The primary structure of the coat protein of the broad-host-range RNA bacteriophage PRR1.

The complete amino acid sequence of the coat protein of RNA bacteriophage PRR1 is presented. After thermolysin digestion, 26 peptides were isolated, covering the complete coat protein chain. Their alignment was established in part using automated Edman degradation on the intact protein, in part with overlapping peptides obtained by enzymic hydrolysis with trypsin, pepsin, subtilisin and Staphylococcus aureus protease, and by chemical cleavage with cyanogen bromide and N-bromosuccinimide. To obtain the final overlaps, a highly hydrophobic, insoluble tryptic peptide was sequenced for seven steps by the currently used manual dansyl-Edman degradation procedure, which was slightly modified for application on insoluble peptides. PRR1 coat protein contains 131 amino acids, corresponding to a molecular weight of 14534. It is highly hydrophobic, and the residues with ionizable side chains are distributed unevenly: acidic residues are absent in the middle third of the sequence, whereas a clustering of basic residues occurs between positions 44 and 62. PRR1 coat protein was compared with the coat proteins of RNA coliphages MS2 and Q beta, and the minimum mutation distance was calculated for both comparisons. It is highly probable that PRR1. Q beta and MS2 share a common ancestor. The basic region present in the three coat proteins is recognized as an essential structural feature of RNA phage coat proteins.     

Translational repression and specific RNA binding by the coat protein of the Pseudomonas phage PP7

PP7 is a single-strand RNA bacteriophage of Pseudomonas aeroginosa and a distant relative to coliphages like MS2 and Qbeta. Here we show that PP7 coat protein is a specific RNA-binding protein, capable of repressing the translation of sequences fused to the translation initiation region of PP7 replicase. Its RNA binding activity is specific since it represses the translational operator of PP7, but does not repress the operators of the MS2 or Qbeta phages. Conditions for the purification of coat protein and for the reconstitution of its RNA binding activity from disaggregated virus-like particles were established. Its dissociation constant for PP7 operator RNA in vitro was determined to be about 1 nm. Using a genetic system in which coat protein represses translation of a replicase-beta-galactosidase fusion protein, amino acid residues important for binding of PP7 RNA were identified.     

Crystal Structure of the Bacteriophage Qβ Coat Protein in Complex with the RNA Operator of the Replicase Gene

The coat proteins of single-stranded RNA bacteriophages specifically recognize and bind to a hairpin structure in their genome at the beginning of the replicase gene. The interaction serves to repress the synthesis of the replicase enzyme late in infection and contributes to the specific encapsidation of phage RNA. While this mechanism is conserved throughout the Leviviridae family, the coat protein and operator sequences from different phages show remarkable variation, serving as prime examples for the co-evolution of protein and RNA structure. To better understand the protein–RNA interactions in this virus family, we have determined the three-dimensional structure of the coat protein from bacteriophage Qβ bound to its cognate translational operator. The RNA binding mode of Qβ coat protein shares several features with that of the widely studied phage MS2, but only one nucleotide base in the hairpin loop makes sequence-specific contacts with the protein. Unlike in other RNA phages, the Qβ coat protein does not utilize an adenine-recognition pocket for binding a bulged adenine base in the hairpin stem but instead uses a stacking interaction with a tyrosine side chain to accommodate the base. The extended loop between β strands E and F of Qβ coat protein makes contacts with the lower part of the RNA stem, explaining the greater length dependence of the RNA helix for optimal binding to the protein. Consequently, the complex structure allows the proposal of a mechanism by which the Qβ coat protein recognizes and discriminates in favor of its cognate RNA.     

