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.     

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.     

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.     

The genome of infectious bursal disease virus consists of two segments of double-stranded RNA.

The RNA of infectious bursal disease virus was reexamined in a detailed analysis. It could be established that its genome consists of two segments of double-stranded RNA. The RNA is RNase resistant and has a sedimentation coefficient of 14S and a buoyant density of 1.62 g/ml. The purine/pyrimidine ratio is nearly 1; the guanine plus cytosine content is 55.3%; the Tm is 95.5 degrees C. The molecular weights of the two double-stranded segments were determined to be 2.2 x 10(6) and 2.5 x 10(6).     

Effect of alkylation on the secondary structure of DNA

S1 nuclease hydrolysis and bezoylated naphthoylated DEAE-cellulose (BND-cellulose) chromatography have been used to demonstrate that alkylation of DNA by dimethyl sulfate at neutral pH leads to the production of partially denatured molecules under conditions where no significant depurination occurs. DNA was alkylated with increasing concentrations of the alkylating agent, and subjected to enzymatic degradation and binding to BND cellulose. An increasing degree of DNA hydrolysis and adherence to BND cellulose was seen. On hydroxyapatite chromatography the alkylated DNA still eluted at the position of double-stranded molecules suggesting the presence of partially denatured regions. The presence of salt had a preventive effect on such denaturation.     

Destabilization of secondary structure in 16S ribosomal RNA by dimethylation of two adjacent adenosines.

Fragments comprising the 49 nucleotides from the 3'-end have been purified from 16S ribosomal RNA of wild-type Escherichia coli and from a kasugamycin-resistant mutant that specifically lacks dimethylation of two adjacent adenines near the 3'-terminus. These fragments, obtained after treatment of ribosomes in vitro with the bacteriocin cloacin DF13, were used to study the effect of the methyl groups on the temperature dependent unfolding of double-stranded regions. Both fragments contain at least 3 independent melting transitions, of which the one with the highest Tm corresponds with the unfolding of a nine-basepair long central hairpin. Dimethylation of the adenines in the loop of this hairpin lowers the melting temperature (Tm) by approximately 2 degrees C at 0.2 M NaCl and by about 5 degrees C at 0.15 M NaCl. It is suggested that m6(2)Am6(2)A is more antagonistic to loop formation that ApA and that the function of the methyl groups is to help to destabilize the 3'-terminal hairpin in 16S rRNA in order to facilitate intermolecular interactions.     

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)].     

How much is secondary structure responsible for resistance of double-stranded RNA to pancreatic ribonuclease A?

1. Double-stranded f2 sus11 or Qbeta RNAs, resistant to bovine pancreatic RNAase A in 0.15 M NaCl/0.015 M sodium citrate (SSC), are quickly and completely degraded at 10-fold lower ionic strength (0.1 X SSC) under otherwise similar conditions. At this ionic strength the secondary structure of double-stranded RNA is maintained, as judged by the following: (a) the unchanged resistance of double-stranded RNA and DNA, under similar low ionic strength conditions, to nuclease S1 from Aspergillus oryzae, in contrast with the sensitivity of the corresponding denatured nucleic acids to this enzyme, specific for single-stranded RNA and DNA; (b) the co-operative pattern of the thermal-transition profile of double-stranded RNA (with a Tm of 89 degrees C) in 0.1 X SSC. 2. Whereas in SSC bovine seminal RNAase (RNAase BS-1) and whale pancreatic RNAase show an activity on double-stranded RNA significantly higher than that of RNAase A, in 0.1 X SSC the activity of the latter enzyme on this substrate becomes distinctly higher than that of RNAase BS-1, and similar to that of whale RNAase. 3. From these results it is deduced that the secondary structure is probably not the only nor the most important variable in determining the susceptibility double-stranded RNA to ribonuclease. Other factors, such as the effect of ionic strength on the enzyme and/or the binding of enzyme to nucleic acids, may play an important role in the process of double-stranded RNA degradation by ribonucleases specific for single-stranded RNA.     

Destabilization of the secondary structure of RNA by ribosomal protein S1 from escherichia coli

S1 is an acidic protein associated with the 3′ end of 16S RNA; it is indispensable for ribosomal binding of natural mRNA. We find that S1 unfolds single stranded stacked or helical polynucleotides (poly rA, poly rC, poly rU). It prevents the formation of poly (rA + rU) and poly (rI + rC) duplexes at 10–25 mM NaCl but not at 50–100 mM NaCl. Partial, salt reversible denaturation is also seen with coliphage MS2 RNA, $\text{E. coli}$ rRNA and tRNA. Generally, only duplex structures with a Tm greater than about 55° are formed in the presence of S1. The protein unfolds single stranded DNA but not poly d(A·T).     

