Chemical modification of guanine residues of mouse 5 S ribosomal RNA with kethoxal. (Nucleosides and nucleotides 46)

Chemical modification of mouse 5 S rRNA with kethoxal was carried out to examine the secondary structure. The guanine residues located at positions 37, 41, 56, 66, 75 and 89 were modified. The relative rates of reaction are in the order G37, G56, G89, G66, G41, G75 at 28 degrees C and G37, G41, G56, G89, G75, G66 at 35 degrees C. These results support a secondary structure model containing 5 helices and 5 loops and indicate that the region around position 37 is the most exposed in higher-order structure.     

Structure and function of 5S ribosomal ribonucleic acid from Torulopsis utilis. III. Detection of single-stranded regions by digestion with nuclease S1

Identification of single-stranded regions in Torulopsis utilis 5S RNA was attempted by the use of Nuclease S1, a single-strand specific endonuclease. When T. utilis 5S RNA was subjected to prolonged incubation with Nuclease S1, about 50% of the substrate 5S RNA remained as large oligonucleotide "cores." Such Nuclease S1-resistant fragments were purified and sequenced by column chromatographic procedures. These analyses revealed that regions around positions 12, 40, 57, and 110 are in exposed single-stranded loops at 37 degrees C and that regions around positions 12 and 40 are most exposed at 20 degrees C. These results are compatible with our secondary structure model for T. utilis 5S RNA (Nishikawa & Takemura (1974) J. Biochem. 76, 935-947) except that the 5' part of the molecule (from the region around position 22 to that around position 57) might have a somewhat looser conformation than our secondary structure model suggests. The implications of such results are also discussed in relation to the presumed function of the sequence C-G-A-U-C (around position 40) as one of the recognition sites for initiator tRNA binding on ribosomes.     

Identification and assignment of base pairs in three helical stems of Torulopsis utilis ribosomal 5S RNA and its RNase T1 and RNase T2 cleaved fragments via 500-MHz proton homonuclear overhauser enhancements

Imino proton resonances in the downfield region (10-14 ppm) of the 500-MHz 1H NMR spectrum of Torulopsis utilis 5S RNA are identified (A X U, G X C, or G X U) and assigned to base pairs in helices I, IV, and V via analysis of homonuclear Overhauser enhancements (NOE) from intact T. utilis 5S RNA, its RNase T1 and RNase T2 digested fragments, and a second yeast (Saccharomyces cerevisiae) 5S RNA whose nucleotide sequence differs at only six residues from that of T. utilis 5S RNA. The near-identical chemical shifts and NOE behavior of most of the common peaks from these four RNAs strongly suggest that helices I, IV, and V retain the same conformation after RNase digestion and that both T. utilis and S. cerevisiae 5S RNAs share a common secondary and tertiary structure. Of the four G X U base pairs identified in the intact 5S RNA, two are assigned to the terminal stem (helix I) and the other two to helices IV and V. Seven of the nine base pairs of the terminal stem have been assigned. Our experimental demonstration of a G X U base pair in helix V supports the 5S RNA secondary structural model of Luehrsen and Fox [Luehrsen, K. R., & Fox, G.E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2150-2154]. Finally, the base-pair proton peak assigned to the terminal G X U in helix V of the RNase T2 cleaved fragment is shifted downfield from that in the intact 5S RNA, suggesting that helices I and V may be coaxial in intact T. utilis 5S RNA.     

Mechanism of ribosomal subunit association: discrimination of specific sites in 16 S RNA essential for association activity.

