Dependence of M1 RNA substrate specificity on magnesium ion concentration.

We have constructed a plasmid expressing E. coli M1 RNA, the catalytic RNA subunit of ribonuclease P, under the control of a phage T7 promoter. The active M1 RNA species synthesized in vitro by T7 RNA polymerase from this vector was reacted with the tRNA(Gln) - tRNA(Leu) precursor RNA (Band K) encoded by phage T4. Only the tRNA(Leu) moiety of this dimeric precursor RNA contains the 3' terminal C-C-A sequence common to all tRNAs. We observed that protein-free M1 RNA was capable of processing the precursor RNA at the 5' ends of both tRNA tRNA sequences. The rate of cleavage of the tRNA(Gln) sequence was more strongly dependent on [Mg2+] than that of tRNA(Leu), increasing severalfold between 100 and 500 mM Mg2+, conditions under which the rate of cleavage at the tRNA(Leu) sequence was constant.     

Product release is a rate-limiting step during cleavage by the catalytic RNA subunit of Escherichia coli RNase P.

The kinetic constants for cleavage of the tRNA(Tyr)Su3 precursor by the M1 RNA of E. coli RNase P were determined in the absence and presence of the C5 protein under single and multiple (steady state) turnover conditions. The rate constant of cleavage in the reaction catalyzed by M1 RNA alone was 5 times higher in single turnover than in multiple turnovers, suggesting that a rate-limiting step is product release. Cleavage by M1 RNA alone and by the holoenzyme under identical buffer conditions demonstrated that C5 facilitated product release. Addition of different product-like molecules under single turnover reaction conditions inhibited cleavage both in the absence and presence of C5. In the presence of C5, the Ki value for matured tRNA was approximately 20 times higher than in its absence, suggesting that C5 also reduces the interaction between the 5'-matured tRNA and the enzyme. In a growing cell the number of tRNA molecules is approximately 1000 times higher than the number of RNase P molecules. A 100-fold excess of matured tRNA over enzyme clearly inhibited cleavage in vitro. We discuss the possibility that RNase P is involved in the regulation of tRNA expression under certain growth conditions.     

Nuclear ligation of RNA 5''-OH kinase products in tRNA.

Mouse L-cell nuclei incorporate gamma-32P from ATP in vitro predominantly in 5'-monophosphoryl termini and internal phosphodiester bonds with a nonrandom nearest-neighbor distribution. In the presence of 1 microgram of alpha-amanitin per ml the gamma-32P showed a time-dependent appearance in RNA bands which migrated with mature tRNA species but not with pre-tRNA and 5S RNA. The gamma-32P was found in internal phosphodiester bonds as shown by alkaline phosphatase resistance and was identified in 3'-monophosphates after RNase T2, T1, and A digestion. The specificity of this incorporation was indicated by a limited number of labeled oligonucleotides from a T1 digest and identification of 70 to 80% of the 32P label as Cp on complete digestion of the eluted tRNA band. We also observed transiently [gamma-32P]ATP-labeled RNA bands (in 5'-monophosphate positions) that were 32 to 45 nucleotides long. The results presented suggest splicing of several mouse L-cell tRNA species in isolated nuclei which involve the RNA 5'-OH kinase products as intermediates.     

On the recognition of helical RNA by cobra venom V1 nuclease

The V1 nuclease from cobra venom preferentially hydrolyzes double helical RNA and has been used extensively for detecting RNA secondary structure. To increase the utility of this enzyme as an RNA structure probe, we have investigated its properties and substrate specificity, using assays for polynucleotide hydrolysis based on fluorescent polynucleotide derivatives. Enzymatic activity requires both Na+ and Mg2+, with optima at 100 and 0.3 mM, respectively. From the sharp decrease in enzyme activity above 100 mM Na+ we estimate that 3-4 ionic interactions between the protein and polynucleotide phosphates take place. Analysis of products remaining after extensive V1 digestion also shows that the minimum size substrate is 4-6 nucleotides long. Helical RNAs and DNAs have Michaelis constants a factor of 3-10 times lower than most single-stranded RNAs. However, poly(epsilon A) has a Michaelis constant equal to the best synthetic double helices tested and is hydrolyzed at a rate comparable to helical RNA. The major V1 cutting sites in yeast tRNAPhe have Michaelis constants lower than any synthetic polymers. These data suggest that V1 nuclease recognizes any 4-6-nucleotide segment of polynucleotide backbone with an approximately helical conformation, but does not require that the bases be paired in a helix. A few single-stranded V1 cleavage sites are known in tRNA and rRNA, and their structures are consistent with the suggested V1 recognition site.     

