Study of the secondary and tertiary structure of phage MS2 RNA using nucleases and fluorescent dyes specific for secondary structure

The binding of ethidium bromide (EtBr) and acridine orange (AO) to RNA in native state or after hydrolysis by S1 and SV nucleases that specifically split single-stranded and double-stranded segments was studied. Nuclease S1 hydrolysis of RNA does not increase the number of EtBr strong binding sites, Tm and hyperchromic effect being also unchanged. Hydrolysis by double-stranded segments accessible to EtBr is followed by the diminishing of Tm and hyperchromism. A supposition is put forward that the main role in stabilization of the RNA tertiary structure is played by double-stranded segments arranged so that some of them are hidden and do not interact with dyes. One of the possible models may be parallel oriented intramolecular "hair-pins" forming compact "rod-like" structures.     

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.     

Detection of periodic patterns in RNA sequences: the first encapsidated region of the TMV RNA

A method is developed to study the periodic properties of nucleotide sequences allowing the favoured pattern of the repeating unit, as well as the length and localization of this periodic segment to be determined simultaneously. The degree of periodicity is evaluated calculating the probabilities for random occurrence of the maximal deviations of the nucleotide composition in each phase, making use of the binomial formula.The nucleotide sequence of the tobacco mosaic virus (TMV) RNA responsible for recognition of the homologous protein (“assembly origin”, AO) (Zimmern & Butler, 1977) was investigated in order to find periodic regions of primary structure which might be essential in the recognition process. As a result the most periodic segments of the AO consisting of 31 and 17 nucleotides corresponding to the schemes GAU or GA1 have been found. However, the periodicities in these regions do not exceed that expected for random sequences. It can be considered as an evidence that in addition to peculiarities of primary structure, some other features such as RNA secondary or tertiary structure are essential in this interaction.For comparison the nucleotide sequences of the other fragments of TMV RNA as well as MS2 RNA, TYMV RNA, 16S rRNA and phage fd DNA were investigated by the same method.     

Rescue of the RNA phage genome from RNase III cleavage.

The secondary structure of the RNA from the single-stranded RNA bacteriophages, like MS2 and Qb, has evolved to serve a variety of functions such as controlling gene expression, exposing binding sites for the replicase and capsid proteins, allowing strand separation and so forth. On the other hand, all of these foldings have to perform in bacterial cells in which various RNA splitting enzymes are present. We therefore examined whether phage RNA structure is under selective pressure by host RNases. Here we show this to be true for RNase III. A fully double-stranded hairpin of 17 bp, which is an RNase III target, was inserted into a non-coding region of the MS2 RNA genome. In an RNase III-host these phages survived but in wild-type bacteria they did not. Here the stem underwent Darwinian evolution to a structure that was no longer a substrate for RNase III. This was achieved in three different ways: (i) the perfect stem was maintained but shortened by removing all or most of the insert; (ii) the stem acquired suppressor mutations that replaced Watson-Crick base pairs by mismatches; (iii) the stem acquired small deletions or insertions that created bulges. These insertions consist of short stretches of non-templated A or U residues. Their origin is ascribed to polyadenylation at the site of the RNase III cut (in the + or - strand) either by Escherichia coli poly(A) polymerase or by idling MS2 replicase.     

Complex-formation of Ethidium Bromide with poly[d(A-T)].poly[d(A-T)

The interaction of Ethidium Bromide (EtBr) with double-stranded (ds-) and single-stranded (ss-) poly[d(A-T)] was studied in different ionic strengths solutions. Optical spectroscopy and Scatchard analysis results indicate that the ligand interacts to both helix and coiled structures of the polynucleotide by "strong" and "weak" binding modes. The association parameters (binding constant -K- and the number of nucleotides corresponding to a binding site -n) of the strong type of interaction were found to be independent of Na+ concentration. Weak interaction occurs at low ionic strength and/or high EtBr concentration. Estimated binding parameters of EtBr with ss- and ds-polynucleotide are in good agreement with those for EtBr-B-DNA complexes. Data obtained provided an evidence for a stacking interaction of EtBr with single stranded poly[d(A-T)].     

