Structural features of Bacillus precursor 5S RNA involved in the interaction with RNAase M5.

Mature 5S (m5S) RNA from Bacillus licheniformis specifically and almost completely inhibits in vitro maturation of bacillus precursor 5S (p5S) RNA, showing that the maturation enzyme RNAase M5 can recognize Bacillus m5S RNA. E. coli m5S RNA is a much less efficient inhibitor, whereas S. carlsbergensis 5S RNA inhibits maturation by about 70%. The differences in inhibition can be correlated with the position of the sequence UAGG (residues 101-104 in B. licheniformis m5S RNA) relative to the double-helical region formed by the 5'- and 3'-terminal sequences (molecular stalk) of m5S RNA. Recent experiments by Meyhack and Pace (Biochemistry 17 (1980) 5804-5810) demonstrated this UAGG sequence to be indispensable for processing of p5S RNA. Other elements of secondary and/or tertiary structure are also required, however. The effect of artificially constructed "5S RNA" molecules having defined disturbances in the base-pairing within the molecular stalk on in vitro maturation shows that base-pairing in the immediate neighbourhood of the bonds to be cleaved during maturation is crucial to recognition of p5S RNA by RNAase M5. G.U pairs are tolerated in this region, however, without loss of efficiency in maturation. Base-pairing does not have to extend throughout the complete molecular stalk. The introduction of an A/C combination at the end of the molecular stalk removed from the bonds cleaved by RNAase M5 does not significantly impair the efficiency of maturation.     

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.     

Base Pairing between U3 Small Nucleolar RNA and the 5′ End of 18S rRNA Is Required for Pre-rRNA Processing

The loop of a stem structure close to the 5' end of the 18S rRNA is complementary to the box A region of the U3 small nucleolar RNA (snoRNA). Substitution of the 18S loop nucleotides inhibited pre-rRNA cleavage at site A(1), the 5' end of the 18S rRNA, and at site A(2), located 1.9 kb away in internal transcribed spacer 1. This inhibition was largely suppressed by a compensatory mutation in U3, demonstrating functional base pairing. The U3-pre-rRNA base pairing is incompatible with the structure that forms in the mature 18S rRNA and may prevent premature folding of the pre-rRNA. In the Escherichia coli pre-rRNA the homologous region of the 16S rRNA is also sequestered, in that case by base pairing to the 5' external transcribed spacer (5' ETS). Cleavage at site A(0) in the yeast 5' ETS strictly requires base pairing between U3 and a sequence within the 5' ETS. In contrast, the U3-18S interaction is not required for A(0) cleavage. U3 therefore carries out at least two functionally distinct base pair interactions with the pre-rRNA. The nucleotide at the site of A(1) cleavage was shown to be specified by two distinct signals; one of these is the stem-loop structure within the 18S rRNA. However, in contrast to the efficiency of cleavage, the position of A(1) cleavage is not dependent on the U3-loop interaction. We conclude that the 18S stem-loop structure is recognized at least twice during pre-rRNA processing.     

Reconstitution of Bacillus stearothermophilus 50 S ribosomal subunits from purified molecular components.

Bacillus stearothermophilus 50 S ribosomal subunits have been reconstituted from a mixture of purified RNA and protein components. The protein fraction of 50 S subunits was separated into 27 components by a combination of various methods including ion exchange and gel filtration chromatography. The individual proteins showed single bands in a variety of polyacrylamide gel electrophoresis systems, and nearly all showed single spots on two-dimensional polyacrylamide gels. The molecular weights of the proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An equimolar mixture of the purified proteins was combined with 23 S RNA and 5 S RNA to reconstitute active 50 S subunits by the procedure of Nomura and Erdmann (Nomura, M., and Erdmann, V. A. (1970) Nature 226, 1214-1218). Reconstituted 52 S subunits containing purified proteins were slightly more active than subunits reconstituted with an unfractionated total protein extract in poly(U)-dependent polyphenylalanine synthesis and showed comparable activity in various assays for ribosomal function. The reconstitution proceeded more rapidly with the mixture of purified proteins than with the total protein extract. Reconstituted 50 S subunits containing purified proteins co-sedimented with native 50 S subunits on sucrose gradients and had a similar protein compsoition. Initial experiments on the roles of the individual proteins in ribosomal structure and function were performed. B. stearothermophilus protein 13 was extracted from 50 S subunits under the same conditions as escherichia coli L7/L12, and the extraction had a similar effect on ribosomal function. When single proteins were omitted from reconstitution mixtures, in most cases the reconstituted 50 S subunits showed decreased activity in polypheylalanine synthesis.     