Yeast-Expressed Bacteriophage-Like Particles for the Packaging of Nanomaterials

Virus-like particles (VLPs) generated by heterologous expression of viral structural genes have become powerful tools in vaccine development. Recently, we and others have reported on the assembly of VLPs of the RNA bacteriophages MS2, Qβ, and GA in yeast. Here, we investigate the formation of VLPs of five additional phages in the yeasts Saccharomyces cerevisiae and Pichia pastoris, namely, the coliphages SP and fr, Acinetobacter phage AP205, Pseudomonas phage PP7, and Caulobacter phage φCb5. In all cases except SP, particle formation was detected, although VLP outcome varied from 0.2 to 8 mg from 1 g of wet cells. We have found that phage φCb5 VLPs easily dissociate into coat protein dimers when applied to strong anion exchangers. Upon salt removal and the addition of nucleic acid or its mimics and calcium ions, the dimers re-assemble into VLPs with high efficiency. A variety of compounds, including RNA, DNA, and gold nanoparticles can be packaged inside φCb5 VLPs. The ease with which phage φCb5 coat protein dimers can be purified in high quantities and re-assembled into VLPs makes them attractive for downstream applications including the internal packaging of nanomaterials and the chemical coupling of peptides of interest on the surface.     

The inhibition of nucleic acid-binding proteins by aurintricarboxylic acid

Qβ replicase, $\text{Escherichia coli}$ RNA polymerase, and T7 RNA polymerase are inhibited by low concentrations of the dye aurintricarboxylic acid (ATA). In each case initiation by the enzyme was preferentially inhibited. The elongation of initiated polynucleotide chains by Qβ replicase was insensitive to ATA in the range of concentrations required to inhibit initiation. Treatment of Qβ replicase, RNA polymerase and $\text{lac}$ repressor with ATA prevented enzymemediated binding of the templates to nitrocellulose filters. We propose that the inhibitor combines with the template binding site of these proteins to prevent initiation.     

Inactivation of Q beta RNA by electrophiles

Methyl, ethyl and isopropyl methanesulfonates (MMS, EMS, iPMS), diethyl pyrocarbonate (DEP) and autoclaved irradiated sucrose and glucose (active principles presumably α,β-unsaturated carbonyl compounds) inactivated the transfectivity of $\text{Q}$β RNA in one-hit processes. In the case of DEP, nealy every carbethoxy group introduced inactivated, whereas several alkyls from the methanesulfonates per RNA molecule seemed te be tolerated. 1,2-Dibromoethane was a relatively strong inhibitor of RNA transfectivity in the presence of thioglycol, probably via the formation of a more reactive “half mustard”.Compared with isolated RNA, the complete $\text{Q}$β phage was somewhat protected against methanesulfonates but slightly more sensitive to the irradiated sugars and distinctly more sensitive to DEP, indicating that the two latter compounds may inactivate in reactions with coat proteins.The negative tests with the strongly mutagenic 2,3,7,8-tetrachlorodibenzdioxin suggest that intercalating agents are probably not active towards RNA.The decrease of the trasnfectivity of $\text{Q}$β RNA may be used as a sensitive system to determine reactivity towards nucleic acids of environmental pollutants.     

Differential translation of turnip yellow mosaic virus mRNAs in vitro

Total TYMV RNA was incubated in a reticulocyte lysate, and the initiation peptides of the main proteins synthesized $\text{in vitro}$ (195 K, 150 K and 20 K daltons) analyzed after tryptic digestion. The 195 K and the 150 K dalton proteins present analogous patterns, different from the one obtained with the 20 K dalton protein (coat protein), suggesting that only one initiation site exists on the genomic RNA for the synthesis of the two high molecular proteins. The results of competition experiments between genomic and coat protein mRNA indicate that the ribosomes have a much greater affinity for the coat protein mRNA. This may represent a regulatory mechanism for the preferential amplification of coat protein synthesis in the infected cells.     

