Crystallization of bacteriophage MS2.

Crystals of bacteriophage MS2 have been obtained by slowly cooling a 1% virus solution from 23 degrees C to 0 degrees C in the presence of poly(ethylene glycol) 6000. The crystals were colorless, needle-like, anisotropic and very fragile. Electron microscopic observation of the crystals revealed a two-dimensional lattice of particles with RNA phage morphology and dimensions. Preliminary X-ray examination of the crystals confirmed their viral nature.     

Localization of Coliphage MS2 A-Protein

The purification of coliphage MS2 dinitrophenol (DNP) conjugates provided a system for localization of the single molecule of A-protein in the capsid of the MS2 phage particle. Three A-protein preparations isolated from unconjugated MS2, overconjugated DNP-MS2, and purified 78S DNP-MS2 were tested for the presence of covalently bound DNP. The binding characteristics to Dowex 1-X8 and rabbit anti-DNP bovine serum albumin (DNP-BSA) immunoglobulin G of the 78S DNP-MS2 and overconjugated DNP-MS2 A-protein preparations indicate that the A-protein is located on the surface of the phage particle where it can be covalently conjugated with hapten. Extensive enzymatic iodination of the A-protein of intact unconjugated MS2 substantiates this conclusion.     

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.     

Comparison of MS/MS methods for protein identification from 2D-PAGE

We have compared the use of a low resolution MALDI-Ion Trap MS/MS and a high-resolution ESI-TOF-MS/MS for the analysis of spots from 2D gels. The main criteria were speed and accuracy of protein identification. The results obtained using the MALDI-MS/MS system are comparable to those from the LC-MS/MS system in terms of accuracy, but less low-level proteins are identified while the time required for the analysis is dramatically reduced.     

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.     

Temperature-sensitive translation of MS2 bacteriophage RNA

A comparison was made of bacteriophage MS2 RNA translation in infected Escherichia coli cells and in a defined cell-free system. A number of temperature-sensitive mutants were used as hosts for viral RNA translation at permissive and restrictive temperatures. The amount of viral coat protein synthesis was determined after gel electrophoresis of proteins from the cell lysates. These results were compared to those obtained with cell-free translation assays conducted with ribosomes isolated from the same mutants. Compared with control cells, a reduced activity in vivo and in vitro was found for each mutant examined at elevated temperatures. A good correlation between the two types of translational assays was observed. These findings are discussed in terms of the translational defects known to be a characteristic of some of these mutant strains.     

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.     

Replication of bacteriophage MS2. X. Phage-specific ribonucleoprotein particles found in MS2-infected Escherichia coli

Phage-specific RNA-protein complexes formed during the MS2 infection process were examined. The fate of ³²P-labeled parental viral RNA was followed to determine what RNA-protein interactions developed early in infection. In order to identify phage-specific ribonucleoprotein complexes at later times in infection, their protein or RNA components were labeled selectively with radioisotopes after suppression of bacterial macromolecular syntheses with Miracil D (Burroughs Wellcome and Co.).     

The regulatory region of MS2 phage RNA replicase cistron. Functional activity of individual MS2 RNA fragments

The present work deals with the structural-functional organization of regulatory regions of messenger RNAs. Some principles of the action of a translational repressor (coat protein) and the formation of the ribosomal initiation complex at the replicase cistron have been studied with MS2 phage RNA. When the complex of MS2 RNA with the coat protein is treated with T₁ ribonuclease, the coat protein selectively protects mainly two fragments (59 and 103 nucleotides in length) from digestion; these fragments contain the intercistronic regulatory region and the beginning of the MS2 replicase cistron. These polynucleotides have been isolated in a pure state and their primary structure has been established.It has been established that both MS2 RNA fragments contain all the necessary information for specific interaction with MS2 coat protein and form a complex with it with an efficiency close to that observed in the case of native MS2 RNA. They also provide the normal polypeptide chain initiation at the replicase cistron. Enzymatic binding of the second aminoacyl-tRNA and electrophoretic analysis of N-terminal dipeptides prove that only the true initiator codon of the replicase cistron is recognized by a ribosome despite the presence of a few additional AUG triplets within the polynucleotides. Under conditions of limited hydrolysis by T₁ ribonuclease, the beginning of the replicase cistron has been removed from the shortest polynucleotide leading to a complete loss of its ability to bind both the coat protein and a ribosome.Some principles of the functioning of the regulatory region in MS2 RNA as well as the nature of the initiator signal of protein biosynthesis are discussed.     