Comparison of the conformation of RNA from phage MS2 and 16S rRNA. Accessibility to nucleases S1 and SV specific to secondary structure and thermal stability

The hydrolysis of E. coli 16S rRNA by nucleases specific to the secondary structure elements (S1 and SV), the melting of the RNA after partial hydrolysis by nuclease S1 and the electrophoretic mobility of hydrolysis products after denaturation-renaturation of RNA were studied. It was shown that the sensitivity of 16S rRNA to nuclease S1 depends on Zn or Mg ions concentration. The melting curves after partial hydrolysis by nuclease S1 were characterized by a decrease of the hyperchromic effect (by approximately 15%) and by a increase of Tm (by 3 degrees). After RNA denaturation followed by slow or fast renaturation the electrophoretic patterns of the hydrolysis products were not changed, as in the case of phage MS2 RNA. It was supposed, that the rRNA molecule has a stable "nucleus" (or "nuclei"), which is organized as an intramolecular association of parallelly oriented double-stranded fragments of this RNA. Previously, such a mode of the spatial organization was proposed by us for phage MS2 RNA.     

A phage RNA-binding protein binds to a non-cognate structured RNA and stabilizes its core structure

Recent studies suggest that some RNA-binding proteins facilitate the folding of non-cognate RNAs. Here, we report that bacteriophage MS2 coat protein (MS2 CP) bound and promoted the catalytic activity of Candida group I ribozyme. Cloning of the MS2-bound RNA segments showed that this protein primarily interacts with the P5ab-P5 structure. Ultraviolet cross-linking and the T1 footprinting assay further showed that MS2 binding stabilized tertiary interactions, including the conserved L9-P5 interaction, and led to a more compact core structure. This mechanism is similar to that of the yeast mitochondrial tyrosyl-tRNA synthetase on other group I introns, suggesting that different RNA-binding proteins may use common mechanisms to support RNA structures.     

Effects of Mg2+ on the free energy landscape for folding a purine riboswitch RNA

There are potentially several ways Mg2+ might promote formation of an RNA tertiary structure: by causing a general "collapse" of the unfolded ensemble to more compact conformations, by favoring a reorganization of structure within a domain to a form with specific tertiary contacts, and by enhancing cooperative linkages between different sets of tertiary contacts. To distinguish these different modes of action, we have studied Mg2+ interactions with the adenine riboswitch, in which a set of tertiary interactions that forms around a purine-binding pocket is thermodynamically linked to the tertiary "docking" of two hairpin loops in another part of the molecule. Each of four RNA forms with different extents of tertiary structure were characterized by small-angle X-ray scattering. The free energy of interconversion between different conformations in the absence of Mg2+ and the free energy of Mg2+ interaction with each form have been estimated, yielding a complete picture of the folding energy landscape as a function of Mg2+ concentration. At 1 mM Mg2+ (50 mM K+), the overall free energy of stabilization by Mg2+ is large, -9.8 kcal/mol, and about equally divided between its effect on RNA collapse to a partially folded structure and on organization of the binding pocket. A strong cooperative linkage between the two sets of tertiary contacts is intrinsic to the RNA. This quantitation of the effects of Mg2+ on an RNA with two distinct sets of tertiary interactions suggests ways that Mg2+ may work to stabilize larger and more complex RNA structures.     

De-intercalation of ethidium bromide and acridine orange by xanthine derivatives and their modulatory effect on anticancer agents: a study of DNA-directed toxicity enlightened by time correlated single photon counting