Ionic conditions for the cleavage of the tRNA-like structure of turnip yellow mosaic virus by the catalytic RNA of RNase P

The 3'-end of the RNA genome of turnip yellow mosaic virus can form a pseudoknotted tRNA-like structure that can be recognized by several tRNA-specific enzymes. We have found that the catalytic RNA component of Bacillus subtilis RNase P can cleave this structure in unusually low ionic strength buffers at a site analogous to the 5'-end of an aminoacyl stem of a tRNA. Most other precursors can only be processed under low ionic strength conditions if the RNase P holoenzyme is used; processing by the catalytic RNA component alone requires a higher ionic strength buffer. The cleavage of the turnip yellow mosaic virus tRNA-like structure demonstrates the importance of the substrate in determining the optimal buffer conditions for this reaction and also shows that high ionic strength buffers are not always necessary for cleavage by the catalytic RNA.     

Processing of a multimeric tRNA precursor from Bacillus subtilis by the RNA component of RNase P

Processing of multimeric precursor tRNAs from Bacillus subtilis by the catalytic RNA component of RNase P was studied in vitro. Previous studies on processing by either Escherichia coli or B. subtilis RNase P-RNA utilized monomeric or dimeric substrates. In the experiments described here, a multimeric precursor tRNA containing six complete tRNA sequences and the partial sequence of a seventh were used. One species did not encode the 3'-terminal CCA sequence and the partial tRNA lacked 3' nucleotides and could form only a 3-base pair instead of a 7-base paired aminoacyl stem. Two species had the potential for forming extended base-paired aminoacyl stems. Processing was studied under varied ionic conditions. Chemical sequencing of the products showed that the RNase P-RNA cleavage produced the proper mature 5' termini for all of the six complete tRNA species, but no 5'-cleavage of the partial species was observed. At suboptimal ionic concentrations, the two species capable of forming extended base-paired aminoacyl stems were not observed. Thus, encoding of the 3'-CCA in a tRNA species is not critical for processing, but the formation of an aminoacyl stem with more than 3 base pairs is necessary. Particularly noteworthy was the observation that all species of the multimeric precursor could be processed at significantly lower ionic conditions than monomeric precursors used previously by ourselves and others. However, a single precursor species produced from the multimeric precursor could also be processed at the same lower ionic conditions as the multimeric precursor. This demonstrates that precursor tRNA species can differ widely in their ionic requirements for processing and that, to a large extent, the optimal conditions of MgCl2 or NH4Cl are a function of the substrate which is used.     

Small Stable RNAs from Escherichia coli: Evidence for the Existence of New Molecules and for a New Ribonucleoprotein Particle Containing 6S RNA

Small stable RNA molecules of Escherichia coli other than 5S (rRNA) and 4S (tRNA) were studied. Two of the molecules corresponded to 4.5S and 6S RNA, which have been reported previously. The third stable RNA molecule, 10S RNA, has not been described before. RNA labeled with (32)P(i) or [(14)C]uracil for a relatively long time, when separated in 5%/12% tandem polyacrylamide gels, displayed three bands corresponding to 10S, 6S, and 4.5S RNA in addition to rRNA and tRNA bands. These RNAs were stable in pulse-chase-labeling experiments. The amount of these RNAs was small, comprising only 0.2 to 0.5% of the total (32)P incorporation. However, this amount represented a large number of molecules; for 6S and 4.5S, it was about 1,000/DNA molecule. These three RNAs were found in the postribosomal supernatant fraction. None of them was found in purified nucleoid fractions in which the tightly coiled DNA molecules were contained. Of these three RNAs, 6S RNA was unique in that it seemed to exist in a ribonucleoprotein particle. All these RNAs, as well as tRNA, were very stable in the cell under various physiological conditions. 5S RNA was less stable. On the other hand, purified 6S RNA was more susceptible than tRNA to cell nucleases when incubated with cell extracts, suggesting that, being in a particle, it is protected from cell nucleases.     

Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions

Folding mechanisms of functional RNAs under idealized in vitro conditions of dilute solution and high ionic strength have been well studied. Comparatively little is known, however, about mechanisms for folding of RNA in vivo where Mg²⁺ ion concentrations are low, K⁺ concentrations are modest, and concentrations of macromolecular crowders and low-molecular-weight cosolutes are high. Herein, we apply a combination of biophysical and structure mapping techniques to tRNA to elucidate thermodynamic and functional principles that govern RNA folding under in vivo–like conditions. We show by thermal denaturation and SHAPE studies that tRNA folding cooperativity increases in physiologically low concentrations of Mg²⁺ (0.5–2 mM) and K⁺ (140 mM) if the solution is supplemented with physiological amounts (∼20%) of a water-soluble neutral macromolecular crowding agent such as PEG or dextran. Low-molecular-weight cosolutes show varying effects on tRNA folding cooperativity, increasing or decreasing it based on the identity of the cosolute. For those additives that increase folding cooperativity, the gain is manifested in sharpened two-state-like folding transitions for full-length tRNA over its secondary structural elements. Temperature-dependent SHAPE experiments in the absence and presence of crowders and cosolutes reveal extent of cooperative folding of tRNA on a nucleotide basis and are consistent with the melting studies. Mechanistically, crowding agents appear to promote cooperativity by stabilizing tertiary structure, while those low molecular cosolutes that promote cooperativity stabilize tertiary structure and/or destabilize secondary structure. Cooperative folding of functional RNA under physiological-like conditions parallels the behavior of many proteins and has implications for cellular RNA folding kinetics and evolution.     

Functional defects in transfer RNAs lead to the accumulation of ribosomal RNA precursors

Normal expression and function of transfer RNA (tRNA) are of paramount importance for translation. In this study, we show that tRNA defects are also associated with increased levels of immature ribosomal RNA (rRNA). This association was first shown in detail for a mutant strain that underproduces tRNA(Arg2) in which unprocessed 16S and 23S rRNA levels were increased several-fold. Ribosome profiles indicated that unprocessed 23S rRNA in the mutant strain accumulates in ribosomal fractions that sediment with altered mobility. Underproduction of tRNA(Arg2) also resulted in growth defects under standard laboratory growth conditions. Interestingly, the growth and rRNA processing defects were attenuated when cells were grown in minimal medium or at low temperatures, indicating that the requirement for tRNA(Arg2) may be reduced under conditions of slower growth. Other tRNA defects were also studied, including a defect in RNase P, an enzyme involved in tRNA processing; a mutation in tRNA(Trp) that results in its degradation at elevated temperatures; and the titration of the tRNA that recognizes rare AGA codons. In all cases, the levels of unprocessed 16S and 23S rRNA were enhanced. Thus, a range of tRNA defects can indirectly influence translation via effects on the biogenesis of the translation apparatus.     

Solution structure of RNase P RNA

The ribonucleoprotein enzyme ribonuclease P (RNase P) processes tRNAs by cleavage of precursor-tRNAs. RNase P is a ribozyme: The RNA component catalyzes tRNA maturation in vitro without proteins. Remarkable features of RNase P include multiple turnovers in vivo and ability to process diverse substrates. Structures of the bacterial RNase P, including full-length RNAs and a ternary complex with substrate, have been determined by X-ray crystallography. However, crystal structures of free RNA are significantly different from the ternary complex, and the solution structure of the RNA is unknown. Here, we report solution structures of three phylogenetically distinct bacterial RNase P RNAs from Escherichia coli, Agrobacterium tumefaciens, and Bacillus stearothermophilus, determined using small angle X-ray scattering (SAXS) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis. A combination of homology modeling, normal mode analysis, and molecular dynamics was used to refine the structural models against the empirical data of these RNAs in solution under the high ionic strength required for catalytic activity.     

The reaction of SLE antibodies with native, single stranded RNA: radioassay and binding specificities.