Solution structure of the E. coli 70S ribosome at 11.5 A resolution

Over 73,000 projections of the E. coli ribosome bound with formyl-methionyl initiator tRNAf(Met) were used to obtain an 11.5 A cryo-electron microscopy map of the complex. This map allows identification of RNA helices, peripheral proteins, and intersubunit bridges. Comparison of double-stranded RNA regions and positions of proteins identified in both cryo-EM and X-ray maps indicates good overall agreement but points to rearrangements of ribosomal components required for the subunit association. Fitting of known components of the 50S stalk base region into the map defines the architecture of the GTPase-associated center and reveals a major change in the orientation of the alpha-sarcin-ricin loop. Analysis of the bridging connections between the subunits provides insight into the dynamic signaling mechanism between the ribosomal subunits.     

Identification of proteins functionally altered by chemical modification of the transfer RNA and polyuridylic acid binding sites of 30 S ribosomal subunits

Previous studies have shown that Rose Bengal-sensitized photo-oxidation of 30 S ribosomal subunits causes inactivation of tRNA binding and partial loss of poly(U) binding activities (Noller et al., 1971). The present studies, reconstitution of 30 S subunits from 16 S RNA, total protein from modified subunits, and purified proteins from untreated subunits, show that proteins S2 and S3 together completely restore these activities to the reconstituted subunits. The modified proteins are capable of in vitro assembly, and give rise to particles with normal sedimentation constants, showing that restoration of activity is not simply due to correction of an assembly defect.Protein S3 restores poly(U) binding and tRNA binding to the same extent, accounting for the lowered mRNA binding activity of the modified particles as well as a corresponding fraction of the tRNA binding activity. Protein S2 restores the remaining fraction of the tRNA binding activity, but has no effect on poly (U) binding. In 50 S-stimulated tRNA binding, proteins S1 and S5 are required in addition to S2 and S3 for full activity.     

Complementarity between the mRNA 5' untranslated region and 18S ribosomal RNA can inhibit translation

In eubacteria, base pairing between the 3' end of 16S rRNA and the ribosome-binding site of mRNA is required for efficient initiation of translation. An interaction between the 18S rRNA and the mRNA was also proposed for translation initiation in eukaryotes. Here, we used an antisense RNA approach in vivo to identify the regions of 18S rRNA that might interact with the mRNA 5' untranslated region (5' UTR). Various fragments covering the entire mouse 18S rRNA gene were cloned 5' of a cat reporter gene in a eukaryotic vector, and translation products were analyzed after transient expression in human cells. For the largest part of 18S rRNA, we show that the insertion of complementary fragments in the mRNA 5' UTR do not impair translation of the downstream open reading frame (ORF). When translation inhibition is observed, reduction of the size of the complementary sequence to less than 200 nt alleviates the inhibitory effect. A single fragment complementary to the 18S rRNA 3' domain retains its inhibitory potential when reduced to 100 nt. Deletion analyses show that two distinct sequences of approximately 25 nt separated by a spacer sequence of 50 nt are required for the inhibitory effect. Sucrose gradient fractionation of polysomes reveals that mRNAs containing the inhibitory sequences accumulate in the fractions with 40S ribosomal subunits, suggesting that translation is blocked due to stalling of initiation complexes. Our results support an mRNA-rRNA base pairing to explain the translation inhibition observed and suggest that this region of 18S rRNA is properly located for interacting with mRNA.     

Structural and functional exchangeability of 5 S RNA species from the eubacterium E.coli and the thermoacidophilic archaebacterium Sulfolobus solfataricus.

The role of 5 S RNA within the large ribosomal subunit of the extremely thermophilic archaebacterium Sulfolobus solfataricus has been analysed by means of in vitro reconstitution procedures. It is shown that Sulfolobus 50 S subunits reconstituted in the absence of 5 S RNA are inactive in protein synthesis and lack 2-3 ribosomal proteins. Furthermore, it has been determined that in the course of the in vitro assembly process Sulfolobus 5 S RNA can be replaced by the correspondent RNA species of E.coli; Sulfolobus reconstituted particles containing the eubacterial 5 S molecule are stable and active in polypeptide synthesis at high temperatures.     

