. This paper shortly summarises new results obtained on the structure and function of 5S rRNA.     

Altered topography of 16S RNA in the inactive form of Escherichia coli 30S ribosomal subunits.

We have studied the topography of 16S RNA in the inactive form of the 30S ribosomal subunit (Ginsburg, I., et al. (1973) J. Mol. Biol. 79, 481), using the guanine-specific reagent kethoxal. Oligonucleotides surrounding reactive guanine residues were isolated and quantitated by means of diagonal electrophoresis and sequenced. Comparison of these results with experiments on active or reactivated subunits reveals the following: (1) Most of the sites which are reactive in active 30S subunits are much more reactive (average 13-fold) in inactive subunits. Upon reactivation, these sites return to a less reactive state. Thus, a reversible increase in accessibility of specific 16S RNA sites parallels the reversible loss of protein synthesis activity of 30S subunits. (2) The number of kethoxal-reactive sites in inactive subunits is about twice that of active subunits. The nucleotide sequences and locations of the additional accessible sites in inactive subunits have been determined. (3) Sites that can be located in the 16S RNA sequence are distributed throughout the RNA chain in inactive subunits, in contrast to the clustering observed in active subunits. (4) The sites of kethoxal substitution are single stranded. Yet, of the 30 sites that can be located, 23 were predicted to be base paired in the proposed secondary structure model for 16S RNA (Ehresmann, C., et al. (1975), Nucleic Acids Res. 2, 265).     

Immune electron microscopic localization of dinitrophenyl-modified ribosomal protein S19 in reconstituted Escherichia coli 30 S subunits using antibodies to dinitrophenol

Escherichia coli small ribosomal subunits have been reconstituted from RNA and high performance liquid chromatography-purified proteins including protein S19 that had been modified at its amino-terminal proline residue with 1-fluoro-2,4-dinitrobenzene. As detailed in the accompanying paper (Olah, T. V., Olson, H. M., Glitz, D. G., and Cooperman, B. S. (1988) J. Biol. Chem. 263, 4795-4800), dinitrophenyl (DNP)-S19 was efficiently incorporated into the site ordinarily occupied by S19. Antibodies to DNP bound effectively to the reconstituted subunits and did not cause dissociation of the modified protein from the subunit. Electron microscopy of the immune complexes was used to localize the modified protein on the subunit surface. More than 95% of the antibody binding sites seen were consistent with a single location of protein S19 on the upper portion or head of the subunit, on the surface that faces the 50 S particle in a 70 S ribosome, and in an area relatively distant from the subunit platform. The S19 site is close to the region in which 30 S subunits are photoaffinity labeled with puromycin. Protein S19 is thus near protein S14 in the small subunit and in proximity to the peptidyl transferase center of the 70 S ribosome.     

Organization of the 16S rRNA around its 5' terminus determined by photochemical crosslinking in the 30S ribosomal subunit

The organization of the 5' terminus region in the 16S rRNA was investigated using a series of RNA constructs in which the 5' terminus was extended by 5 nt or was shortened to give RNA molecules that started at positions -5, +1, +5, +8, +14, or +21. The structural and functional effects of the 5' extension/truncations were determined after the RNAs were reconstituted. 30S subunits containing 16S rRNA with 5' termini at -5, +1, +5, +8 and +14 had similar structures (judged by UV-induced crosslinking) and exhibited a gradual reduction in tRNA binding activity compared to that seen with 30S subunits reconstituted with native 16S rRNA. To create the 5' terminal site-specific photocrosslinking agent, the reagent azidophenacylbromide (APAB) was attached to the 5' terminus of 16S rRNA through a guanosine monophosphorothioate and the APA-16S rRNAs were reconstituted. Crosslinking carried out with the APA revealed sites in six regions around positions 300-340, 560, 900, 1080, the 16S rRNA decoding region, and at 1330. Differences in the pattern and efficiency of crosslinking for the different constructs allow distance estimates for the crosslinked sites from nucleotide G9. These measurements provide constraints for the arrangement of the RNA elements in the 30S subunit. Similar experiments carried out in the 70S ribosome resulted in a five- to tenfold lower frequency of crosslinking. This is most likely due to a repositioning of the 5' terminus upon subunit association.