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RNA-modifying machines in archaea.

It has been known for nearly half a century that coding and non-coding RNAs (mRNA, and tRNAs and rRNAs respectively) play critical roles in the process of information transfer from DNA to protein. What is both surprising and exciting, are the discoveries in the last decade that cells, particularly eukaryotic cells, contain a plethora of non-coding RNAs and that these RNAs can either possess catalytic activity or can function as integral components of dynamic ribonucleoprotein machines. These machines appear to mediate diverse, complex and essential processes such as intron excision, RNA modification and editing, protein targeting, DNA packaging, etc. Archaea have been shown to possess RNP complexes; some of these are authentic homologues of the eukaryotic complexes that function as machines in the processing, modification and assembly of rRNA into ribosomal subunits. Deciphering how these RNA-containing machines function will require a dissection and analysis of the component parts, an understanding of how the parts fit together and an ability to reassemble the parts into complexes that can function in vitro. This article summarizes our current knowledge about small-non-coding RNAs in Archaea, their roles in ribosome biogenesis and their relationships to the complexes that have been identified in eukaryotic cells.

Amino Acid Sequence↗

23S rRNA positions essential for tRNA binding in ribosomal functional sites.

rRNA plays an important role in function of peptidyl transferase, the catalytic center of the ribosome responsible for the peptide bond formation. Proper placement of the peptidyl transferase substrates, peptidyl-tRNA and aminoacyl-tRNA, is essential for catalysis of the transpeptidation reaction and protein synthesis. In this report, we define a small set of rRNA nucleotides that are most likely directly involved in binding of tRNA in the functional sites of the large ribosomal subunit. By binding biotinylated tRNA substrates to randomly modified large ribosomal subunits from Escherichia coli and capturing resulting complexes on the avidin resin, we identified four nucleotides in the large ribosomal subunit rRNA (positions G2252, A2451, U2506, and U2585) whose modifications prevent binding of a peptidyl-tRNA analog in the P site and one residue (U2555) whose modification interferes with transfer of peptidyl moiety to puromycin. These nucleotides represent a subset of positions protected by tRNA analogs from chemical modification and significantly narrow the number of 23S rRNA nucleotides that may be directly involved in tRNA binding in the ribosomal functional sites.

Binding Sites↗

Structure of a class II TrmH tRNA-modifying enzyme from Aquifex aeolicus.

Biological RNAs contain a variety of post-transcriptional modifications that facilitate their efficient function in the cellular environment. One of the two most common forms of modification is methylation of the 2'-hydroxyl group of the ribose sugar, which is performed by a number of S-adenosylmethionine (SAM) dependent methyltransferases. In bacteria, many of these modifications in tRNA and rRNA are carried out by the alpha/beta-knot superfamily of enzymes, whose SAM-binding pocket is created by a characteristic deep trefoil knot. TrmH, an enzyme found throughout all three kingdoms of life, modifies the universally conserved guanosine 18 position of tRNA. The crystal structure of TrmH from the thermophilic bacterium Aquifex aeolicus has been determined at 1.85 A resolution using data collected from a synchrotron-radiation source. The protein reveals a fold typical of members of the SpoU clan of proteins, a subfamily of the alpha/beta-knot superfamily, with alpha-helical extensions at the N- and C-termini that are likely to be involved in tRNA binding.

Bacteria↗

A novel erythromycin resistance methylase gene (ermTR) in Streptococcus pyogenes.

Erythromycin resistance among streptococci is commonly due to target site modification by an rRNA-methylating enzyme, which results in coresistance to macrolide, lincosamide, and streptogramin B antibiotics (MLSB resistance). Genes belonging to the ermAM (ermB) gene class are the only erythromycin resistance methylase (erm) genes in Streptococcus pyogenes with MLSB resistance that have been sequenced so far. We identified a novel erm gene, designated ermTR, from an erythromycin-resistant clinical strain of S. pyogenes (strain A200) with an inducible type of MLSB resistance. The nucleotide sequence of ermTR is 82.5% identical to ermA, previously found, for example, in Staphylococcus aureus and coagulase-negative staphylococci. Our finding provides the first sequence of an erm gene other than ermAM that mediates MLSB resistance in S. pyogenes.