Alteration of 23 S ribosomal RNA and erythromycin-induced resistance to lincomycin and spiramycin in Staphylococcus aureus

Functionally active “hybrid” 50 S ribosomal subunits can be reconstituted using 23 S RNA from Staphylococcus aureus (strain 1206) and 5 S RNA, as well as 50 S ribosomal proteins from Bacillus stearothermophilus. Using this system, resistance of S. aureus 50 S subunits to lincomycin and spiramycin was analyzed. When 23 S RNA from either phenotypically resistant (“induced resistance”) S. aureuscells or derived genetically resistant (“constitutive resistance”) S. aureus cells, were used, the reconstituted 50 S subunits showed the resistant phenotype similar to that seen in native 50 S subunits obtained from resistant cells; only very weak inhibition by the antibiotics was observed in poly (U) - directed polyphenylalanine synthesis involving these 50 S subunits. In contrast, the 50 S particles reconstituted using 23 S RNA from uninduced (sensitive) S. aureus were subject to greater inhibition by the antibiotics in cell-free poly-peptide synthesis. It is concluded that modification of 23 S RNA, presumably the previously observed methylation to form dimethyladenine, is responsible for the resistance to the antibiotics in this strain of S. aureus.     

Characterization of the Low-Molecular-Weight RNAs Associated with the 70S RNA of Rous Sarcoma Virus

Two low-molecular-weight RNAs are associated with the 70S RNA complex of Rous sarcoma virus: a previously described 4S RNA and a newly identified 5S RNA. The 4S RNA constitutes 3 to 4% of the 70S RNA complex or the equivalent of 12 to 20 molecules per 70S RNA. It exhibits a number of structural properties characteristic of transfer RNA as revealed by two-dimensional electrophoresis of oligonucleotides obtained from a T1 ribonuclease digest of the 4S RNA species. The 5S RNA is approximately 120 nucleotides in length, constitutes 1% of the 70S RNA complex or the equivalent of 3 to 4 molecules per molecules of 70S RNA, and is identical in nucleotide composition and structure to 5S RNA from uninfected chicken embryo fibroblasts. Melting studies indicate that the 5S RNA is released from the 70S RNA complex at the same temperature required to dissociate 70S RNA into its constituent 35S subunits. In contrast, greater than 80% of the 4S RNA is released from 70S RNA prior to its conversion into subunits. The possible biological significance of these 70S-associated RNAs is discussed.     

Topography of 16 S RNA in 30 S subunits and 70 S ribosomes accessibility to cobra venom ribonuclease

A ribonuclease extracted from the venom of the cobra Naja oxiana, which shows an unusual specificity for double-stranded RNA regions, was used to obtain new insight on the topography of Escherichia coli ribosomal 16 S RNA in the 30 S subunit and in the 70 S couple. ³²P-labeled 30 S subunits or reconstituted 70 S tight couples containing ³²P-labeled 16 S RNA have been digested under progressively stronger conditions. The cleavage sites have been precisely localized and the chronology of the hydrolysis process studied.The enzyme cleaves the 16 S RNA within 30 S subunits at 21 different sites, which are not uniformly distributed along the molecule. These results provide valuable information on the 16 S RNA topography and evidence for secondary structure features.The binding of the 50 S subunit markedly reduces the rate of the 16 S RNA hydrolysis and provides protection for several cleavage sites. Four of them are clustered in the 3′-terminal 200 nucleotides of the molecule, one in the middle (at position 772) and one in the 5′ domain (at position 336). Our results provide further evidence that the 3′-terminal and central regions of the RNA chain are close to each other in the ribosome structure and lie at the interface of the two subunits. They also suggest that the 5′ domain is probably not involved exclusively in structure and assembly.     

The initial U3 snoRNA:pre-rRNA base pairing interaction required for pre-18S rRNA folding revealed by in vivo chemical probing

The synthesis of ribosomal subunits in the nucleolus is a conserved, essential process that results in cytoplasmic ribosomes with precisely processed and folded rRNAs assembled with ribosomal proteins. It has been proposed, but never directly demonstrated, that the U3 small nucleolar RNA (snoRNA), a nucleolar component required for ribosome biogenesis, is a chaperone for pre-18S rRNA folding. To test this, we used in vivo chemical probing with dimethyl sulfate to detect changes in pre-rRNA structure upon genetic manipulation of the yeast, Saccharomyces cerevisiae. Based on changes in nucleotide reactivity, we found that the U3 snoRNA is indeed required for folding of the pre-18S rRNA. Furthermore, we detected a new essential base pairing interaction that is likely the initial anchor that recruits the U3 snoRNA to the pre-rRNA, is a prerequisite for the subsequent interactions, and is required for the small subunit processome formation. Substitution of the 5'-ETS nucleotides of the pre-rRNA involved in this initial base pairing interaction is lethal, but growth is restored when a complementary U3 snoRNA is expressed. The U3 snoRNP, via base pairing, and its associated proteins, are part of the required machinery that orchestrates the folding of pre-rRNA that results in the assembly of the small ribosomal subunit.     