Time Correlated Single Photon Counting (TCSPC) was used for the first time to analyze the effect/changes in the mode of intercalation of ethidium bromide (EtBr) and acridine orange (AO) to calf thymus DNA brought about due to interaction of naturally occurring methylxanthines such as theophylline (X1), theobromine (X2) and caffeine (X3). UV absorption and fluorescence studies were also carried to observe the behaviour of these xanthines on the modulation of the binding mode of anticancer agents (cisplatin, novantrone, and actinomycin D) and certain intercalating dyes (EtBr and AO) to DNA. In TCSPC analysis we found that when the concentration of the drugs (X1, X2 and X3) increased from 0.025 mM to 2 mM i.e. P/D 2.4 to P/D 0.03 reduction in intercalation of EtBr and AO was observed, suggesting that xanthine derivatives could play very important role in reducing the DNA-directed toxicity in a dose dependent manner. In TCSPC, the amplitude of smaller lifetime component A(1) and higher lifetime component A(2) are attributed to free and intercalated dye concentration and their variation could indicate the process of intercalation or reduced intercalation of EtBr and AO by xanthine derivatives. We found that at the maximum drug concentration the smaller lifetime component A(1) was increased by 7-8% and 17-37% in EtBr and AO intercalated complex respectively. Also the changes in lifetime and fluorescence decay profile were observed for the DNA-intercalated dyes before and after treatment with xanthines. Especially, at maximum P/D 0.03 the lifetime of DNA-intercalated EtBr and AO reduced by 1-2 ns. The present analysis reveals that xanthines are able to interact with free dyes and also with intercalated dyes, suggesting that when they interact with free dyes they might inhibit the further intercalation of dye molecules to DNA and the interaction with intercalated dyes might lead to displacement of the dyes resulting in de-intercalation. The results obtained from UV and fluorescence spectroscopy also support the present investigation of probable interaction of xanthines with the DNA damaging agents in modulating/reducing the DNA-directed toxicity.     

Study of 2.5S RNA of 1,4-alpha-glucan branching enzyme by fluorescent methods using ethidium bromide

The fluorescence yield and lifetime of ethidium bromide complexes with 1,4-alpha-glucan branching enzyme and its free nucleic acid component 2.5S RNA were measured. Both fluorescence parameters showed a 10-fold increase in comparison with those characteristics for the free dye. This increase allows to suggest the existence of double-stranded regions in 2.5S RNA both in the free as well as in the protein bound state. The coefficients of fluorescence polarization were also determined for ethidium bromide complexed with free and protein bound 2.5S RNA. They proved to be 13 and 18% respectively. No concentration depolarization was observed in both types of ethidium bromide and ethidium bromide--enzyme--RNA complexes. This proves that the double-stranded regions are rather short and that two ethidium bromide molecules can't be bound to each of them. The binding isotherms were measured for ethidium bromide absorbed on 2.5S RNA and on the holoenzyme. Their parameters napp and rmax are identical in the cases of free and protein bound 2,5S RNA (rmax = 0.046 +/- 0.001). However the binding constants of ethidium bromide complexes with free and protein bound 2.5S RNA differ significantly (Kapp = 2.2 X 10(6) M-1 for free 2.5S RNA and Kapp = 1.6 X 10(6) M-1 for the holoenzyme). The quantity of nucleotides involved in the two double-stranded regions accessible for ethidium binding is estimated to be about 28%. Increasing of Mg2+ ion concentration up to 10(-3) results in a decrease of ethidium bromide binding with double stranded regions. It may be due to a more compact tertiary structure of 2.5S RNA in the presence of Mg2+ in the free as well as in protein bound state.     

Complex-Formation of Ethidium Bromide with Poly[d(A-T)]·Poly[d(A-T)]

Abstract

The interaction of Ethidium Bromide (EtBr) with double-stranded (ds-) and single-stranded (ss-) poly[d(A-T)] was studied in different ionic strengths solutions. Optical spectroscopy and Scatchard analysis results indicate that the ligand interacts to both helix and coiled structures of the polynucleotide by “strong” and “weak” binding modes. The association parameters (binding constant—K—and the number of nucleotides corresponding to a binding site—n) of the strong type of interaction were found to be independent of Na+ concentration. Weak interaction occurs at low ionic strength and/or high EtBr concentration. Estimated binding parameters of EtBr with ss- and ds-polynucleotide are in good agreement with those for EtBr-B-DNA complexes. Data obtained provided an evidence for a stacking interaction of EtBr with single stranded poly[d(A-T)].     