Modification of 30 S ribosomal subunits with kethoxal causes loss of their ability to associate with 50 S subunits under tight couple conditions. To identify those 16 S RNA sequences important for the association. ³²P-labeled 30 S subunits were partially inactivated by reaction with kethoxal. The remaining association-competent 30 S subunits were selected from the modified population by their ability to form 70 S ribosomes. Comparison of kethoxal diagonal maps of the association-competent subunits with those of the total population of modified subunits reveals nine sites in 16 S RNA whose modification leads to loss of association activity. Eight of these sites were previously found to be protected from kethoxal attack and one was shown to have enhanced reactivity in 70 S ribosomes (Chapman &; Noller, 1977). As before, these sites are not distributed thoughout the molecule, but are found to be clustered in two regions, at the middle and at the 3′ terminus of the 16 S RNA chain.We interpret these findings in terms of a simple preliminary model for the functional organization of 16 S RNA, supported by the observations of other investigators, in which we divide the molecule into four domains. (1) Residues 1 to 600 are involved mainly in structural organization and assembly. (2) Residues 600 to 850 include sites which make contact with the 50 S subunit and are essential for subunit association. (3) Sites from the domain comprising residues 850 to 1350 line a pocket at the interface between the two ribosomal subunits. and contribute to the binding site(s) for transfer RNA. (4) Residues 1350 to 1541 also contain sequences which bind the 50 S subunit, but some sites in this domain alternatively participate in the initiation of protein synthesis.     

The effect of tRNA binding on the structure of 5 S RNA in Escherichia coli. A chemical modification study

The structure of 5 S RNA within the 70 S ribosome from Escherichia coli was studied using the chemical reagent kethoxal (alpha-keto-beta-ethoxybutyraldehyde) to modify accessible guanosines. The modification pattern of 5 S RNA from free 70 S ribosomes was compared with that of poly(U) programmed ribosomes where tRNA had been bound to both the A- and P-sites. Binding to the ribosomal A-site was achieved enzymatically using the elongation factor Tu and GTP in the presence of deacylated tRNA which blocks the ribosomal P-site. Modified guanosines were identified after partial RNase T1 hydrolysis and separation of the hydrolysis products on sequencing gels. Binding of tRNA to the ribosome leads to a strong protection of 5 S RNA guanosine G-41 and to some degree G-44 from kethoxal modification. The limited RNase T1 hydrolysis pattern provides evidence for the existence of a 5 S RNA conformation different from the known 5 S RNA A- and B-forms which are characterized by their gel electrophoretic mobility. The importance of 5 S RNA for the binding of tRNA to the ribosome is discussed.     

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).     

Protection of ribosomal RNA from kethoxal in polyribosomes: Implication of specific sites in ribosome function

In an attempt to probe the topography of 5 S, 16 S and 23 S RNAs in a functionally engaged ribosome, polysomes were probed using the structure-sensitive, guanine-specifie reagent kethoxal. Reactivities of guanine residues at 38 specific ribosomal RNA sites in polysomes were compared with their corresponding reactivities in vacant 70 S ribosomes. No polysome-specific protection was seen for 5 S RNA. In 16 S RNA, positions 530, 693 or 1079, 966, 1338 and 1517 showed protection in polysomes; all of these sites have highly conserved primary and secondary structures, and include several methylated nucleotides. In 23 S RNA, polysome protection is seen at positions 277, 1071, 1475 or 2112, 2116 and 2751. We attribute polysome-specific protection either to direct contact of transfer RNA and/or messenger RNA with the protected sites or to tRNA and/or mRNA-induced changes in ribosome conformation involving the protected sites.     

Physical consequences of chemical modification: circular dichroism of the kethoxalated oligoribonucleotides

Alterations in CD spectra are found in G-containing oligoribonucleotides after modification with kethoxal (beta-ethoxy--alpha-ketobutyraldehyde). Stacking interactions in kethoxalated oligomers are followed by temperature dependence of their CD amplitudes. It is shown that for oligomers with nucleosides in anti-conformation adduct formation destroys the stacking interaction with 3'-neighbour but not with a 5'-neighbour. For nucleosides in non-standard conformation (i.e. syn-conformation of guanine in GpGpCp) the physical alteractions may be seen in those cases, when the substituting group affects the initial conformation or the interplane base contacts via, for instance, blocking NH(2)-group of guanine in GpUp.The results demonstrated that even a single monomer modification in a polymer chain could not be considered as a local event having no influence on the three-dimensional structure. The degree of conformational disorders depends both on the conformation of single nucleotides in the stack and on the nature of the nearest neighbours of the modified base.     