Translation of Pseudomonas aeruginosa Bacteriophage PP7 RNA by a Cell-Free Amino Acid Incorporating System from Escherichia coli

We have compared the activities of the RNA genomes of Pseudomonas aeruginosa phage PP7 and coliphages Qbeta and f2 in a cell-free amino acid incorporating system derived from Escherichia coli. The rate of incorporation of [(14)C]leucine in the PP7 RNA-directed system is greater than in the systems directed by either Qbeta or f2 RNA. The response to changes in phage RNA concentrations is similar in all the systems, reaching a saturation level at 0.75 to 1.0 mg of RNA per ml of reaction mixture. Analysis of complete reaction mixtures of the PP7 RNA and of the Qbeta RNA systems by sucrose gradient centrifugation shows generally similar patterns for both RNAs. The principal differences are that in the PP7 system a slightly higher percentage of RNA forms ribosome complexes and that the polysomes are somewhat smaller. PP7 RNA is also degraded more extensively during the reaction than is Qbeta RNA. Analysis of the products of the reactions by acrylamide gel electrophoresis shows that PP7 coat protein is the only identifiable product of the PP7 RNA-directed system, suggesting that only the coat protein cistron is translated by E. coli ribosomes.     

Translation of bacteriophage R17 and Qbeta RNA in a mammalian cell-free system

The polycistronic RNAs from both bacteriophage R17 and Qβ are translated in a mammalian cell-free system of purified and partially purified components. The requirement of one of the partially purified initiation factors (IF-E₃ from rabbit reticulocytes) for the phage RNA translation is strikingly different from that for rabbit globin messenger RNA translation. The phage RNA-directed products are characterized by acrylamide gel electrophoresis and compared with those synthesized in an Escherichia coli cell-free system. There is good agreement between the respective coat proteins and the presumptive synthetase proteins. R17 RNA directs the synthesis of two additional defined polypeptides. However, their possible relationship with the A-protein cistron has not yet been investigated. The RNA from the amB2 mutant of R17, which carries an amber triplet at position 6 in the coat protein cistron, directs the synthesis of the same polypeptides as the wild-type RNA with the exception of the coat protein which is completely abolished. This identifies the product made with wild-type RNA as coat protein and provides a direct in vitro assay for the suppression of nonsense mutations in eukaryotic cells.     

Enhancing effect of magnesium ion on cell-free synthesis of read-through protein of bacteriophage Qbeta.

This report describes the enhancing effect of magnesium ion on the synthesis of read-through protein of bacteriophage Qβ in a cell-free protein synthesizing system from $\text{E. coli}$. At 6 mM of magnesium acetate, the major product was coat protein. At 12 mM of magnesium, it was replaced by read-through protein. This enhanced synthesis was substituted by the addition of 0.25 mM of spermine or 1 mM of spermidine to 6 mM of magnesium. These results suggest that magnesium or combination of magnesium and polyamines causes leaky termination at the end of the coat protein cistron of Qβ-RNA.     

Differential effect of Ca+2 on the translation of yeast mitochondrial and some viral RNA'S in an E.coli cell-free system.

Addition of 6mM CaCl₂ to an $\text{E. coli}$ cell-free system resulted in a several-fold enhancement of yeast mt RNA translation and in a severe inhibition of protein synthesis directed by MS2, Qβ and T₅ RNA's. CaCl₂ did not alter the Mg⁺² optimum or the time-course of protein synthesis and had no apparent effect on RNA degradation. Formaldehyde treatment of MS2 RNA markedly diminished the CaCl₂-mediated inhibition of its translation. Addition of equimolar amounts of EGTA, together with CaCl₂, abolished the effect of the latter on cell-free protein synthesis. FMet tRNA binding to ribosomes was enhanced by CaCl₂ in the presence of mt RNA, inhibited in the presence of MS2 RNA, and unaffected in the presence of formaldehyde-treated MS2 RNA. Maximal effect on initiation complex formation was observed with 0.1 mM CaCl₂.     

RNA recognition site of PP7 coat protein

The coat proteins of different single-strand RNA phages use a common protein tertiary structural framework to recognize different RNA hairpins and thus offer a natural model for understanding the molecular basis of RNA-binding specificity. Here we describe the RNA structural requirements for binding to the coat protein of bacteriophage PP7, an RNA phage of Pseudomonas. Its recognition specificity differs substantially from those of the coat proteins of its previously characterized relatives such as the coliphages MS2 and Qbeta. Using designed variants of the wild-type RNA, and selection of binding-competent sequences from random RNA sequence libraries (i.e. SELEX) we find that tight binding to PP7 coat protein is favored by the existence of an 8 bp hairpin with a bulged purine on its 5' side separated by 4 bp from a 6 nt loop having the sequence Pu-U-A-G/U-G-Pu. However, another structural class possessing only some of these features is capable of binding almost as tightly.     