Highly Selective Acridine and Ethidium Staining of Bacterial DNA and RNA

The acridine dyes acridine orange (AO) and coriphosphine O (CPO) and ethidium bromide (EtBr) were used to stain bacterial digests after electrophoresis in native and denaturing (SDS) polyacrylamide gels and were shown to stain DNA and RNA preferentially over other subcellular components in the gels. Vegetative cell digests of Bacillus subtilis, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus showed intense staining of DNA with AO and CPO near the top of the gel, but little or no staining of other cellular constituents. EtBr stained both DNA and RNA in the gels. Protein standards and non-nucleic acid cellular constituents stained faintly with high concentrations (> 100 μM) of AO, lower concentrations (13.9 μM) of CPO, and did not stain with 0.5 μg/ml EtBr in denaturing gels. The complete set of cellular biochemicals was visualized by silver staining, while the protein subset was detected by Coomassie blue staining. The highest concentrations of AO (120 μM) and CPO (13.9 μM) were shown to detect purified DNA in gels with a sensitivity in the range of 25–50 ng per band. This work demonstrates the specificity of acridine and ethidium dyes for nucleic acids, while illustrating the level of non-nucleic acid-specific interactions with other cellular components by staining of electrophoretically separated cellular components in a gel matrix.     

Secondary structure of the central region of bacteriophage MS2 RNA. Conservation and biological significance

The RNA of the Escherichia coli RNA phages is highly structured with 75% of the nucleotides estimated to take part in base-pairing. We have used enzymatic and chemical sensitivity of nucleotides, phylogenetic sequence comparison and the phenotypes of constructed mutants to develop a secondary structure model for the central region (900 nucleotides) of the group I phage MS2. The RNA folds into a number of, mostly irregular, helices and is further condensed by several long-distance interactions. There is substantial conservation of helices between the related groups I and II, attesting to the relevance of discrete RNA folding. In general, the secondary structure is thought to be needed to prevent annealing of plus and minus strand and to confer protection against RNase. Superimposed, however, are features required to regulate translation and replication. The MS2 RNA section studied here contains three translational start sites, as well as the binding sites for the coat protein and the replicase enzyme. Considering the density of helices along the RNA, it is not unexpected to find that all these sites lie in helical regions. This fact, however, does not mean that these sites are recognized as secondary structure elements by their interaction partners. This holds true only for the coat protein binding site. The other four sites function in the unfolded state and the stability of the helix in which they are contained serves to negatively control their accessibility. Mutations that stabilize helices containing ribosomal binding sites reduce their efficiency and vice versa. Comparison of homologous helices in different phage RNAs indicates that base substitutions have occurred in such a way that the thermodynamic stability of the helix is maintained. The evolution of individual helices shows several distinct size-reduction patterns. We have observed codon deletions from loop areas and shortening of hairpins by base-pair deletions from either the bottom, the middle or the top of stem structures. Evidence for the coaxial stacking of some helical segments is discussed.     

Translational initiation at the coat-protein gene of phage MS2: native upstream RNA relieves inhibition by local secondary structure

Maximal translation of the coat-protein gene from RNA bacteriophage MS2 requires a contiguous stretch of native MS2 RNA that extends hundreds of nucleotides upstream from the translational start site. Deletion of these upstream sequences from MS2 cDNA plasmids results in a 30-fold reduction of translational efficiency. By site-directed mutagenesis, we show that this low level of expression is caused by a hairpin structure centred around the initiation codon. When this hairpin is destabilized by the introduction of mismatches, expression from the truncated messenger increases 20-fold to almost the level of the full-length construct. Thus, the translational effect of hundreds of upstream nucleotides can be mimicked by a single substitution that destabilizes the structure. The same hairpin is also present in full-length MS2 RNA, but there it does not Impair ribosome binding. Apparently, the upstream RNA somehow reduces the inhibitory effect of the structure on translational initiation. The upstream MS2 sequence does not stimulate translation when cloned in front of another gene, nor can unrelated RNA segments activate the coat-protein gene. Several possible mechanisms for the activation are discussed and a function in gene regulation of the phage is suggested.     

