Structure of a U.U pair within a conserved ribosomal RNA hairpin.

A conserved hairpin corresponding to nt 1057-1081 of large subunit rRNA (Escherichia coli numbering) is part of a domain targeted by antibiotics and ribosomal protein L11. The stem of the hairpin contains a U.U juxtaposition, found as either U.U or U.C in virtually all rRNA sequences. This hairpin has been synthesized and most of the aromatic and sugar protons were assigned by two-dimensional proton NMR. Distances and sugar puckers deduced from the NMR data were combined with restrained molecular dynamics calculations to deduce structural features of the hairpin. The two U residues are stacked in the helix, form one NH3-O4 hydrogen bond and require an extended backbone conformation (trans alpha and gamma) at one of the U nucleotides. The hairpin loop, UAGAAGC closed by a U-A pair, is the same size as tRNA anticodon loops, but not as well ordered.     

Secondary methylation of yeast ribosomal precursor RNA.

The timing of methylation of the ribosomal sequences of ribosomal precursor RNA (pre-rRNA) from the yeast Saccharomyces carlsbergensis was investigated by fingerprint analysis of the methylated oligonucleotides derived from the various precursors. From the total of 37 ribose and 6 base-methyl groups found in 26-S rRNA, the two copies of the base-methylated nucleoside m3U as well as the doubly methylated sequence Um-Gm psi are not yet present in 37-S RNA, the predominant common precursor of 26-S and 17-S rRNA. Introduction of these methyl groups into the ribosomal sequences appears to take place at the level of 29-S pre-rRNA, the immediate precursor to 26-S rRNA. From the total of 18 ribose-methylated and 6 base-methylated nucleosides found in 17-S rRNA, the latter group (one copy of m7G, the m62A-m62A- sequence and the hypermodified methylated nucleoside "mX") is completely missing in 37-S pre-rRNA. The methyl group of m7G is introduced into 18-S pre-rRNA, the direct precursor of 17-S rRNA, in the nucleus. The -m62A-m62A- sequence is methylated after transport of the 18-S pre-rRNA to the cytoplasm prior to the final maturation into 17-S rRNA.     

Variable methylation of the ribosomal RNA genes of the rat.

Both the pattern and level of rRNA gene methylation vary in the rat. This variation reflects stages in the maturation process and perhaps the level of gene expression in different tissues. We studied methylation at a common site, the inner cytosine of the sequence CCGG, by hybridizing 32P-rRNA to DNA digests obtained with endonuclease Msp I (which cleaves CCGG and CMCGG) and its isochizomer, HpaII (which cleaves only CCGG). In the liver, the changing pattern of rRNA gene methylation reflected the late stages of development: the rRNA genes were mostly unmethylated at 14 days gestation; by 18 days gestation, about 30% of them were methylated, and this level persisted into adulthood. In 18-day DNA, the methylation was uniform, but in adult DNA, the methylation pattern was discontinuous, because otherwise methylated genes contained a demethylated region. Similar developmental changes were observed in brain DNA. In a tissue culture cell line, the change from the continuous to the discontinuous pattern of the methylation could be induced by transformation with Kirsten sarcoma virus. And, in adult tissues, the lowest level of rRNA gene methylation was found in rapidly growing jejunal epithelium, and the highest level, in non-growing spermatozoa.     

Crystal structure of RumA, an iron-sulfur cluster containing E. coli ribosomal RNA 5-methyluridine methyltransferase

RumA catalyzes transfer of a methyl group from S-adenosylmethionine (SAM) specifically to uridine 1939 of 23S ribosomal RNA in Escherichia coli to yield 5-methyluridine. We determined the crystal structure of RumA at 1.95 A resolution. The protein is organized into three structural domains: The N-terminal domain contains sequence homology to the conserved TRAM motif and displays a five-stranded beta barrel architecture characteristic of an oligosaccharide/oligonucleotide binding fold. The central domain contains a [Fe(4)S(4)] cluster coordinated by four conserved cysteine residues. The C-terminal domain displays the typical SAM-dependent methyltransferase fold. The catalytic nucleophile Cys389 lies in a motif different from that in DNA 5-methylcytosine methyltransferases. The electrostatic potential surface reveals a predominately positively charged area that covers the concave surface of the first two domains and suggests an RNA binding mode. The iron-sulfur cluster may be involved in the correct folding of the protein or may have a role in RNA binding.     