Escherichia coli 3H-tRNA and MS2 phage 125I-RNA were prepared and used in a sensitive nitrocellulose filter assay. Antibodies that bound these RNA ligands occurred in the sera of several patients with SLE, but not in sera of patients with other connective tissue diseases. The antibody populations that bound polyribonucleotides (largely IgG) were distinct from antibody populations that bound polydeoxyribonucleotides. Competition experiments showed that the anti-RNA antibodies preferentially bound native ssRNA as compared with synthetic single and double stranded polyribonucleotides. There was increasing affinity with increasing m.w. of the ssRNA. The anti-tRNA population was of restricted heterogeneity (Sips index 0.83) and bound tRNA with an average association constant (Ko) of 9 x 10(6) l/mole at 4 degrees C. The anti-MS2 RNA population was much more heterogeneous (Sips index 0.67) and bound MS2 RNA with a Ko of about 3 x 10(9) l/mole at 4 degrees C. Whereas NZB/NZW mice spontaneously produce RNA reactive antibodies with conformation specificity for native tRNA, human SLE anti-RNA antibodies appear to have very little of this type of conformation specificity.     

Induced circular dichroism of acridine orange bound to double-stranded RNA and transfer RNA

The induced circular dichroism (CD) in the visible region of acridine orange bound to the double-stranded RNA from cytoplasmic polyhedrosis virus and to yeast tRNA has been measured as a function of RNA phosphate-to-dye ratio (P/D), under the conditions of 0.01 M Na⁺ at pH 7.0. The shape of the CD spectrum of acridine orange bound to the double-stranded RNA was quite different from the spectrum of the dye bound to DNA. The CD spectral features of acridine orange bound to the double-stranded regions in tRNA closely resembled those of the double-stranded RNA-dye complex, suggesting that the dyes bind similarly to the two RNA's. It was further found that the CD spectrum of the tRNA-dye complex at sufficiently high P/D ratios, which is assignable to monomeric, intercalated dye to the base-paired parts in tRNA, is also distinct from the corresponding spectrum of the DNA-dye complex.     

Evidence for tertiary structure in natural single stranded RNAs in solution.

Binding isotherms (20 degrees C) of ethidium bromide to a number of tRNA species at various ionic strengths indicate that i) the number ni of intercalation sites is high 7 to 11 per molecule, in the low salt form III, but small, 2 to 1, at high Mg2+ or Na+ when form I predominates. ii) modification of tRNA at strategic positions for 3D folding prevents full expression of intercalation restriction iii) maximal restriction is obtained at salt concentrations higher than needed for full conversion to form I. It is inferred that restriction, which is not observed with bihelical RNA (or DNA), requires the native tRNA 3D structure but also some physical coupling between the region of 3D folding and bihelical arms. Ribosomal RNAs, some viral RNAs, mRNA from sheep mammary gland as well as the random copolymers Poly UG, Poly AUG, Poly AUCG all exhibit intercalation restriction. Hence 3D folding of the polyribonucleotide chains appears to be a feature common to single-stranded RNAs when free in solution under physiological conditions.     

Comparison of the conformation of RNA from phage MS2 and 16S rRNA. Interaction with dyes specific for the secondary structure of native RNA and RNA subjected to hydrolysis by nuclease S1

The interaction of ethidium bromide (EtBr) with double-stranded (ds), and acridine orange (AO) with single-stranded (ss) fragments of 16S rRNA Escherichia coli in a wide range of ionic strength, at various pH, Zn2+ ion concentrations and partial hydrolysis by nuclease S1 was investigated. It was shown that about 90% of the RNA molecule is accessible to both dyes, when the ionic strength is near of 0.01 (pH 7). Approximately half of the RNA becomes inaccessible to dyes, when the ionic strength was increased up to 0.08-0.24 (pH 4.7-7), independent on the presence of Zn2+ ions (10(-3) M). About a half of the ds-, and a quarter of the ss-segments of the RNA, deduced from the secondary structure model were protected from the interaction with EtBr and AO. The hydrolysis of about a half of ss-segments upon addition of the Zn2+ (10(-3) M) ions did not affect the RNA tertiary structure. The experimental data obtained confirm the idea of the existence of some "nucleus" (or "nuclei") within the 16S rRNA molecule. The "nucleus" seems to be inaccessible to the dyes and is very stable to heat denaturation. It was supposed that this structure is organized by means of interaction of some of the parallelly oriented ds-segments, as it was suggested earlier for the phage MS2 RNA structure.     

