Directed hydroxyl radical probing of 16S ribosomal RNA in 70S ribosomes from internal positions of the RNA

Directed hydroxyl radical probing of 16S ribosomal RNA from Fe(II) tethered to specific sites within the RNA was used to determine RNA-RNA proximities in 70S ribosomes. We have transcribed 16S ribosomal RNA in vitro as two separate fragments, covalently attached an Fe(II) probe to a 5'-guanosine-alpha-phosphorothioate at the junction between the two fragments, and reconstituted 30S subunits with the two separate pieces of RNA and the small subunit proteins. Reconstituted 30S subunits capable of association with 50S subunits were selected by isolation of 70S ribosomes. Hydroxyl radicals, generated in situ from the tethered Fe(II), cleaved sites in the 16S rRNA backbone that were close in three-dimensional space to the Fe(II), and a primer extension was used to identify these sites of cleavage. Two sets of 16S ribosomal RNA fragments, 1-360/361-1542 and 1-448/449-1542, were reconstituted into active 30S subunits. Fe(II) tethered to position 361 results in cleavage of 16S rRNA around nucleotides 34, 160, 497, 512, 520, 537, 552, and 615, as well as around positions 1410, 1422, 1480, and 1490. Fe(II) tethered to position 449 induces cleavage around nucleotide 488 and around positions 42 and 617. Fe(II) tethered to the 5' end of 16S rRNA induces cleavage of the rRNA around nucleotides 5, 601, 615, and 642. These results provide constraints for the positioning of these regions of 16S rRNA, for which there has previously been only limited structural information, within the 30S subunit.     

Interaction of yeast elongation factor 3 with polynucleotides, ribosomal RNA and ribosomal subunits

In addition to the two usual eukaryotic elongation factors (EF-1 alpha and EF-2) fungal ribosomes need a third protein, elongation factor 3, for translation. EF-3 is essential for in vivo and in vitro protein synthesis. Functionally, EF-3 stimulates EF-1 alpha dependent binding of aminoacyl-tRNA to the ribosomal A site when E site is occupied by deacylated tRNA. EF-3 has intrinsic ATPase activity which is regulated by the functional state of the ribosome. EF-3 ATPase is activated by both 40S and 60S ribosomal subunits. However intact 80S ribosomes are needed for efficient activation of EF-3 ATPase. EF-3 appears to be an RNA binding protein with high affinity for polynucleotides containing guanosine rich sequences. To determine whether guanosine rich sequence of ribosomal RNA is involved in EF-3 binding, an antisense oligonucleotide dC6 was used to block EF-3 interaction with the ribosome. The oligonucleotide suppresses activation of EF-3 ATPase by 40S ribosomal subunit and not by the 60S or the 80S particles. Poly(U)-directed polyphenylalanine synthesis by yeast ribosomes is inhibited by dC6. To define the binding site of the oligonucleotide and presumably of EF-3 on 18S ribosomal RNA, hydrolysis of rRNA by RNase H was followed in the presence of dC6. These experiments reveal an RNase H cleavage site at 1094GGGGGG1099 sequence of 18S ribosomal RNA. This guanosine rich sequence of rRNA is suggested to be involved in EF-3 binding to yeast ribosome. Data presented in this communication suggest that the activity of EF-3 involved a direct interaction with the guanosine rich sequence of rRNA.     

Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies

We have investigated protein-rRNA cross-links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2-iminothiolane as cross-linking agents. We identified amino acids cross-linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross-linked to rRNA have been sequenced in which no direct cross-linked amino acid could be detected. The cross-linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross-linked peptides in ribosomal protein S3 shows a common primary sequence motif--the KH motif--directly involved in interaction with rRNA, and the cross-linked amino acid in ribosomal protein L36 lies within the zinc finger-like motif of this protein. The cross-linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three-dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein-antibiotic cross-linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.     