Structure of 4.5S RNA in the signal recognition particle of Escherichia coli as studied by enzymatic and chemical probing.

The structure of 4.5S RNA, the Escherichia coli homologue of the signal recognition particle (SRP) RNA, alone and in the SRP complex with protein P48 (Ffh) was probed both enzymatically and chemically. The molecule is largely resistant against single strand-specific nucleases, indicating a highly base paired structure. Reactivity appears mainly in the apical tetraloop and in one of the conserved internal loops. Although some residues are found reactive toward dimethylsulphate and kethoxal in regions predicted to be unpaired by the phylogenetic secondary structure model of 4.5S RNA, generally the reactivity is low, and some residues in internal loops are not reactive at all. RNase V1 cleaves the RNA at multiple sites that coincide with predicted helices, although the cleavages show a pronounced asymmetry. The binding of protein P48 to 4.5S RNA results in a protection of residues in the apical part of the molecule homologous to eukaryotic SRP RNA (domain IV), whereas the cleavages in the conserved apical tetraloop are not protected. Hydroxyl radical treatment reveals an asymmetric pattern of backbone reactivity; in particular, the region encompassing nucleotides 60-82, i.e., the 3' part of the conserved domain IV, is protected. The data suggest that a bend in the domain IV region, most likely at the central asymmetric internal loop, is an important element of the tertiary structure of 4.5S RNA. Hyperchromicity and lead cleavage data are consistent with the model as they reveal the unfolding of a higher-order structure between 30 and 40 degrees C. Protection by protein P48 occurs in this region of the RNA and, more strongly, in the 5' part of domain IV (nt 26-50, most strongly from 35 to 49). It is likely that P48 binds to the outside of the bent form of 4.5S RNA.     

Transcription of polyoma virus DNA after interaction with nuclear proteins in vitro.

Analysis of the nucleotide sequence at the 5′-triphosphate termini of RNA chains synthesized by T7 RNA polymerase from T7 DNA template indicates that nearly all RNA chains synthesized in this polymerase reaction contain the sequence, pppGpGp. In addition, studies carried out on T7 DNA-dependent ³²PP_i exchange into ribonucleoside triphosphates suggest that immediately following the guanine residues at the 5′-end of RNA formed in the T7 RNA polymerase reaction, there is one or more adenine residues. These results indicate a high degree of specificity of initiation of RNA synthesis by T7 RNA polymerase.     

Interconversion of tight and loose couple 50 S ribosomes and translocation in protein synthesis

On incubation of 50 S ribosomes, isolated from either tight couple (TC) or loose couple (LC) 70 S ribosomes, with elongation factor G (EG-G) and guanosine 5'-triphosphate, a mixture of TC and LC 50 S ribosomes is formed. There is almost complete conversion of LC 50 S ribosomes to TC 50 S ribosomes on treatment with EF-G, GTP, and fusidic acid. Similarly, TC 50 S ribosomes are converted to LC 50 S ribosomes, although partially, by treatment with EF-G and a GTP analogue like guanyl-5'-yl methylenediphosphate (GMP-P(CH2)P) or guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) and including a polymer of 5'-uridylic acid (poly(U] in the incubation mixture. Furthermore, LC 23 S RNA isolated from LC 50 S ribosomes is converted to TC 23 S RNA on heat treatment, but similar treatment does not affect TC 23 S RNA. The interconversion was followed by several physical and biological characteristics of TC and LC 50 S ribosomes, like association capacities with 30 S ribosomes before and after kethoxal treatment, susceptibility to RNase I and polyphenylalanine-synthesizing capacity in association with 30 S ribosomes, as well as thermal denaturation profiles, circular dichroic spectra, and association capacity of isolated 23 S RNAs. These data strongly support the proposition that TC and LC 50 S ribosomes are the products of translocation during protein synthesis. The conformational change of 23 S RNA induced by EF-G and GTP is most probably responsible for the interconversion, and L7/L12 proteins play an important role in the process. A two-site model based on kethoxal data has also been proposed to explain the tightness and looseness of 70 S couples.     