Base pairing between U3 and the pre-ribosomal RNA is required for 18S rRNA synthesis.

The nucleolus, the site of pre-ribosomal RNA (pre-rRNA) synthesis and processing in eukaryotic cells, contains a number of small nucleolar RNAs (snoRNAs). Yeast U3 snoRNA is required for the processing of 18S rRNA from larger precursors and contains a region complementary to the pre-rRNA. Substitution mutations in the pre-rRNA which disrupt this base pairing potential are lethal and prevent synthesis of 18S rRNA. These mutant pre-rRNAs show defects in processing which closely resemble the effects of genetic depletion of components of the U3 snoRNP. Co-expression of U3 snoRNAs which carry compensatory mutations allows the mutant pre-rRNAs to support viability and synthesize 18S rRNA at high levels. Pre-rRNA processing steps which are blocked by the external transcribed spacer region mutations are largely restored by expression of the compensatory U3 mutants. Pre-rRNA processing therefore requires direct base pairing between snoRNA and the substrate. Base pairing with the substrate is thus a common feature of small RNAs involved in mRNA and rRNA maturation.     

Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA

Dynamic changes in secondary structure of the 16S rRNA during the decoding of mRNA are visualized by three-dimensional cryo-electron microscopy of the 70S ribosome. Thermodynamically unstable base pairing of the 912-910 (CUC) nucleotides of the 16S RNA with two adjacent complementary regions at nucleotides 885-887 (GGG) and 888-890 (GAG) was stabilized in either of the two states by point mutations at positions 912 (C912G) and 885 (G885U). A wave of rearrangements can be traced arising from the switch in the three base pairs and involving functionally important regions in both subunits of the ribosome. This significantly affects the topography of the A-site tRNA-binding region on the 30S subunit and thereby explains changes in tRNA affinity for the ribosome and fidelity of decoding mRNA.     

Basepairing potential of the 3' terminus of 16S RNA: dependence on the functional state of the 30S subunit and the presence of protein S21.

The deoxyoctanucleotide 5'd (AAGGAGGT) which is complementary to the 3' terminus of 16S RNA has been used as a probe to measure the potential of this rRNA region to engage in intermolecular basepairing. The site specific binding of the octanucleotide is shown by labeling 16S RNA in situ at its 3' end with [32P]pCp and T4 RNA ligase (EC 6.5.1.3.). The label can be released as pA[32P]pCp by the simultaneous action of RNAse H (EC 3.1.4.34) and 5'd(AAGGAGGT). WE show that (1) 30S subunits prepared according to standard procedures, bind less than one copy of 5'd(AAGGAGGT); (2) isolated 16S RNA and 30S subunits inactivated by transcient exposure to 0.5 mM Mg2+ do not bind the octanucleotide; (3) binding to inactive subunits can be restored by a brief heat treatment; (4) 30S subunits lacking protein S21 do not bind 5'd(AAGGAGGT) even when submitted to heat treatment; (5) addition of protein S21 to subunits lacking S21 restores octamer binding; (6) the apparent exposure of the 16S RNA 3' terminus brought about by protein S21 is accompanied by the potential of the subunits to accept MS2 RNA as messenger; (7) the presence or absence of S1 on 30S subunits has no effect on their octanucleotide binding property.     

The 5S rRNA loop E: chemical probing and phylogenetic data versus crystal structure.