Study of the structural organization of RNA from phage MS2 using fluorescent dyes

The spatial organization of phage MS2 RNA by binding to ethidium bromide (EtBr) and acridine orange (AO) to RNA was studied. The analyses of dye interaction by spectrophotometric and fluorometric methods have demonstrated that only about a half of 65-70% nucleotides of double-stranded segments can interact with AO and EtBr. On the other hand all the single-stranded segments appear to be accessible to AO binding. These interactions did not practically change when ionic strength (0.01-0.3), Mg2+ and Zn2+ concentrations (10(-3) M) or pH (4.7-7.4) varied. The data permit to suppose that phage MS2 RNA has a very stable tertiary structure which makes part of double-stranded segments unaccessible to inter calating dyes. Taking these and other facts into consideration we suppose that double-stranded segments play an important role in stabilization of the RNA tertiary structure. One of the most possible structure is a compact "rod-like" intramolecular aggregate of double-stranded hairpin-like segments of RNA with parallel orientation.     

The decoding region of 16S RNA; a cross-linking study of the ribosomal A, P and E sites using tRNA derivatized at position 32 in the anticodon loop.

A photo-reactive diazirine derivative was attached to the 2-thiocytidine residue at position 32 of tRNA(Arg)I from Escherichia coli. This modified tRNA was bound under suitable conditions to the A, P or E site of E.coli ribosomes. After photo-activation of the diazirine label, the sites of cross-linking to 16S rRNA were identified by our standard procedures. Each of the three tRNA binding sites showed a characteristic pattern of cross-linking. From tRNA at the A site, a major cross-link was observed to position 1378 of the 16S RNA, and a minor one to position 936. From the P site, there were major cross-links to positions 693 and to 957 and/or 966, as well as a minor cross-link to position 1338. The E site bound tRNA showed major cross-links to position 693 (identical to that from the P site) and to positions 1376/1378 (similar, but not identical, to the cross-link observed from the A site). Immunological analysis of the concomitantly cross-linked ribosomal proteins indicated that S7 was the major target of cross-linking from all three tRNA sites, with S11 as a minor product. The results are discussed in terms of the overall topography of the decoding region of the 30S ribosomal subunit.     

DNase I-tRNA interaction

Deoxyribonuclease I (DNase I) binds right-handed DNA duplex via a minor groove and the backbone phosphate group with no contact to the major groove. It hydrolyses double-stranded DNA predominantly by a single-stranded nicking mechanism under physiological conditions, in the presence of divalent Mg and Ca cations. Even though DNase-RNA interaction was observed, less is known about the protein-RNA binding mode and the effect of such complexation on both protein and RNA conformations. The aim of this study was to examine the effects of DNase I-tRNA interaction on tRNA and protein conformations. The interaction of DNase I with tRNA is monitored under physiological conditions, in the absence of Mg2+, using constant DNA concentration of 12.5 mM (phosphate) and various protein contents (10 microM to 250 microM). FTIR, UV-visible, and CD spectroscopic methods were used to analyze the protein binding mode, the binding constant, and the effects of polynucleotide-enzyme interaction on both tRNA and protein conformations. Spectroscopic evidence showed major DNase-PO2 and minor groove interactions with overall binding constant of K = 2.1 (+/-0.7) x 10(4) M(-1). The DNase I-tRNA interaction alters protein secondary structure with major reduction of the alpha-helix, and increases the random coil, beta-anti and turn structures, while tRNA remains in the A-conformation. No digestion of tRNA by DNase I was observed in the protein-tRNA complexes.     

Both yeast W double-stranded RNA and its single-stranded form 20S RNA are linear.

Most yeast strains carry a cytoplasmic double-stranded RNA (dsRNA) molecule called W, of 2.5 kb in size. We have cloned and sequenced most of W genome (1), and we proposed that W (+) strands were identical to 20S RNA, a single-stranded RNA (ssRNA) species, whose copy number is highly induced under stress conditions. Recently it was proposed that 20S RNA was circular (2). In this paper, however, we demonstrate that both W dsRNA and 20S RNA are linear. Linearity of W dsRNA is shown by the stoichiometric labelling of both strands of W with 32P-pCp and T4 RNA ligase. The last 3' end nucleotide of both strands is about 70 to 80% C and 20 to 30% A. Linearity of 20S RNA is directly demonstrated by a site-specific cleavage of 20S RNA with RNase H, using an oligodeoxynucleotide complementary to an internal site of 20S RNA. The cleavage produced not one but two RNA fragments expected from the linearity of 20S RNA.