Amino Acid Sequence↗

cDNA cloning and characterization of the human U3 small nucleolar ribonucleoprotein complex-associated 55-kilodalton protein.

The eukaryotic nucleolus contains a large number of small RNA molecules (snoRNAs) which, in the form of small nucleolar ribonucleoprotein complexes (snoRNPs), are involved in the processing and modification of pre-rRNA. The most abundant and one of the best-conserved snoRNAs is the U3 RNA. So far, only one human U3 snoRNA-associated protein, fibrillarin, has been characterized. Previously, the U3 snoRNPwas purified from CHO cells, and three proteins of 15, 50, and 55 kDa were found to copurify with the U3 snoRNA (B. Lübben, C. Marshallsay, N. Rottmann, and R. Lührmann, Nucleic Acids Res. 21:5377-5385, 1993). Here we report the cDNA cloning and characterization of the human U3 snoRNP-associated 55-kDa protein. The isolated cDNA codes for a novel nucleolar protein which is specifically associated with the U3 snoRNA. This protein, referred to as hU3-55k, is the first characterized U3 snoRNP-specific protein from humans. hU3-55k is a new member of the family of WD-40 repeat proteins and is conserved throughout evolution. It appears that the C-terminal end of hU3-55k is required for nucleolar localization and U3 snoRNA binding.

Amino Acid Sequence↗

A systematic, ligation-based approach to study RNA modifications.

Over 100 different chemical types of modifications have been identified in thousands of sites in tRNAs, rRNAs, mRNAs, small nuclear RNAs, and other RNAs. Some modifications are highly conserved, while others are more specialized. They include methylation of bases and the ribose backbone, rotation, and reduction of uridine, base deamination, elaborate addition of ring structures, carbohydrate moieties, and more. We have developed a systematic approach to detect and quantify the extent of known RNA modifications. The method is based on the enzymatic ligation of oligonucleotides using the modified or unmodified RNA as the template. The efficiency of ligation is very sensitive to the presence and the type of modifications. First, two oligo pairs for each type of modification are identified. One pair greatly prefers ligation using the unmodified RNA template over the modified RNA template or vice versa. The other pair has equal reactivity with unmodified and modified RNA. Second, separate ligations with each of the two oligo pairs and the total RNA mixture are performed to detect the presence or absence of modifications. Multiple modification sites can be examined in the same ligation reaction. The feasibility of this method is demonstrated for three 2'O-methyl modification sites in yeast rRNA.

DNA Methylation↗

[Affinity labeling of the ribosomes from Escherichia coli by 4-(N-2-chloroethyl-N-methyl-aminobenzyl-5'-phosphoamide of heptauridylic acid].

4-(N-2-chloroethyl-N-methyl)aminobenzyl-5'-phosphoamide of the heptauridylic acid was used to localize the structures organizing mRNA-binding site of ribosomes from E. coli. Both rRNA and proteins undergo modification in the ribosome 10-18 and 82-90% of the total ribosomal subunit modification, respectively. Using the method of two-dimensional electrophoresis in the acrylamide gel the proteins S5, S11, S13 and L2, L7/L12, L31 which are labelled by the analog of mRNA were identified. We assume these proteins to be located in the vicinity of mRNA-binding centre of ribosomes.

Affinity Labels↗

A sparsomycin-resistant mutant of Halobacterium salinarium lacks a modification at nucleotide U2603 in the peptidyl transferase centre of 23 S rRNA.

Sparsomycin, a broad-spectrum antibiotic, acts at the peptidyl transferase centre of the ribosome, stabilizing peptidyl-tRNA binding at the P-site and weakening ternary complex binding. A sparsomycin-resistant mutant was isolated for the archaeon Halobacterium salinarium and shown to lack a post-transcriptional modification of U2603 (Escherichia coli numbering U2584), which is a universally conserved uridine base located within the peptidyl transferase loop of 23 S rRNA. This mutant also exhibited altered sensitivities to the peptidyl transferase antibiotics anisomycin, chloramphenicol and puromycin. Several lines of evidence indicate that the unmodified uridine base lies within the P-substrate site of the peptidyl transferase centre.