Directed hydroxyl radical probing of 16S ribosomal RNA in ribosomes containing Fe(II) tethered to ribosomal protein S20

The 16S ribosomal RNA neighborhood of ribosomal protein S20 has been mapped, in both 30S subunits and 70S ribosomes, using directed hydroxyl radical probing. Cysteine residues were introduced at amino acid positions 14, 23, 49, and 57 of S20, and used for tethering 1-(p-bromoacetamidobenzyl)-Fe(II)-EDTA. In vitro reconstitution using Fe(II)-derivatized S20, together with the remaining small subunit ribosomal proteins and 16S ribosomal RNA (rRNA), yielded functional 30S subunits. Both 30S subunits and 70S ribosomes containing Fe(II)-S20 were purified and hydroxyl radicals were generated from the tethered Fe(II). Hydroxyl radical cleavage of the 16S rRNA backbone was monitored by primer extension. Different cleavage patterns in 16S rRNA were observed from Fe(II) tethered to each of the four positions, and these patterns were not significantly different in 30S and 70S ribosomes. Cleavage sites were mapped to positions 160-200, 320, and 340-350 in the 5' domain, and to positions 1427-1430 and 1439-1458 in the distal end of the penultimate stem of 16S rRNA, placing these regions near each other in three dimensions. These results are consistent with previous footprinting data that localized S20 near these 16S rRNA elements, providing evidence that S20, like S17, is located near the bottom of the 30S subunit.     

Studies on the formation and stability of aminoacyl-tRNA synthetase complexes from Ehrlich ascites cells

Nine aminoacyl-tRNA synthetases from Ehrlich ascites cells were examined with respect to their ability to be isolated as high molecular weight complexes, soluble enzymes, and ribosome-bound enzymes. Several different methods were employed for cell homogenization and enzyme isolation, with particular attention paid to the effects of hypotonic, isotonic, and hypertonic buffers on enzyme isolation. The binding of all synthetases to ribosomes was eliminated if the low ionic strength of the isolation buffer was raised to isotonic levels. In contrast, neither the ionic strength or composition of the buffers, nor the procedures used for cell homogenization or enzyme isolation had any significant effect on the isolation of the high molecular weight synthetase complex. Certain enzymes (lysyl-, methionyl- and isoleucyl-tRNA synthetases) formed very stable complexes and high molecular weight species were the predominant forms of these enzymes under all conditions of cell homogenization and enzyme isolation. Other enzymes (glycyl-, tyrosinyl- and threonyl-tRNA synthetases) formed complexes very weakly, if at all, and always appeared predominately in the soluble enzyme fraction. Isolated soluble forms of the lysyl-, methionyl- and isoleucyl-tRNA synthetases did not associate to form significant amounts of complex upon re-isolATION, SUGGESTING THAT A COMPONENT NECESSARY FOR COMPLEX FORMATION WAS MISSING FROM THE SOLUBLE ENZYME FRACTION. However, the soluble forms of these enzymes, but not the glycyl-, tyrosinyl- and threonyl-tRNA synthetases, did for complexes when mixed with ribosomal RNA or polyuridylic acid. Preliminary experiments showed no significant differences between the complexed and soluble forms of the lysyl-, methionyl- and isoleucyl-tRNA synthetases with respect to Km values or ability to charge different isoaccepting tRNAs.     

Ectopic pairing and evolution of 5S ribosomal RNA genes in the chromosomes of Drosophila funebris

5S ribosomal RNA from Drosophila melanogaster labeled with 125I was used to locate the 5S rRNA genes in chromosomes of D. funebris by means of in situ hybridization. Silver grains were observed at three distinct sites, one of which was a recognized reverse repeat. Only one half of the reverse repeat, however, hybridizes with 5S rRNA and the significance of this phenomenon is discussed. A case of ectopic pairing between two different 5S sites in the genome is reported, and the significance of ectopic pairing is considered.     