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.     

Effect of Fluorophenylalanine on Bacteriophage MS2 Replication

A concentration of 10 mug of fluorophenylalanine per ml added to a chemically defined medium reduced by 100-fold the number of bacteriophage MS2 produced on Escherichia coli C3000 and increased the latent period. Fluorophenylalanine was most effective when added concurrent with infection. Addition of a 10-fold greater concentration of phenylalanine reversed the inhibition caused by fluorophenylalanine. Radioactive fluorophenylalanine was incorporated into the coat protein. The four phenylalanine-containing chymotryptic peptides are not equally accessible to fluorophenylalanine. Only two of the peptides are highly labeled by fluorophenylalanine. Incorporation of fluorophenylalanine decreased the specific infectivity and the rate of adsorption but did not increase the sensitivity of the whole virus to ribonuclease. MS2 ribonucleic acid (RNA) functioned as messenger RNA for the incorporation of both phenylalanine and fluorophenylalanine in a cell-free incorporating system from E. coli.     

Automatic validation of phosphopeptide identifications by the MS2/MS3 target-decoy search strategy

Manual checking is commonly employed to validate the phosphopeptide identifications from database searching of tandem mass spectra. It is very time-consuming and labor intensive as the number of phosphopeptide identifications increases greatly. In this study, a simple automatic validation approach was developed for phosphopeptide identification by combining consecutive stage mass spectrometry data and the target-decoy database searching strategy. Only phosphopeptides identified from both MS2 and its corresponding MS3 were accepted for further filtering, which greatly improved the reliability in phosphopeptide identification. Before database searching, the spectra were validated for charge state and neutral loss peak intensity, and then the invalid MS2/MS3 spectra were removed, which greatly reduced the database searching time. It was found that the sensitivity was significantly improved in MS2/MS3 strategy as the number of identified phosphopeptides was 2.5 times that obtained by the conventional filter-based MS2 approach. Because of the use of the target-decoy database, the false-discovery rate (FDR) of the identified phosphopeptides could be easily determined, and it was demonstrated that the determined FDR can precisely reflect the actual FDR without any manual validation stage.     

Intermediates of Bacteriophage MS2 Assembly In Vivo

The in vivo process of virion assembly was studied in rifampin-treated, MS2-infected Escherichia coli during late times of infection-after 18 min postinfection. Differential sucrose gradient sedimentation of infected-cell lysates taken at various times after radioactive labeling indicated a definite temporal order of appearance of phage-specific protein in assembly-related structures. Labeled MS2 protein appears first as a low-molecular-weight peak at the tops of gradients, then as a peak at 40S and as a large number of almost unseparable structures between 40 and 80S, and finally as 80S mature phage particles. During the chase of a short labeling period, radioactive phage protein was found to disappear from gradients in the same temporal order as it appeared; the soluble peak disappears first, followed by the 40 to 70S region. The chased label appears quantitatively in the 80S phage peak. Labeled phage RNA was found to appear first in the 40S peak, then in the structures between 40 and 70S, and finally in 80S phage particles. The order of disappearance of labeled phage RNA during a chase is the same as its appearance. Resedimentation of the 40 to 70S region indicated the presence of distinct structures at 60 and 70S and many indistinct ones between 40 and 60S. The smaller intermediates exhibit separable maturation protein-rich and coat protein-rich segments, indicating nonrandom binding of the two proteins during the initial steps of assembly. Larger, discrete intermediates appear at 60 and 70S. Treatment of the various structures with pancreatic RNase results in destruction of those from 40 through 60S; treatment of the 70S structure results in the conversion of some of it to a 45S peak, presumably the complete capsid. A small fraction of the 80S phage peak is also sensitive to RNase, resulting in a similar 45S peak. Pulse-chase experiments indicate that structures from 40 through 60S as well as the RNase-sensitive 70S structure are assembly intermediates, but that the RNase-insensitive 70S and the RNase-sensitive 80S structures are not.