Anti-Bacterial Agents↗

Implications of a functional large ribosomal RNA with only three modified nucleotides.

The sequence and structure of the peptidyl transferase region of large subunit ribosomal RNA is highly conserved and specific modified nucleotides could be important structural or functional elements in the catalytic center responsible for peptide bond formation. In fact, it has not been possible to reconstitute active E coli 50S subunits from in vitro transcripts of 23S rRNA and total 50S proteins. It is significant therefore, that the PET56 gene of yeast encodes an essential ribose methyltransferase that specifically modifies a universally conserved nucleotide, G2270, in the peptidyl transferase center of the mitochondrial large ribosomal RNA (21S). Since the loss of this modification in yeast mitochondrial 21S rRNA severely affects the assembly of 54S subunits, it is likely that the analogous 2'-O-methylguanosine at position 2251 (Gm2251) in E coli 23S rRNA is also required for the assembly of 50S subunits. Gm could be a critical structural determinant for the correct folding of the rRNA, the binding of one or more ribosomal proteins, or the interaction of the rRNA with tRNA. Previous work has shown that the mitochondrial large rRNAs are minimally modified relative to the E coli and eukaryotic cytoplasmic rRNAs. By direct chemical analysis using combined high performance liquid chromatography-mass spectrometry, the modification status of the yeast mitochondrial rRNAs was reexamined, revealing the presence of Gm, Um and pseudouridine (psi) in 21S rRNA. The Um was mapped to nucleotide 2791, which corresponds to the ribose methylated and universally conserved U2552 in E coli 23S rRNA, and the psi has been recently mapped to position 2819, which corresponds to psi 2580 in E coli 23S rRNA. The retention of Um and psi nucleotides in the peptidyl transferase center of the otherwise minimally modified mitochondrial rRNAs suggests that these modifications, like Gm2270, might be essential for ribosome assembly or function or both.

Base Sequence↗

RecA-Mediated gene conversion and aminoglycoside resistance in strains heterozygous for rRNA.

Clinical resistance to aminoglycosides in general is due to enzymatic drug modification. Mutational alterations of the small ribosomal subunit rRNA have recently been found to mediate acquired resistance in bacterial pathogens in vivo. In this study we investigated the effect of 16S rRNA heterozygosity (wild-type [wt] and mutant [mut] operons at position 1408 [1408wt/1408mut]) on aminoglycoside resistance. Using an integrative vector, we introduced a single copy of a mutated rRNA operon (1408 A-->G) into Mycobacterium smegmatis, which carries two chromosomal wild-type rRNA operons; the resultant transformants exhibited an aminoglycoside-sensitive phenotype. In contrast, introduction of the mutated rRNA operon into an M. smegmatis rrnB knockout strain carrying a single functional chromosomal wild-type rRNA operon resulted in aminoglycoside-resistant transformants. Subsequent analysis by DNA sequencing and RNase protection assays unexpectedly demonstrated a homozygous mutant genotype, rRNAmut/rRNAmut, in the resistant transformants. To investigate whether RecA-mediated gene conversion was responsible for the aminoglycoside-resistant phenotype in the rRNAwt/rRNAmut strains, recA mutant strains were generated by allelic exchange techniques. Transformation of the recA rrnB M. smegmatis mutant strains with an integrative vector expressing a mutated rRNA operon (Escherichia coli position 1408 A-->G) resulted in transformants with an aminoglycoside-sensitive phenotype. Subsequent analysis showed stable heterozygosity at 16S rRNA position 1408 with a single wild-type allele and a single resistant allele. These results demonstrate that rRNA-mediated mutational resistance to aminoglycosides is recessive.

Alleles↗

Changing nucleosome positions in vivo through modification of the DNA rotational information.

