Addressed enzymatic fragmentation of RNA molecules

RNase H has been used for selective cleavage of RNA of MS2 and R17 bacteriophages and 16S RNA from E. coli ribosomes in the region of formation of heteroduplex composed of RNA and an oligodeoxyribonucleotide complementary to a certain part of it. The oligonucleotides used--d(C-T-C-A-T-G-T-T-), d(C-C-A-T-C-T-T-T-T) and d(T-T-T-C-C-A-T-C-T-T-T-T)--were synthesized by chemical methods. The molecular weight of the fragments produced on cleavage of the RNA of MS2 and R17 were estimated with the use of gel electrophoresis under denaturating conditions. The dependence of the enzyme activity on Mg2+ and Na+ concentration and of RNA cleavage on the RNA: oligodeoxyribonucleotide ratio was investigated.     

Defective bacteriophage MS2 particles containing specific RNA FRAGMENTS]

Two types of MS2 particle are revealed when phage lysates are banded in CsCl density gradient. The lower band contain normal phage particles with a density of 1.46 g/cm3. The upper band with a density of 1.44 g/cm3 containes uninfective incomplete MS2 particles. Both phage types reveal no abnormalities in the content of the coat protein and A-protein. They are nearly identical in RNA content. RNA in the normal buoyant density phage particles is native. RNA in the defective particles consists of three specific fragments with molecular weights 6.5-10(5), 5.5-10(5) and 4.4-10(5) and molar ratios 5:4:9 respectively. THE 5'-TERMINAL ANALYSIS OF RNA from defective MS2 particles reveals the presence of native pppGp. THE 3'-TERMINAL ANALYSIS OF THE INDIVIDUAL RNA fragments reveals the presence of adenosine only in the shortest fragment. RNA fragmentation in defective particles can be explained by the action of intracellular RNAses on the unprotected regions on RNA chain in structurally incomplete virions.     

The RNA binding site of bacteriophage MS2 coat protein.

The coat protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis. In order to identify the structural components of coat protein required for its RNA binding function, a series of repressor-defective mutants has been isolated. To ensure that the repressor defects were due to substitution of binding site residues, the mutant coat proteins were screened for retention of the ability to form virus-like particles. Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of protein folding or stability defects. Each of the variant coat proteins was purified and its ability to bind operator RNA in vitro was measured. DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity. Localization of the substituted sites in the three-dimensional structure of coat protein reveals that amino acid residues on three adjacent strands of the coat protein beta-sheet are required for translational repression and RNA binding. The sidechains of the affected residues form a contiguous patch on the interior surface of the viral coat.     

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.     

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.     

Tritium labeling of RNA and protein of bacteriophage MS2

Thermal activation of tritium gas is used for labeling of the nucleoprotein, phage MS 2. The obtained preparation of tritiated phage has a specific radioactivity of 20-50 Ci/mmole, is considerably infectious and appears suitable for a wide range of studies. The radioactivity is distributed between intraphage RNA and phage outer protein (approximately 1:3 ratio). Consequently, phage capsid is porous and sufficiently permeable for activated tritium atoms.     

Functional expression of individual plasmid-coded RNA bacteriophage MS2 genes

The genes of the RNA-containing bacteriophage MS2 were individually inserted into thermoinducible expression plasmids under control of the phage λ P_L promoter. Three phage-coded proteins (A-protein, coat protein, and replicase) were expressed at high efficiency. Induced cultures specifically complemented superinfecting amber mutants of phage MS2. Regulatory mechanisms operative during the natural infection cycle of the phage were reproduced by the plasmid expression system.     

Self cleavage of a precursor RNA from bacteriophage T4

We found that a precursor of an RNA molecule from T4-infected Escherichia coli cells (p2Spl; precursor of species 1) has the capacity to cleave itself in a specific position. This cleavage is similar to a cleavage carried out by the aid of a protein, RNase F, that has been previously identified. This cleavage could lead to the maturation of an RNA (species 1) found in T4-infected E. coli cells. The reaction is time and temperature-dependent and is relatively slow as compared to the protein-dependent reaction. It requires at least a monovalent cation and is aided by non-ionic detergents. In the absence of detergent the cleavage can occur but at a reduced rate. The substrate does not contain hidden nicks and a variety of experiments suggest that it does not contain a protein. Moreover, we found no indication that the cleavage is due to contaminating nucleases in the substrate or in the reagents. The intact secondary and tertiary structures of the molecule are necessary for the cleavage to occur. The finding of a self cleaving RNA molecule has interesting evolutionary implications.     

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.     

Effect of deleterious mutation-accumulation on the fitness of RNA bacteriophage MS2

Abstract.— RNA viruses show the highest mutation rate in nautre. It has been extensively demonstrated that, in the absence of purifying selection, RNA viruses accumulate deleterious mutations at a high rate. However, the parameters describing this accumulation are, in general, poorly understood. The present study reports evidences for fitness declines by the accumulation of deleterious mutations in the bacteriophage MS2. We estimated the rate of fitness decline to be as high as 16% per bottleneck transfer. In addition, our results agree with an additive model of fitness effects.     

Studies on the secondary structure of single-stranded RNA from the bacteriophage MS2. II Analysis of the RNase IV cleavage products

Single-stranded RNA from the bacteriophage MS2 was cleaved into two unequal fragments using the Escherichia coli endonuclease RNase IV. The fragments were purified by sucrose gradient centrifugation and secondary structure maps of the purified fragments were prepared after spreading the RNAs in 0·5 mmMgCl₂. Comparison of these maps with those of native RNA permitted the identification of the 5′ and 3′ ends of the maps of native single-stranded RNA. In addition, the location of the cleavage site with respect to the secondary and tertiary structure of the RNA suggests that the conformation of the RNA around this site may be important in determining the specificity of cleavage by the enzyme.The approximate location of individual viral genes within the secondary structure map has been obtained by comparing the map of native RNA with known sequence data. A new model is proposed to explain the role of secondary structure, as seen in the electron microscope, in the regulation of the synthesis of coat protein and the viral subunit of the MS2 replicase.