Structure of 5 S ribosomal RNA from Escherichia coli: identification of kethoxal-reactive sites in the A and B conformations.

The topographies of the A and B conformers of free 5 S RNA have been examined using kethoxal as a probe of single-stranded, accessible guanine residues. Each of the kethoxal-reactive guanines has been identified using diagonal electrophoresis, and the relative rate of modification at each site has been studied.Free 5 S RNA in the A form has several reactive guanines in addition to G₁₃ and G₄₁, which are the only two available for reaction in the intact 50 S ribosomal subunit (Noller &; Herr, 1974). The relative reactivities of these sites are G₄₁ ? G₁₃ > G₆₉ > G₂₄ > G₈₆ > G₁₀₇ > G₁₆, G₂₃, G₄₄. Modification at G₂₃ and G₄₄ reaches maximum values of only about 0.05 mol per mol 5 S RNA, suggesting that these residues are unreactive in the major conformer of the A form population. These results are compatible with a secondary structure model based on phylogenetic sequence conservation (Fox &; Woese, 1975), but imply that 12 of the 18 unpaired guanines in this model are involved in further molecular interactions.The modification pattern of the B conformer demands a different base-pairing arrangement and shows that the B form contains less structure than the A form. The relative reactivities in the B form are G₁₃ > G₁₀₂ > G₁₆ > G₂₄, G₄₄ > G₆₁, G₁₀₀ > G₂₃, G₅₁, G₁₀₇ > G₅₄, G₅₆. Several sites show plateaux at submolar modification levels, indicating the existence of some conformational heterogeneity in preparations of the B form of 5 S RNA. Heat-denatured 5 S RNA appears to contain a mixture of conformers including the A and B form.These results place limitations on certain structural and functional models for 5 S RNA. For example, G₄₄, which has often been implicated in base-pairing with tRNA, is accessible in the B form but not in the A form. Yet the B form does not bind the 5 S RNA-specific ribosomal proteins, nor is there evidence for its existence in the ribosome.     

The structure of the RNA binding site of ribosomal proteins S8 and S15.

Proteins S8 and S15 from the 30 S ribosomal subunit of Escherichia coli were bound to 16 S RNA and digested with ribonuclease A. A ribonucleoprotein complex was isolated which contained the two proteins and three noncontiguous RNA subfragments totaling 93 nucleotides, that could be unambiguously located in the 16 S RNA sequence. We present a secondary structural model for the RNA moiety of the binding site complex, in which the two smaller fragments are extensively base-paired, respectively, to the two halves of the large fragment, to form two disconnected duplexes. Each of the two duplexes is interrupted by a small internal loop. This model is supported by (i) minimum energy considerations, (ii) sites of cleavage by ribonuclease A, and (iii) modification by the single strand-specific reagent kethoxal. The effect of protein binding on the topography of the complex is reflected in the kethoxal reactivity of the RNA moiety. In the absence of the proteins, 5 guanines are modified; 4 of these, at positions 663, 732, 733, and 741, are strongly protected from kethoxal when protein S15 is bound.     

Isolation and anaylsis of (Gp)nXp sequences of rat liver 5S RNA by means of restricted ribonucleas T2 hydrolysis

Essentual difficulties arise when base number in oligoguanylic blocks and location of these blocks along the polynucleotide chain need to be determined in the course of determination of the nucleotide sequences in ribonucleic acids. To overcome this difficulty it is suggested to take advantage of a recently discovered resistance of phosphodiester bond between kethoxalated G and its 3'-neighbour against T(2) RNase hydrolysis 1,2. The approach is illustrated by analysis of 5S RNA from rat liver. Sequences of general formula (Gp)(n)Xp were isolated from T(2) RNase hydrolysate of 5 S RNA rapidly and quantitatively. The information obtained greatly facilitates the whole procedure of sequencing. It is expected that the method proposed would be effective for analysis of 5 S and 4 S RNA and for highmolecular weight fragments of ribosomal and viral RNAs.     