A significant fraction of the bases in a folded, structured RNA molecule participate in noncanonical base pairing interactions, often in the context of internal loops or multi-helix junction loops. The appearance of each new high-resolution RNA structure provides welcome data to guide efforts to understand and predict RNA 3D structure, especially when the RNA in question is a functionally conserved molecule. The recent publication of the crystal structure of the "Loop E" region of bacterial 5S ribosomal RNA is such an event [Correll CC, Freeborn B, Moore PB, Steitz TA, 1997, Cell 91:705-712]. In addition to providing more examples of already established noncanonical base pairs, such as purine-purine sheared pairings, trans-Hoogsteen UA, and GU wobble pairs, the structure provides the first high-resolution views of two new purine-purine pairings and a new GU pairing. The goal of the present analysis is to expand the capabilities of both chemical probing and phylogenetic analysis to predict with greater accuracy the structures of RNA molecules. First, in light of existing chemical probing data, we investigate what lessons could be learned regarding the interpretation of this widely used method of RNA structure probing. Then we analyze the 3D structure with reference to molecular phylogeny data (assuming conservation of function) to discover what alternative base pairings are geometrically compatible with the structure. The comparisons between previous modeling efforts and crystal structures show that the intricate involvements of ions and water molecules in the maintenance of non-Watson-Crick pairs render the process of correctly identifying the interacting sites in such pairs treacherous, except in cases of trans-Hoogsteen A/U or sheared A/G pairs for the adenine N1 site. The phylogenetic analysis identifies A/A, A/C, A/U and C/A, C/C, and C/U pairings isosteric with sheared A/G, as well as A/A and A/C pairings isosteric with both G/U and G/G bifurcated pairings. Thus, each non-Watson-Crick pair could be characterized by a phylogenetic signature of variations between isosteric-like pairings. In addition to the conservative changes, which form a dictionary of pairings isosterically compatible with those observed in the crystal structure, concerted changes involving several base pairs also occur. The latter covariations may indicate transitions between related but distinctive motifs within the loop E of 5S ribosomal RNA.     

Prediction of secondary structures of 16S and 23S rRNA fromEscherichia coli

Small and large subunits ofEscherichia coli ribosome have three different rRNAs, the sequences of which are known. However, attempts by three groups to predict secondary structures of 16S and 23S rRNAs have certain common limitations namely, these structures are predicted assuming no interactions among various domains of the molecule and only 40% residues are involved in base pairing as against the experimental observation of 60 % residues in base paired state. Recent experimental studies have shown that there is a specific interaction between naked 16S and 23S rRNA molecules. This is significant because we have observed that the regions (oligonucleotides of length 9–10 residues), in 16S rRNA which are complementary to those in 23S rRNA do not have internal complementary sequences. Therefore, we have developed a simple graph theoretical approach to predict secondary structures of 16S and 23S rRNAs. Our method for model building not only uses complete sequence of 16S or 23S rRNA molecule along with other experimental observations but also takes into account the observation that specific recognition is possible through the complementary sequences between 16S and 23S rRNA molecules and, therefore, these parts of the molecules are not used for internal base pairing. The method used to predict secondary structures is discussed. A typical secondary structure of the complex between 16S and 23S rRNA molecules, obtained using our method, is presented and compared Briefly with earlier model Building studies.     

New features of 23S ribosomal RNA folding: the long helix 41-42 makes a "U-turn" inside the ribosome.

23S rRNA from Escherichia coli was cleaved at single internucleotide bonds using ribonuclease H in the presence of appropriate chimeric oligonucleotides; the individual cleavage sites were between residues 384 and 385, 867 and 868, 1045 and 1046, and 2510 and 2511, with an additional fortuitous cleavage at positions 1117 and 1118. In each case, the 3'' terminus of the 5'' fragment was ligated to radioactively labeled 4-thiouridine 5''-,3''-biphosphate ("psUp"), and the cleaved 23S rRNA carrying this label was reconstituted into 50S subunits. The 50S subunits were able to associate normally with 30S subunits to form 70S ribosomes. Intra-RNA crosslinks from the 4-thiouridine residues were induced by irradiation at 350 nm, and the crosslink sites within the 23S rRNA were analyzed. The rRNA molecules carrying psUp at positions 867 and 1117 showed crosslinks to nearby positions on the opposite strand of the same double helix where the cleavage was located, and no crosslinking was detected from position 2510. In contrast, the rRNA carrying psUp at position 384 showed crosslinking to nt 420 (and sometimes also to 416 and 425) in the neighboring helix in 23S rRNA, and the rRNA with psUp at position 1045 gave a crosslink to residue 993. The latter crosslink demonstrates that the long helix 41-42 of the 23S rRNA (which carries the region associated with GTPase activity) must double back on itself, forming a "U-turn" in the ribosome. This result is discussed in terms of the topography of the GTPase region in the 50S subunit, and its relation to the locations of the 5S rRNA and the peptidyl transferase center.