Calorimetric measurements of the destabilisation of a ribosomal RNA hairpin by dimethylation of two adjacent adenosines.

Fragments of 16S ribosomal RNA from E. coli and B. stearothermophilus, respectively comprising the 49 and 52 3' terminal nucleotides have been studied thermodynamically using high sensitivity differential scanning calorimetry. The fragments were isolated after cleavage of 16S rRNA in the ribosome by the bacteriocin cloacin DF13. Comparison of the thermal properties of the E. coli fragments with those derived from a kasugamycin resistant mutant, which specifically lacks dimethylation of two adjacent adenosines was employed to study the effect of the methylgroups on the thermal stability. Both E. coli species exhibit similar complex melting patterns with several transitions. Overall molar transition enthalpies are equal and do not depend significantly on buffer conditions (120 kcal/mol at 15 mM Na+ to 136 kcal/mol at 215 mM Na+). However, the transition with the highest Tm, corresponding to unfolding of a nine basepair central helix is lowered by the dimethylation of the adenines in the four-membered loop. This decrease amounts to 4 degrees C at 15 mM Na+ and 2 degrees C at 215 mM Na+. The corresponding nine basepair long hairpin in the Bacillus fragment melts at a temperature of 70 degrees C at 15 mM Na+. This Tmax is much higher than expected on the basis of the sequence in the hairpin.     

Mutants lacking individual ribosomal proteins as a tool to investigate ribosomal properties.

We have isolated and characterized mutants which lack one or two of sixteen of the proteins of the Escherichia coli ribosome. The mutation responsible in each case mapped close to, and probably in, the corresponding gene. A conditional lethal phenotype and a variable degree of impairment in growth was observed. The missing protein was readily restored to the organelle if E coli or other eubacterial ribosomal proteins were added to a suspension of the mutant particles. The mutants have been used to investigate the role of individual proteins in ribosome function and assembly. They have also aided in the topographic pinpointing of proteins on the surface of the organelle.     

Synthesis and characterization of modified nucleotides in the 970 hairpin loop of Escherichia coli 16S ribosomal RNA

The synthesis of the 6-O-DPC-2-N-methylguanosine (m²G) nucleoside and the corresponding 5′-O-DMT-2′-O-TOM-protected 6-O-DPC-2-N-methylguanosine phosphoramidite is reported [DPC, diphenyl carbamoyl; DMT, 4,4′-dimethoxytrityl; TOM, [(triisopropylsilyl)oxy]methyl]. The availability of the phosphoramidite allows for syntheses of hairpin RNAs with site-selective incorporation of 2-N-methylguanosine modification. Four 18-nt hairpin RNA analogues representing the 970-loop region (helix 31 or h31; U960–A975) of Escherichia coli 16S rRNA were synthesized with and without modifications in the loop region. Subsequently, stabilities and conformations of the singly and doubly modified RNAs were examined and compared with the corresponding unmodified RNA. Thermodynamic parameters and circular dichroism spectra are presented for the four helix 31 RNA analogues. Surprisingly, methylations in the loop region of helix 31 slightly destabilize the hairpin, which may have subtle effects on ribosome function. The hairpin construct is suitable for future ligand-binding experiments.     

Structure of a small RNA hairpin.