Oligonucleotide directed mutagenesis of Escherichia coli 5S ribosomal RNA: construction of mutant and structural analysis.

The ribosomal 5S RNA gene from the rrnB operon of E. coli was mutagenised in vitro using a synthetic oligonucleotide hybridised to M13 ssDNA containing that gene. The oligonucleotide corresponded to the 5S RNA sequence positions 34 to 51 and changed the guanosine at position 41 to a cytidine. The DNA containing the desired mutation was identified by dot blot hybridisation and introduced back into the plasmid pKK 3535 which contains the total rrnB operon in pBR 322. Plasmid coded 5S rRNA was selectively labeled with 32p using a modified maxi-cell system, and the replacement of guanosine G41 by cytidine was confirmed by RNA sequencing. The growth of cells containing mutant 5S rRNA was not altered by the base change, and the 5S rRNA was processed and incorporated into 50S ribosomal subunits and 70S ribosomes. The structure of wildtype and mutant 5S rRNA was compared by chemical modification of accessible guanosines with kethoxal and limited enzymatic digestion using RNase T1 and nuclease S1. These results showed that the wildtype and mutant 5S rRNA do not differ significantly in their structure. Furthermore, the formation, interconversion and stability of the two 5S rRNA A- and B-conformers are unchanged.     

Application of a rapid gel method to the sequencing of fragments of 16S ribosomal RNA from Escherichia coli.

A gel sequencing method has been applied to two 5' end-labelled fragments of the 16S ribosomal RNA from E. coli. The procedure involves partial enzymatic hydrolysis by ribonucleases T1, U2 or A, in order to generate series of end-labelled subfragments terminating in guanine, adenine, or pyrimidine residues, respectively. The two fragments concerned were approximately 75 and 90 nucleotides in length, and both arose from the 3' region of the 16S RNA. The sequences deduced are compared with the published sequence of 16S RNA, and contribute information to the final ordering of the ribonuclease T1 oligonucleotides in the latter, as well as revealing some probable errors.     

Secondary structure model for bacterial 16S ribosomal RNA: phylogenetic, enzymatic and chemical evidence.

We have derived a secondary structure model for 16S ribosomal RNA on the basis of comparative sequence analysis, chemical modification studies and nuclease susceptibility data. Nucleotide sequences of the E. coli and B. brevis 16S rRNA chains, and of RNAse T1 oligomer catalogs from 16S rRNAs of over 100 species of eubacteria were used for phylogenetic comparison. Chemical modification of G by glyoxal, A by m-chloroperbenzoic acid and C by bisulfite in naked 16S rRNA, and G by kethoxal in active and inactive 30S ribosomal subunits was taken as an indication of single stranded structure. Further support for the structure was obtained from susceptibility to RNases A and T1. These three approaches are in excellent agreement. The structure contains fifty helical elements organized into four major domains, in which 46 percent of the nucleotides of 16S rRNA are involved in base pairing. Phylogenetic comparison shows that highly conserved sequences are found principally in unpaired regions of the molecule. No knots are created by the structure.     

Three-dimensional model of Escherichia coli ribosomal 5 S RNA as deduced from structure probing in solution and computer modeling.

