Studies on the bacteriophage MS2. 23. Fixation of the MS2 RNA acid structure by formaldehyde

When MS2 RNA is heated at low p^H in the presence of formaldehyde, a fast-sedimenting conformation is irreversibly formed. This species is homogeneous and stable at neutral p^H. Its formation further requires Mg⁺⁺ ions and low ionic strength. The most compact form sediments at 46S and is obtained after short reaction times at high temperature or after long reaction times at 35°C. Melting curves suggest that the specific acid conformation is not destabilized by the formaldehyde addition reaction. The p^H at which this acid conformation is formed depends on the MgC1₂ concentration. At 10^?2M MgCl₂ the midpoint is p^H 5.3. Removal of more than half of the bound formaldehyde has no effect on the compactness of the molecule, although most of the original secondary structure has not yet re-formed.     

Studies on the bacteriophage MS2. XXII. Conformation of MS2 RNA in acid medium

MS2 RNA, which sediments at 27S in a neutral buffer, can be converted to a compact 57S conformation at pH 3.8. Requirements for this conversion, besides protonation, are small concentrations of Mg⁺⁺ ions and a low ionic strength. On the other hand, after heating in the presence of EDTA and at low ionic strength, the RNA can be unfolded to an 11.7S form at pH 6.8 and to 10.5S at pH 3.8. The compact 57S form has lost at least 50% of its secondary structure, as determined by its hypochromicity. It corresponds to a monomer species, as will be shown in a following paper (XXIV). Comparative studies with the homopolymers poly A and poly C and with the heteropolymers poly A,U, poly A,C, and poly A,G indicate that the interactions involved in the acid RNA conformation are not simply explainable by the known interactions of the A–A⁺, C–C⁺, and/or A–C⁺ type.     

Site-specific cleavage of MS2 RNA by a thermostable DNA-linked RNase H

A series of DNA-linked RNases H, in which the 15-mer DNA is cross-linked to the Thermus thermophilus RNase HI (TRNH) variants at positions 135, 136, 137 and 138, were constructed and analyzed for their abilities to cleave the complementary 15-mer RNA. Of these, that with the DNA adduct at position 135 most efficiently cleaved the RNA substrate, indicating that position 135 is the most appropriate cross-linking site among those examined. To examine whether DNA-linked RNase H also site-specifically cleaves a highly structured natural RNA, DNA-linked TRNHs with a series of DNA adducts varying in size at position 135 were constructed and analyzed for their abilities to cleave MS2 RNA. These DNA adducts were designed such that DNA-linked enzymes cleave MS2 RNA at a loop around residue 2790. Of the four DNA-linked TRNHs with the 8-, 12-, 16- and 20-mer DNA adducts, only that with the 16-mer DNA adduct efficiently and site-specifically cleaved MS2 RNA. Primer extension revealed that this DNA-linked TRNH cleaved MS2 RNA within the target sequence.     

Improvement of RNA secondary structure prediction using RNase H cleavage and randomized oligonucleotides

RNA secondary structure prediction using free energy minimization is one method to gain an approximation of structure. Constraints generated by enzymatic mapping or chemical modification can improve the accuracy of secondary structure prediction. We report a facile method that identifies single-stranded regions in RNA using short, randomized DNA oligonucleotides and RNase H cleavage. These regions are then used as constraints in secondary structure prediction. This method was used to improve the secondary structure prediction of Escherichia coli 5S rRNA. The lowest free energy structure without constraints has only 27% of the base pairs present in the phylogenetic structure. The addition of constraints from RNase H cleavage improves the prediction to 100% of base pairs. The same method was used to generate secondary structure constraints for yeast tRNA^Phe, which is accurately predicted in the absence of constraints (95%). Although RNase H mapping does not improve secondary structure prediction, it does eliminate all other suboptimal structures predicted within 10% of the lowest free energy structure. The method is advantageous over other single-stranded nucleases since RNase H is functional in physiological conditions. Moreover, it can be used for any RNA to identify accessible binding sites for oligonucleotides or small molecules.     

Studies on the bacteriophage MS2. XXIV. Hydrodynamic properties of the native and acid MS2 RNA structures

The molecular weight of native 26.6S MS2 RNA, of acidic 53.1S MS2 RNA and of formaldehyde-treated 36.1S MS2 RNA was determined by light-scattering studies. The three forms examined correspond to monomers. Also νv values were determined, and in conjunction with our previous estimates for S_20,W and [η], led again to the conclusion that formaldehyde-cross-linked 37S MS2 RNA is a monomer. On the basis of the experimentally observed R_G, [η], S_20,W, and νv values, and taking a prolate ellipsoid as a model, the hydrodynamic volume, the solvation water and the axial ratio could be deduced for the three conformations. In the “native” 26S form the RNA occupies only 8.2% of the hydrodynamic volume. The formation of the 36S and the 53S structures correspond largely to a loss of solvation water.     

Studies on the bacteriophage MS 2

Summary The viral proteins synthesized in non-suppressor cells by amber mutants in the A protein cistron of the RNA bacteriophage MS2 were analyzed. Protein synthesis was studied in rifampicin-inhibited cultures and the labeled, viral proteins were separated on sodium dodecyl sulphate containing polyacrylamide gels. We found that 7 out of 19 mutants synthesized an A protein-fragment corresponding in length to 88% of the wild-type A protein. This fragment was not incorporated into the defective particles formed by the mutants. 12 mutants synthesized no detectable amount of fragment. It was shown that the absence of fragment is not due to selective proteolytic breakdown.     

Computer method for predicting the secondary structure of single-stranded RNA.