The secondary structure of ribosomal ribonucleic acid in solution

1. The u.v.-absorption spectrum of ribosomal RNA from rabbit reticulocytes was studied as a function of temperature at different pH values. The changes in the spectrum over the range 220-320mmu were interpreted on the basis of the assumption that the effect of denaturation and ionization are additive. The results suggest that in neutral salt solutions the secondary structure of the ribosomal RNA samples studied is due to two species of helical segments stabilized principally, if not solely, by complementary base pairs but differing in nucleotide composition: each species appears to be heterogeneous in other respects in view of the breadth of the melting ranges. 2. The number of base pairs per helical segment was estimated to be small (between 4 and 17) on the basis of the relation between melting temperature and chain length previously established by Lipsett and others for model compounds. Small fragments (about 2s) obtained by alkaline hydrolysis appeared to form the same helical segments as the intact molecule in accord with the estimated size of these segments. 3. Specific nucleotide sequences appear necessary to account for the hysteresis observed on titrating ribosomal RNA with acid or alkali within the range pH3.0-7.0 since this phenomenon was less pronounced for Escherichia coli transfer RNA and for RNA from turnip yellow-mosaic virus.     

Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly

YqeH, a circularly permuted GTPase, is conserved among bacteria and eukaryotes including humans. It was shown to be essential for the assembly of small ribosomal (30S) subunit in bacteria. However, whether YqeH interacts with 30S ribosome and how it may participate in 30S assembly are not known. Here, using co-sedimentation experiments, we report that YqeH co-associates with 30S ribosome in the GTP-bound form. In order to probe whether YqeH functions as RNA chaperone in 30S assembly, we assayed for strand dissociation and annealing activity. While YqeH does not exhibit these activities, it binds a non-specific single and double-stranded RNA, which unlike the 30S binding is independent of GTP/GDP binding and does not affect intrinsic GTP hydrolysis rates. Further, S5, a ribosomal protein which participates during the initial stages of 30S assembly, was found to promote GTP hydrolysis and RNA binding activities of YqeH.     

The action of ribonuclease T1 on reovirus double-stranded RNA.

A small number of nucleotides are released from highly purified reovirus double-stranded RNA by ribonuclease T₁ in the presence of 0.3 m NaCl. These nucleotides include ppGp, which is quantitatively released from the RNA, and lesser amounts of ApUpGp, Gp, and ApGp. The same products are released from each of the three size classes of double-stranded RNA segments. In experiments involving specific labeling of termini, the only demonstrable sites of hydrolysis were at the 5′ termini of the minus strands. The limited extent of ribonuclease T₁ hydrolysis and localization of its action at the 5′ termini of the minus strands are compatible with a perfect duplex structure for the double-stranded RNA segments wherein the secondary structure of the termini is less stable than that of internal regions.     

Cucumber mosaic virus-associated RNA 5. VI. Characterization and denaturation-renaturation behavior of the double-stranded form.

The double-stranded form of cucumber mosaic virus-associated RNA 5 has been purified and further characterized. Its molecular weight determined by sedimentation equilibrium is 2.15 . 10(5). The buoyant density calculated from its symmetrical distribution in Cs2SO4, following isopycnic ultracentrifugation, is 1.615 g/cm3. The sedimentation rate of double-stranded cucumber mosaic virus-associated RNA 5 is slightly greater than that of cucumber mosaic virus-associated RNA 5; its electrophoretic mobility in polyacrylamide gel (2.4%) is less than that of cucumber mosaic virus-associated RNA 5. By the above standards the double-stranded cucumber mosaic virus-associated RNA 5 preparations used were found to be nomogeneous in size as well as density. Thermal denaturation monitored by means of ultraviolet light absorption produced multitransitional denaturation profiles. The average melting temperature (Tm) was 88 degrees C in 0.1 x SSC. Monotransitional denaturation profiles and slightly higher Tm values were obtained when resistance against ribonuclease digestion was measured. These denaturation experiments and other propertied led to the conclusion that double-stranded cucumber mosaic virus-associated RNA 5 and the double-stranded form of peanut stunt virus-associated RNA 5 are small double-stranded nucleic acids with several homostable base-pair regions, characterized by distinct G + C contents and Tm values.     

Dynamics of the muscle thin filament regulatory switch: the size of the cooperative unit.