The effects of the rotational information of DNA in determining the in vivo localization of nucleosomal core particles (ncps) have been studied in the Saccharomyces cerevisiae 5 S rRNA repeat gene. The distribution of the phased series of flexibility signals present in this DNA has been altered by inserting in its centre a 25 bp tract. The effects of such alteration on the in vivo distribution of the helically phased, alternatively located ncps have been determined relative to a reference 21 bp insertion mutant. The results show that the answers provided in vitro and in vivo by the yeast 5 S rRNA gene sequence to specific modifications of the DNA rotational frame are similar, thus pointing to the relevance of DNA rotational information in vivo.

Chromosomes, Fungal↗

In vitro evidence that eukaryotic ribosomal RNA transcription is regulated by modification of RNA polymerase I.

We have utilized a cell-free transcription system from Acanthamoeba castellanii to test the functional activity of RNA polymerase I and transcription initiation factor I (TIF-I) during developmental down regulation of rRNA transcription. The results strongly suggest that rRNA transcription is regulated by modification, probably covalent, of RNA polymerase I: (1) The level of activity of TIF-I in extracts from transcriptionally active and inactive cells is constant. (2) The number of RNA polymerase I molecules in transcriptionally active and inactive cells is also constant. (3) In contrast, though the specific activity of polymerase I on damaged templates remains constant, both crude and purified polymerase I from inactive cells have lost the ability to participate in faithful initiation of rRNA transcription. (4) Polymerase I purified from transcriptionally active cells has the same subunit architecture as enzyme from inactive cells. However, the latter is heat denatured 5 times faster than the active polymerase.

Amoeba↗

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.

Base Sequence↗

Conformational changes in the structure of domains II and V of 28S rRNA in ribosomes treated with the translational inhibitors ricin or alpha-sarcin.

Ricin and alpha-sarcin modify neighbouring sites in the so-called sarcin/ricin (S/R) loop of 28S rRNA, thereby destroying the necessary dynamic flexibility of the ribosome, and inhibiting the elongation factor assisted steps of the elongation cycle. The effects of the two translational inhibitors on the conformation of domains II and V of 28S rRNA were investigated by chemical modification of programmed mouse ribosomes pretreated with ricin or alpha-sarcin. The results showed that the two ribosome-inactivating proteins (RIP) influenced the structure of the ribosomal RNA. Inhibitor-affected sites were located at or near sites previously proposed to be involved in functional domains. The modification patterns obtained after ricin or alpha-sarcin treatment of ribosomes were partially overlapping. However, there were several inhibitor-specific structural changes in 28S rRNA. Such changes were found at positions located at the GTPase activating centre of the ribosome and in the S/R domain, indicating that the structure in these regions of the ribosomes differed after treatment with the two inhibitors. These changes are consistent with ricin and alpha-sarcin having specific effects on eEF-2 and eEF-1 interaction with the ribosome, respectively.

Animals↗

Characterization of the secondary structure features of Escherichia coli, Caldariella acidophila and mammalian ribosomal RNA species by chemical modification of sterically exposed bases.

The helix content of rRNA species (Escherichia coli, Caldariella acidophila, rat liver) and the G . C content of their bihelical domains have been investigated by chemical modification of uracil and cytosine residues with probes specific for sterically exposed bases. By using radioactively labelled rRNA, G . C base pairs and the sum of A . U plus G . U base pairs have been quantified assuming that they are numerically identical with the unreactive cytosine and uracil rings, respectively. Exposed uracil bases were probed by their conversion to alkali-labile, nonultraviolet-absorbing sulphonated adducts, with 1.33 M bisulfite pH 7, at 20 degrees C; the adducts can be separated from unreacted uracil, and quantified, by cation-exchange chromatography of RNase T2 plus pancreatic RNase digests of bisulfite-modified rRNA. Exposed cytosines were probed by their conversion to methoxyaminated, alkali-stable, derivatives with 1 M methoxyamine, pH 5.5, at 37 degrees C, and quantified by monitoring the CMP/AMP radioactivity ratio after alkaline hydrolysis of modified rRNA. Exposed uracil rings can also be estimated spectrophotometrically by the alkali-catalyzed reversal of the non-ultraviolet-absorbing sulphonated adducts after separation of the latter from unreacted uracil. The cytosine deamination reaction, catalyzed by bisulfite at pH 6, has also been investigated and found to exhibit little specificity for sterically exposed bases of rRNA, the (G + C)-richer rRNA species of C. acidophila being considerably less susceptible to non-specific deamination than the (G + C)-poorer rRNA of E. coli. A high degree of congruence is shown to exist between results obtained by chemical modification and melting hyperchromicity experiments.