The hairpin stem-loop form of the RNA oligonucleotide rCGC(UUU)GCG has been studied by NMR spectroscopy. In 10 mM phosphate buffer this RNA molecule forms a unimolecular hairpin with a stem of three base pairs and a loop of three uridines, as judged by both NMR and UV absorbance melting behavior. Distance and torsion angle restraints were determined using homonuclear proton-proton and heteronuclear proton-phosphorus 2-D NMR. These values were used in restrained molecular dynamics to determine the structure of the hairpin. The stem has characteristics of A-form geometry, although distortion from A-form occurs in the 3'-side of the stem, presumably to aid in accommodating the small loop. The loop nucleotides adopt C2'-endo conformations. NOE's strongly suggest stacking of the uracils with the stem, especially the first uracil on the 5'-side of the loop. The reversal of the chain direction in the loop seems to occur between U5 and U6. Loop structures produced by molecular dynamics simulations had a wide range of conformations and did not show stacking of the uracils. A flexible loop with significant dynamics is consistent with all the data.     

Cytoplasmic methylation of mature 5.8 S ribosomal RNA

Levels of 2-O-methylation were determined in ribosomal 5·8 S RNAs from whole cells and both the nuclear and cytoplasmic fractions of rat liver, rat kidney cells in culture (NRK) and HeLa cells. All 5·8 S RNA molecules contained the alkali stable Gm-Cp dinucleotide at position 77 but only whole cell rat liver RNA contained large amounts (0·7 mol) of Um at position 14. All nuclear 5·8 S RNA fractions were largely undermethylated at this site. In contrast, cytoplasmic 5.8 S RNA from rat liver and, to a lesser degree, NRK cells contained significantly more Um; up to 80% of the molecules from rat liver contained the methylated residue. These results indicate that mature 5·8 S RNA can be methylated in the cytoplasm. When labeling kinetics were examined in NRK cells, the methylation at residue 14 was found to increase as a function of the time spent in the cytoplasm, confirming that this modification is, unlike other ribosomal RNA methylations, in part or largely cytoplasmic.     

The role of 5-S RNA in temperature-sensitive ribosomal RNA maturation.

The synthesis of 5-S RNA was found to be unchanged at both the permissive (33.5 degrees C) and non-permissive (38.5 degrees C) temperatures in a temperature-sensitive Baby Hamster Kidney cell line (BHK 21 ts 422 E) as measured relative to synthesis of 18-S rRNA. The 5-S RNA is shown to be associated with nucleolar ribonucleoprotein particles even though rRNA processing does not yield a functional 28-S rRNA at the non-permissive temperature. The amount of 5-S RNA found associated with the 80-S ribonucleoprotein particles was the same at the permissive and non-permissive temperatures, indicating that an aberrant 5-S RNA contribution to rRNA processing is not a primary cause for the temperature-sensitive lesion of rRNA maturation in this mutant cell line. The amount of 5-S RNA in nucleolar 80-S RNA particles indicated that the association of 5-S RNA with the rRNA precursor particle occurs before the cleavage step at which 32-S precursor RNA is produced.     

Conformational and thermodynamic effects of naturally occurring base methylations in a ribosomal RNA hairpin of Bacillus stearothermophilus.

The 3'-terminal colicin fragments of 16S ribosomal RNA were isolated from Bacillus stearothermophilus and from its kasugamycin-resistant (ksgA) derivative lacking N6-dimethylation of the two adjacent adenosines in a hairpin loop. The fragment from the ksgA strain still contains a naturally occurring N2-methylguanosine in the loop. An RNA molecule resembling the B. stearothermophilus colicin fragment but without modified nucleosides was synthesized in vitro using a DNA template and bacteriophage T7 RNA polymerase. Proton-NMR spectra of the RNAs were recorded at 500 MHz. The imino-proton resonances of base-paired G and U residues could be assigned on the basis of previous NMR studies of the colicin fragment of Escherichia coli and by a combination of methylation-induced shifts and thermal melting of base pairs. The assignments were partly confirmed by NOE measurements. Adenosine dimethylation in the loop has a distinct conformational effect on the base pairs adjoining the loop. The thermal denaturation melting curve of the enzymatically synthesized RNA fragment was also determined and the transition midpoint (tm) was found to be 73 degrees C at 15 mM Na+. A comparison with previously determined thermodynamic parameters for various colicin fragments demonstrates that base methylations in the loop lead to a relatively strong destabilization of the hairpin helix. In terms of free energy the positive contribution of the methylations are in the order of the deletion of one base pair from the stem. Other data show that recently published free-energy parameters do not apply for certain RNA hairpins.     