Comparative properties of bacteriophage phi6 and phi6 nucleocapsid.

Nonionic detergent treatments released a nucleocapsid from the enveloped bacteriphage phi6. The nucleocapsid sedimented at nearly the same rate as the whole phage in sucrose density gradients, but the buoyant density in Cs2S04 changed from 1.22 g/cm3 for the whole phage to 1.33 g/cm3 for the nucleocapsid. The detergent completely removed the lipid and 5 of the 10 proteins from the phage. Surface labeling of the phage and nucleocapsid with 125I revealed that protein P3 was on the outer surface of the whole phage and P8 was on the surface of the nucleocapsid. Both the phage and the nucleocapsid were stable between pH 6.0 and 9.5. Low concentrations of EDTA (10-4 M) dissociated the nucleocapsid but had no effect on the whole phage. The nucleocapsid contained all three double-stranded RNA segments, as well as RNA polymerase activity.     

Discriminative effect of rifampin of RNA replication of various RNA bacteriophages.

Rifampin interferes exclusively with RNA replication in vivo of the group I phages MS2, f2, and R17, whereas QbetaRNA replication is unaffected by the drug. In addition, rifampin has a discriminative effect of group I phage RNA replication. In the experimental system employed by us the antibiotic differentially interferes with the synthesis of minus RNA strands in f2, whereas it has almost no effect on the synthesis of progeny plus strands. In MS2, the drug differentially arrests the synthesis of progeny plus strands and almost fails to affect the synthesis of minus RNA strands. In R17 both steps of its RNA replication are affected by rifampin, although each step is only partially (approximately 50%) inhibited. The relation of the present results to the possible role of bacterial proteins and tertiary structure of phage RNA in the process of template recognition is discussed.     

Sequence homologies between ribosomal and phage RNAs: A proposed molecular basis for RNA phage parasitism

An extensive nucleotide sequence homology between the 3′-end of the 16 S ribosomal RNA and segments of bacteriophage MS2 or Qβ RNA is described. In addition, a notable sequence homology of coliphage RNAs with several other segments of ribosomal RNA is shown. The role of bacterial proteins in the recognition of phage RNA, and the resemblance of phage and host RNAs as the molecular basis of RNA phage parasitism is discussed.The problem of quantifying the degree of homology is discussed in the Appendix with a preliminary attempt towards a solution. A relative measure of homology is described, and used to analyze statistically the data obtained.     

Construction and characterization of a plasmid containing a nearly full-size DNA copy of bacteriophage MS2 RNA.

An apparently full-length complementary DNA copy of in vitro polyadenylated MS2 RNA was synthesized with avian myeloblastosis virus RNA-dependent DNA polymerase. After the MS2 RNA template was removed from the complementary DNA strand with T₁ and pancreatic RNase digestion, the complementary DNA became a good template for the synthesis of double-stranded MS2 DNA with Escherichia coli DNA polymerase I. We then constructed molecular chimeras by inserting the double-stranded MS2 DNA into the PstI restriction endonuclease cleavage site of the E. coli plasmid pBR322 by means of the poly(dA)· poly(dT) tailing procedure. An E. coli transformant carrying a plasmid with a nearly full-length MS2 DNA insertion, called pMS2-7, was chosen for further study. Correlation between the restriction cleavage site map of pMS2-7 DNA and the cleavage map predicted from the primary structure of MS2 RNA, and nucleotide sequence analysis of the 5′ and 3′ end regions of the MS2 DNA insertion, showed that the entire MS2 RNA had been faithfully copied, and that, except for 14 nucleotides corresponding to the 5′-terminal sequence of MS2 RNA, the fulllength DNA copy of the viral genetic information had been inserted into the plasmid. Restriction endonuclease analysis of the chimera plasmid DNA also revealed the presence of an extra DNA insertion which was identified as the translocatable element IS1³ (see following paper).