Kinetic analysis of the RNA-dependent adenosinetriphosphatase activity of DbpA, an Escherichia coli DEAD protein specific for 23S ribosomal RNA

The adenosinetriphosphatase (ATPase) activity of the Escherichia coli DEAD protein DbpA is unusual in that it is specifically stimulated by 23S ribosomal RNA (rRNA). A coupled spectroscopic ATPase assay was used to investigate the interaction of DbpA with RNA and ATP. A 153-base fragment of domain V of 23S rRNA is kinetically identical to intact, native rRNA in activating DbpA: kcat = 600 min-1, Kapp(RNA) = 10 nM, and Km(ATP) = 120 microM. The ATPase turnover in the absence of RNA is 0.25 min-1. Fragments of 23S rRNA lacking this site (nucleotides 2454-2606) are essentially inactive, as are other RNAs such as poly(A) and tRNA. The relative RNA specificity of DbpA ranges from 10(3) to 10(6) [kmax/Kapp(RNA)]. The interaction with this small RNA fragment was further investigated with regard to stoichiometry, pH, salt and temperature. DbpA is not activated by E. coli ribosomes, nor by large subunits, while denatured ribosomes stimulate full ATPase activity. Taken together with the tight, site-specific binding to naked, unmodified 23S rRNA, this suggests a role for DbpA in ribosome biogenesis rather than translation.     

Mapping the inside of the ribosome with an RNA helical ruler

The structure of ribosomal RNA (rRNA) in the ribosome was probed with hydroxyl radicals generated locally from iron(II) tethered to the 5' ends of anticodon stem-loop analogs (ASLs) of transfer RNA. The ASLs, ranging in length from 4 to 33 base pairs, bound to the ribosome in a messenger RNA-dependent manner and directed cleavage to specific regions of the 16S, 23S, and 5S rRNA chains. The positions and intensities of cleavage depended on whether the ASLs were bound to the ribosomal A or P site, and on the lengths of their stems. These data predict the three-dimensional locations of the rRNA targets relative to the positions of A- and P- site transfer RNAs inside the ribosome.     

Endotoxinemia in liver cirrhosis]

Ribosomes isolated from either dry viable or non-viable pea embryonic axis tissue were equally effective in the support of polyphenylalanine synthesis in a poly(U)-directed cell-free protein-synthesising system. Ribosomes isolated from imbibed non-viable axis tissue were impaired in their ability to support polyphenylalanine synthesis in the cell-free system. RNA isolated from ribosomes and 40-S ribosomal subunits of dry or imbibed viable axis tissue was found not to be degraded, whereas the equivalent RNA species isolated from non-viable axis tissue showed an increased degree of breakdown as imbibition proceeded. Even though rRNA of imbibed non-viable axis tissue was degraded, the ribosomes and ribosomal subunits of these embryos appeared intact. In viable embryonic axis tissue the percentage of ribosomes present in the cell in the form of polysomes increased during imbibition whereas no polysomes could be detected in ribosomal preparations from dry or imbibed non-viable axis tissue. The breakdown of rRNA in ribosomal particles from non-viable axis tissue may be a contributory factor to senescence and loss of viability in Pisum arvense.     

Experience of intravascular malignant lymphomatosis with urinary incontinence and gait disturbance

Nucleotide residue U89 in the D loop of Escherichia coli 5S rRNA is adjacent to two domains of 23S rRNA in the large ribosomal subunit [Dokudovskaya et al., RNA 2 (1996) 146-152]. 50S ribosomal subunits were reconstituted containing U89(C, G or A) mutants of 5S rRNAs and the activities of the corresponding 70S ribosomes were studied. The U89C mutant behaves similarly to the wild-type 5S rRNA. Replacement of the pyrimidine base at position U89 by more bulky purine bases impairs the incorporation of 5S rRNA into 50S subunits, whereas the particles formed showed full activities in poly(U)-dependent poly(Phe) synthesis in the presence of either U89G or U89A 5S rRNA mutants. The activity of the reconstituted particles depends on the incorporation of 5S rRNA in agreement with early observations.     

Diagnostic groups and depressed mood as predictors of 22-month mortality in medical inpatients

An undescribed rickettsia was directly analyzed with specific rickettsial molecular biology tools on Ixodes ricinus L. collected in different localities of the province of Cadiz (southwestern Spain). On the basis of the results of the citrate synthase (glta) gene, 190 kD-outer membrane protein (rOmpA) gene, and 16S ribosomal RNA (16S rRNA) gene partial sequence data, it was found that this rickettsia is sufficiently genetically distinct from other Rickettsia to be considered a distinct taxonomic entity. The isolation and culture of this organism, as well as comparative antigenic analysis, are required to ensure its conclusive taxonomic placement among spotted fever rickettsiae. The epidemiologic role of this new rickettsial agent and its possible pathogenicity to wild and domestic animals or humans is still unknown and needs to be investigated.     