Actin thin filaments containing bound tropomyosin (Tm) or tropomyosin troponin (Tm.Tn) exist in two states ("off" and "on") with different affinities for myosin heads (S1), which results in the cooperative binding of S1. The rate of S1 binding to, and dissociating from, actin, Tm.actin, and Tm.Tn.actin, monitored by light scattering (LS), was compared with the rate of change in state, monitored by the excimer fluorescence (Fl) of a pyrene label attached to Tm. The ATP-induced S1 dissociation showed similar exponential decreases in LS for actin.S1, Tm.actin.S1, and Tm.Tn.actin.S1 +/- Ca2+. The Fl change, however, showed a delay that was greater for Tm.Tn.actin than Tm.actin, independent of Ca2+. The S1 binding kinetics gave observed rate constants for the S1-induced change in state that were 5-6 times the observed rate constants of S1 binding to Tm.actin, which were increased to 10-12 for Tm.Tn.actin, independent of Ca2+. The rate of the Fl signals showed that the on/off states were in rapid equilibrium. These data indicate that the apparent cooperative unit for Tm.actin is 5-6 actin subunits rather than the minimum structural unit size of 7, and is increased to 10-12 subunits for Tm.Tn.actin, independent of the presence of Ca2+. Thus, Tm appears semi-flexible, and Tn increases communication between neighboring structural units. A general model for the dynamic transitions involved in muscle regulation is presented.     

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)].     

Theoretical basis for stabilizing messenger RNA through secondary structure design

RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term ‘superfolder’ mRNAs. These designs exhibit a wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity. Furthermore, their folding is robust to temperature, computer modeling method, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1 and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable.     

Importance of internal regions and the overall length of tropomyosin for actin binding and regulatory function

Tropomyosin (Tm) binds along actin filaments, one molecule spanning four to seven actin monomers, depending on the isoform. Periodic repeats in the sequence have been proposed to correspond to actin binding sites. To learn the functional importance of length and the internal periods we made a series of progressively shorter Tms, deleting from two up to six of the internal periods from rat striated alpha-TM (dAc2--3, dAc2--4, dAc3--5, dAc2--5, dAc2--6, dAc1.5--6.5). Recombinant Tms (unacetylated) were expressed in Escherichia coli. Tropomyosins that are four or more periods long (dAc2--3, dAc2--4, and dAc3--5) bound well to F-actin with troponin (Tn). dAc2--5 bound weakly (with EGTA) and binding of shorter mutants was undetectable in any condition. Myosin S1-induced binding of Tm to actin in the tight Tm-binding "open" state did not correlate with actin binding. dAc3--5 and dAc2--5 did not bind to actin even when the filament was saturated with S1. In contrast, dAc2--3 and dAc2--4 did, like wild-type-Tm, requiring about 3 mol of S1/mol of Tm for half-maximal binding. The results show the critical importance of period 5 (residues 166--207) for myosin S1-induced binding. The Tms that bound to actin (dAc2--3, dAc2--4, and dAc3--5) all fully inhibited the actomyosin ATPase (+Tn) in EGTA. In the presence of Ca(2+), relief of inhibition by these Tms was incomplete. We conclude (1) four or more actin periods are required for Tm to bind to actin with reasonable affinity and (2) that the structural requirements of Tm for the transition of the regulated filament from the blocked-to-closed/open (relief of inhibition by Ca(2+)) and the closed-to-open states (strong Tm binding to actin-S1) are different.     

Sequence-specific single-strand RNA binding protein encoded by the human LINE-1 retrotransposon.

Previous experiments using human teratocarcinoma cells indicated that p40, the protein encoded by the first open reading frame (ORF) of the human LINE-1 (L1Hs) retrotransposon, occurs in a large cytoplasmic ribonucleoprotein complex in direct association with L1Hs RNA(s), the p40 RNP complex. We have now investigated the interaction between partially purified p40 and L1Hs RNA in vitro using an RNA binding assay dependent on co-immunoprecipitation of p40 and bound RNA. These experiments identified two p40 binding sites on the full-length sense strand L1Hs RNA. Both sites are in the second ORF of the 6000 nt RNA: site A between residues 1999 and 2039 and site B between residues 4839 and 4875. The two RNA segments share homologous regions. Experiments involving UV cross-linking followed by immunoprecipitation indicate that p40 in the in vitro complex is directly associated with L1Hs RNA, as it is in the p40 RNP complex found in teratocarcinoma cells. Binding and competition experiments demonstrate that p40 binds to single-stranded RNA containing a p40 binding site, but not to single-stranded or double-stranded DNA, double-stranded RNA or a DNA-RNA hybrid containing a binding site sequence. Thus, p40 appears to be a sequence-specific, single-strand RNA binding protein.