Animals↗

A human mitochondrial GTP binding protein related to tRNA modification may modulate phenotypic expression of the deafness-associated mitochondrial 12S rRNA mutation.

Human mitochondrial 12S rRNA A1555G mutation has been found to be associated with deafness. However, putative nuclear modifier gene(s) has been proposed to regulate the phenotypic expression of this mutation. In yeast cells, mutant alleles of MSS1, encoding a mitochondrial GTP-binding protein, manifest a respiratory-deficient phenotype only when coupled with mitochondrial 15S rRNA P(R)(454) mutation corresponding to human A1555G mutation. This suggests that an MSS1-like modifier gene may influence the phenotypic expression of the A1555G mutation. We report here the identification and characterization of human MSS1 homolog, GTPBP3, the first identified vertebrate gene related to mitochondrial tRNA modification. The Gtpbp3 is the mitochondrial GTPase evolutionarily conserved from bacteria to mammals. Functional conservation of this protein is supported by the observation that isolated human GTPBP3 cDNA can complement the respiratory-deficient phenotype of yeast mss1 cells carrying P(R)(454) mutation. GTPBP3 is ubiquitously expressed in various tissues as multiple transcripts, but with a markedly elevated expression in tissues of high metabolic rates. We showed that Gtpbp3 localizes in mitochondrion. These observations suggest that the human GTPBP3 is a structural and functional homolog of yeast MSS1. Thus, allelic variants in GTPBP3 could, if they exist, modulate the phenotypic manifestation of human mitochondrial A1555G mutation.

Alleles↗

Probing the structure of mouse Ehrlich ascites cell 5.8S, 18S and 28S ribosomal RNA in situ.

The secondary structure of mouse Ehrlich ascites 18S, 5.8S and 28S ribosomal RNA in situ was investigated by chemical modification using dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulphonate. These reagents specifically modify unpaired bases in the RNA. The reactive bases were localized by primer extension followed by gel electrophoresis. The three rRNA species were equally accessible for modification i.e. approximately 10% of the nucleotides were reactive. The experimental data support the theoretical secondary structure models proposed for 18S and 5.8/28S rRNA as almost all modified bases were located in putative single-strand regions of the rRNAs or in helical regions that could be expected to undergo dynamic breathing. However, deviations from the suggested models were found in both 18S and 28S rRNA. In 18S rRNA some putative helices in the 5'-domain were extensively modified by the single-strand specific reagents as was one of the suggested helices in domain III of 28S rRNA. Of the four eukaryote specific expansion segments present in mouse Ehrlich ascites cell 28S rRNA, segments I and III were only partly available for modification while segments II and IV showed average to high modification.

Animals↗

Chemical modification studies and the secondary structure of HeLa cell 5.8S rRNA.

Various secondary structure models have been proposed for 5.8 S rRNA. In this paper HeLa cell 5.8 S rRNA is shown to possess several sites that are reactive to carbodiimide at 25 degrees, and other regions that are unreactive. Previous work has established the distribution of reactive and unreactive cytidine residues along the primary structure (11). The secondary structure model of Nazar et al. (7) is fully compatible with the chemical reactivity data whereas other models are partly incompatible. We conclude that the model of Nazar et al. provides the best approximation so far available to the conformation of isolated 5.8 S rRNA. Findings on the effect temperature on the chemical reactivity of different parts of the structure are summarized. The findings described in this paper should provide a basis for examining the specific interaction of 5.8 S rRNA with 28 s rRNA.

Base Sequence↗