We present a computer method utilizing published values for base pairing energies to compute the most energetically favorable secondary structure of an RNA from its primary nucleotide sequence. After listing all possible double-helical regions, every pair of mutally incompatible regions (whose nucleotides overlap) is examined to determine whether parts of those two regions can be combined by branch migration to form a pair of compatible new subregions which together are more stable than either of the original regions separately. These subregions are added to the list of base pairing regions which will compete to form the best overall structure. Then, a 'hyperstructure matrix' is generated, containing the unique topological relationship between every pair of regions. We have shown that the best structure can be chosen directly from this matrix, without the necessity of creating and examing every possible secondary structure. We have included the results from our solution of the 5S rRNA of the cyanobacterium Anacystis nidulans as an example of our program's capabilities.     

Codon usage and secondary structure of MS2 phage RNA.

MS2 is an RNA bacteriophage (3569 bases). The secondary structure of the RNA has been determined, and is known to play an important role in regulating translation. Paired regions of the genome have a higher G+C content than unpaired regions. It has been suggested that this reflects selection for high G+C content to encourage pairing, but a re-analysis of the data together with computer simulation suggest that it is an automatic consequence in any RNA sequence of the way it folds up to minimise its free energy. It has also been suggested that the three registers in which pairing can occur in a coding region are used differentially to optimise the use of the redundancy of the genetic code, but re-analysis of the data shows only weak statistical support for this hypothesis.     

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.     

Studies of virus structure by laser-Raman spectroscopy. II. MS2 phage, MS2 capsids and MS2 RNA in aqueous solutions.

Laser-Raman spectra of the bacteriophage MS2, and of its isolated coat-protein and RNA components, have been obtained as a function of temperature in both H₂O and D₂O (deuterium oxide) solutions. The prominent Raman lines in the spectra are assigned to the amino acid residues and polypeptide backbone of the viral coat protein and to the nucleotide residues and ribosyl-phosphate backbone of the viral RNA. The Raman frequencies and intensities, and their temperature dependence, indicate the following features of MS2 structure and stability. Coat-protein molecules in the native phage maintain a conformation determined largely by regions of β-sheet (~60%) and random-chain (~40%) structures. There are no disulfide bridges in the virion and all sulfhydryl groups are accessible to solvent molecules. Protein-protein interactions in the virion are stable up to 50 °C. Release of viral RNA from the virion does not affect either the conformation of the coat-protein molecules or the thermal stability of the capsid. MS2 RNA within the virion contains a highly ordered secondary structure in which most (~85%) of the bases are either paired or stacked or both paired and stacked and in which the RNA backbone assumes a geometry of the A-type. When RNA is partially or fully released from the virion its overall secondary structure at 32 °C is unchanged. However, the exposed RNA is more susceptible to changes in secondary structure promoted by increasing the temperature. Thus the viral capsid exerts a significant stabilizing effect on the secondary structure of MS2 RNA. This stabilization is ionic-strength dependent, being more pronounced in solutions containing high concentrations of KCl. Raman intensity profiles as a function of temperature reveal that disordering of the MS2 RNA backbone and rupture of hydrogen-bonding between complementary bases are gradual processes, the major portions of which occur above 40 °C. However, the unstacking of purine and pyrimidine bases is a more co-operative phenomenon occurring almost exclusively above 55 °C.     

Studies on bacteriophage fd DNA. II. Localization of RNA initiation sites on the cleavage map of the fd genome.

In an in vitro RNA synthesizing system, a single size of A-start RNA and three different sizes of G-start RNA are predominantly transcribed on the doubly closed replicative form (RFI) DNA of phage fd. When the RFI DNA was cleaved into three fragments (HinH-A, HinH-B and HinH-C) by a restriction endonuclease from Haemophilus influenzae H-I, the A-start RNA was predominantly initiated on HinH-B and the three G-start RNAs on HinH-A. RFI DNA was further cleaved into smaller pieces by two other restriction endonucleases from H. aphirophilus and H. gallinarum. Upon mixing the digests with RNA polymerase, two specific fragments derived from HinH-A were bound to the polymerase with GTP present. G-start RNA was efficiently initiated on the fragments isolated by this procedure. On the basis of these observations and estimates of the size of RNA formed on each fragment, the initiation sites for major RNA species were localized on the cleavage map of the phage fd genome previously constructed.     

Effect of spermidine on the RNA-A protein complex isolated from the RNA bacteriophage MS2.

The polyamine spermidine has recently been reported to be a substantial component of the RNA phage particle. Its effect on the isolated RNA-A protein complex of the phage MS2 is investigated here. This complex infects intact Escherichia coli cells via F-pili, as does the whole phage. It is shown that the infectivity of the complex on intact E. coli cells was enhanced by incubation with spermidine. Optimal stimulation (20-fold) of the complex infectivity was achieved by incubation with 3 x 10(-4) M spermidine for 20 to 30 min at 37 degrees C. This gave a more compact structure to the complex, as could be seen by its faster sedimentation in sucrose gradients. Although spermidine and Mg2+ are known to partially replace one another in several systems, no enhancement of the infectivity of the complex, but only its considerably faster sedimentation in sucrose gradients, occurred after incubation with 3 x 10(-4) M Mg2+. Only if the Mg2+ concentration was raised by more than one order of magnitude could increased infectivity of the complex be observed. At concentrations of spermidine and Mg2+ that maximally stimulated the infectivity of the complex on intact E. coli cells, no increase in infectivity of phenol-extracted RNA to E. coli spheroplasts was detected. From these in vitro results, the role of the polyamine spermidine in the RNA phage particle for the infecting, RNA-A protein complex molecules in phage infection is discussed.