Divergence towards a dead end? Cleavage of the divergent domains of ribosomal RNA in apoptosis

In several cases of apoptotic death the large ribosomal subunit 28S rRNA is specifically cleaved. The cleavages appear at specific sites within those domains of the rRNA molecule that have shown exceptional high divergence in evolution (D domains). The cleavages accompany rather than precede apoptosis, and there is a positive, but not complete, correlation between rRNA cleavage and internucleosomal DNA fragmentation. Most cell types studied so far show two alternative cleavage pathways that are mutually exclusive. Cleavage can either start in the D8 domain with secondary cuts within a subdomain of D2 (D2c), or in the D2 domain with subsequent excision of the D2c subdomain. The latter pathway is of particular interest since D2 (unlike D8) is normally inaccessible for RNase attack. That apoptosis specifically affects the ribosomal divergent domains suggests that these domains, which make up roughly 25% of total cellular RNA, might have evolved to serve functions related to apoptosis. Future studies will be directed to test the hypothesis that rRNA fragmentation may be part of an apoptotic program directed against the elimination of illegitimate (viral?) polynucleotides.     

RNA sequence determinants for aminoglycoside binding to an A-site rRNA model oligonucleotide

The codon-anticodon interaction on the ribosome occurs in the A site of the 30 S subunit. Aminoglycoside antibiotics, which bind to ribosomal RNA in the A site, cause misreading of the genetic code and inhibit translocation. Biochemical studies and nuclear magnetic resonance spectroscopy were used to characterize the interaction between the aminoglycoside antibiotic paromomycin and a small model oligonucleotide that mimics the A site of Escherichia coli 16 S ribosomal RNA. Upon chemical modification, the RNA oligonucleotide exhibits an accessibility pattern similar to that of 16 S rRNA in the 30 S subunit. In addition, the oligonucleotide binds specifically aminoglycoside antibiotics. The antibiotic binding site forms an asymmetric internal loop, caused by non-canonical base-pairs. Nucleotides that are important for binding of paromomycin were identified by performing quantitative footprinting on oligonucleotide sequence variants and include the C1407.G1494 base-pair, and A.U base-pair at positions 1410/1490, and nucleotides A1408, A1493 and U1495. The asymmetry of the internal loop, which requires the presence of a nucleotide in position 1492, is also crucial for antibiotic binding. Introduction into the oligonucleotide of base changes that are known to confer aminoglycoside resistance in 16 S rRNA result in weaker binding of paromomycin to the oligonucleotide. Oligonucleotides homologous to eukaryotic rRNA sequences show reduced binding of paromomycin, suggesting a physical origin for the species-specific action of aminoglycosides.     

Estimating substitution rates in ribosomal RNA genes

A model is introduced describing nucleotide substitution in ribosomal RNA (rRNA) genes. In this model, substitution in the stem and loop regions of rRNA is modeled with 16- and four-state continuous time Markov chains, respectively. The mean substitution rates at nucleotide sites are assumed to follow gamma distributions that are different for the two types of regions. The simplest formulation of the model allows for explicit expressions for transition probabilities of the Markov processes to be found. These expressions were used to analyze several 16S-like rRNA genes from higher eukaryotes with the maximum likelihood method. Although the observed proportion of invariable sites was only slightly higher in the stem regions, the estimated average substitution rates in the stem regions were almost two times as high as in the loop regions. Therefore, the degree of site heterogeneity of substitution rates in the stem regions seems to be higher than in the loop regions of animal 16S-like rRNAs due to presence of a few rapidly evolving sites. The model appears to be helpful in understanding the regularities of nucleotide substitution in rRNAs and probably minimizing errors in recovering phylogeny for distantly related taxa from these genes.     

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.