The conformation of Escherichia coli 5 S rRNA was investigated using chemical and enzymatic probes. The four bases were monitored at one of their Watson-Crick positions with dimethylsulfate (at C(N-3) and A(N-1], with a carbodiimide derivative (at G(N-1) and U(N-3] and with kethoxal (at G(N-1, N-2]. Position N-7 of purine was probed with diethylpyrocarbonate (at A(N-7] and dimethylsulfate (at G(N-7]. Double-stranded or stacked regions were tested with RNase V1 and unpaired guanine residues with RNase T1. We also used lead(II) that has a preferential affinity for interhelical and loop regions and a high sensitivity for flexible regions. Particular care was taken to use uniform conditions of salt, magnesium, pH and temperature for the different enzymatic chemical probes. Derived from these experimental data, a three dimensional model of the 5 S rRNA was built using computer modeling which integrates stereochemical constraints and phylogenetic data. The three domains of 5 S rRNA secondary structure fold into a Y-shaped structure that does not accommodate long-range tertiary interactions between domains. The three domains have distinct structural and dynamic features as revealed by the chemical reactivity and the lead(II)-induced hydrolysis: domain 2 (loop B/helix III/loop C) displays a rather weak structure and possesses dynamic properties while domain 3 (helix V/region E/helix IV/loop D) adopts a highly structured and overall helical conformation. Conserved nucleotides are not crucial for the tertiary folding but maintain an intrinsic structure in the loop regions, especially via non-canonical pairing (A.G, G.U, G.G, A.C, C.C), which can close the loops in a highly specific fashion. In particular, nucleotides in the large external loop C fold into an organized conformation leading to the formation of a five-membered loop motif. Finally, nucleotides at the hinge region of the Y-shape are involved in a precise array of hydrogen bonds based on a triple interaction between U14, G69 and G107 stabilizing the quasi-colinearity of helices II and V. The proposed tertiary model is consistent with the localization of the ribosomal protein binding sites and possesses strong analogy with the model proposed for Xenopus laevis 5 S rRNA, indicating that the Y-shape model can be generalized to all 5 S rRNAs.     

Secondary structure of the 5' end of bacteriophage MS2 RNA Methoxyamine and kethoxal modification.

To refine the secondary structure model of the 5' end of the bacteriophage MS2 genome, 32P-labeled MS2 RNA was partially digested with T1 RNase or with Cm-RNase and the 5'-end fragment was isolated, renatured and submitted to treatment with methoxyamine or kethoxal. The resulting modified RNA was digested with T1 RNase and the products were separated by minifingerprinting. Methoxyamine-induced modification of exposed cytidines was detected by differential mobility of modified oligonucleotides, while kethoxal-induced alteration of exposed guanosines was monitored by resistance to T1 ribonuclease digestion. The positions of the modified residues are discussed in terms of an improved secondary structure model proposed for the 5' end of the viral RNA. The structure itself is discussed in relation to sequence conservation and biological function.     

Direct covalent mercuration of nucleotides and polynucleotides.

Nucleotides of cytosine and uracil are readily mercurated by heating at 37-50 degrees in buffered aqueous solutions (pH 5.0-8.0) containing mercuric acetate. Proton magnetic resonance, elemental, electrophoretic, and chromatographic analyses have shown the products to be 5-mercuricytosine and 5-mercuriuracil derivatives, where the mercury atom is covalently bonded. Polynucleotides can be mercurated under similar conditions. Cytosine and uracil bases are modified in RNA while only cytosine residues in DNA are substituted. There is little, if any, reaction with adenine, thymine, or guanine bases. The rate of polymer mercuration is, unlike that of mononucleotides, markedly influenced by the ionic strength of the reaction mixture: the lower the ionic strength the faster the reaction rate. Pyrimidine residues in single- and double-stranded polymers react at essentially the same rate. Although most polynucleotides can be extensively mercurated at pH 7.0 in sodium or Trisacetate buffers, tRNA undergoes only limited substitution in Tris buffers. The mild reaction conditions give minimal single-strand breakage and, unlike direct iodination procedures, do not produce pyrimidine hydrates. Mercurated polynucleotides can be exploited in a variety of ways, particularly by crystallographic and electron microscopic techniques, as tools for studying polynucleotide structure.