Inducible ribosomal RNA methylation in Streptomyces lividans, conferring resistance to lincomycin

Streptomyces lividans TK21 possesses inducible ribosomal RNA methylase activity that confers high-level resistance to lincomycin and lower levels of resistance to certain macrolides. The methylase gene (designated lrm) is inducible by erythromycin and other macrolides and also by celesticetin (a lincosamide) but not by lincomycin. The lrm enzyme monomethylates the N6-amino group of adenosine at position 2058 within 23S-like ribosomal RNA.     

Using phosphorothioate-substituted RNA to investigate the thermodynamic role of phosphates in a sequence specific RNA-protein complex

Part of the binding affinity and specificity in RNA-protein complexes is often contributed by contacts between the protein and backbone phosphates that are held in position by the RNA structure. This study focuses on the well-characterized interaction between a dimer of the MS2 coat protein and a small RNA hairpin. Using a short oligoribonucleotide which contains all the necessary sequence elements required for tight protein binding, a single phosphorothioate linkage was introduced at 13 different positions. In each case, the R(P) and S(P) stereoisomers were separated and their affinities to the MS2 coat protein were determined. Comparison of these biochemical data with the crystal structure of the protein-hairpin complex indicates that introduction of a phosphorothioate only affects binding at sites where a protein-phosphate contact is observed in the crystal structure. This means that phosphorothioate-containing oligoribonucleotides should also be useful for mapping phosphate contacts in RNA-protein complexes for which no crystal structure is available.     

Studies on the methylation of cytoplasmic ribosomal RNA from cultured higher plant cells.

The methylation of cytoplasmic ribosomal RNA of cultured sycamore cells (Acer pseudoplatanus L.) was investigated. Labelled 17-S and 26-S rRNA were prepared from cells that had been incubated with either [32P]phosphate, [Me-3H]methionine or [Me-14C]methionine. Ion-exchange resin chromatography of 0.3 M KOH or 1 M HCl hydrolysates and two-dimensional chromatographic analyses of phosphodiesterase plus phosphatase digests of 17-S and 26-S rRNA were performed. 17-S and 26-S rRNA contain 49 and 91 methyl groups per molecule, respectively. These values were verified in sevemral ways. The high degree of methylation of sycamore rRNA, particularly for the 26-S rRNA, contrasts with the situation in all other investigated organisms. Several methylated bases were identified. 7-Methylguanine and 5-methylcytosine both occur in 17-S and 26-S rRNA. N6-Methyladenine and N6,N6-dimethyladenine are restricted to the 17-S rRNA while 3-methyluracil and 1-methyladenine occur in the 26-S rRNA. One hypermodified uridine was also tentatively identified in the small rRNA. In 17-S rRNA, there is one copy of 7-methylguanine, N6-methyladenine and hypermodified uridine and two copies of N6,N6-dimethyladenine. 3-Methyluracil, 1-methyladenine and 5-methylcytosine occur twice, twice and three times, respectively, in 26-S rRNA. 7-Methylguanine and 5-methylcytosine are only in submolar amounts in the 26-S and 17-S rRNA, respectively. There are 40 +/- 2 and 83 +/- 3 2'-O-methylriboses per 17-S and 26-S rRNA molecule, respectively. In addition to the four 2'-O-methylnucleosides, one 2'-O-methylpseudouridine is present in the 17-S rRNA. Several lines of evidence argues for a non-random distribution of the methylriboses. In particular, one and seven Nm-Nm-Np structures occur in the 17-S and 26-S rRNA, respectively. The data are discussed comparatively with the methylation pattern of Escherichia coli, yeast and HeLa cell rRNA.