Localization of Qβ Maturation Cistron Ribosome Binding Site

THE RNA of phage Qβ consists of about 3,500 nucleotides¹ and comprises three known cistrons coding for maturation protein (a structural component of the viral particle also designated as A or A₂ protein), coat protein and the β subunit of the viral replicase². When ribosomes are bound to intact Qβ RNA they attach predominantly to one binding site, namely that at the beginning of the coat cistron³. This binding site is located at about the 1,400th nucleotide from the 5′ end¹.     

Binding of the structural protein soc to the head shell of bacteriophage T4

Qβ plus strands with a 70 S ribosome bound to the coat cistron initiation site were used as template for Qβ replicase. Minus strand synthesis proceeded until the replicase reached the ribosome. The ribosome was removed and elongation was continued in a substrate-controlled, stepwise fashion. The nucleotide analog N⁴-hydroxyCMP was introduced into the positions complementary to the third and fourth nucleotides of the coat cistron. The minus strands were elongated to completion, purified and used as template for Qβ replicase. The final plus strand preparation consisted of four species, with the sequences -A-U-G-G- (wild type), -A-U-A-G- (mutant C₃), -A-U-G-A- (mutant C₄) and -A-U-A-A- (mutant $\text{C}{}_3\text{C}{}_4$) at the coat initiation site. The ribosome binding capacity of the mutant RNAs relative to wild type was <0.1 (C₃), 3.2 (C₄) and 0.3 ($\text{C}{}_3\text{C}{}_4$). The finding that mutant C₃ no longer formed an initiation complex suggests that the interaction of the ribosome binding site with fMet-tRNA plays an essential role in the formation of the 70 S initiation complex. The fact that mutant C₄ RNA bound more efficiently than wild type, and that mutant $\text{C}{}_3\text{C}{}_4$ RNA showed substantial ribosome binding capacity whereas the single mutant C₃ did not, can be explained by assuming that an A residue following the A-U-G triplet interacts with a complementary U residue in the anticodon loop sequence. In the case of $\text{C}{}_3\text{C}{}_4$ this additional base-pair may offset the reduced codon-anticodon interaction resulting from the modification of the A-U-G codon.     

Nondiscrimination of RNA viral message in binding to 30S ribosomes derived from T4 phage infected Escherichia coli

The effect of T4 phage on ribosomes in terms of their ability to bind RNA viral template is examined. It is found that the 30S subunits of T4 ribosomes bind MS2 RNA as efficiently as do the subunits of uninfected $\text{E. coli}$ ribosomes. On the other hand, analyses of the formation of 70S initiation complex, presumably from MS2 RNA-30S ribosome complex, using both labeled MS2 RNA and initiator tRNA, reveal that T4 ribosomes are only about half as active as $\text{E. coli}$ ribosomes. The latter phenomenon has been reported previously. These results suggest that, following T4 infection, ribosomes are modified in such a way that the attachment of fMet-tRNA_f to MS2 RNA-30S subunit complex is impaired.     

Inhibition of exonuclease V after infection of E. coli by bacteriophage T7

Exonuclease V ($\text{recBC}$ DNase) is inactivated in $\text{E. coli}$ between 4 and 7 min after infection by T7. This process requires protein sythesis. The inactivation does not occur when T7 is deficient for its RNA polymerase and thus does not express the genes involved in DNA replication and phage maturation. Some implications of this new function of T7 are discussed with respect to the processes of infection and DNA replication.     

Oligonucleotide Sequence of Replicase Initiation Site in Qβ RNA

THE single stranded RNA genome of bacteriophage Qβ has been variously estimated to consist of from 3,500¹ to 4,500² nucleotides. It contains three known cistrons³, which correspond to three of the four Qβ-specific proteins synthesized in vivo and in vitro^4–6. These are: (1) the gene for the maturation or A protein (molecular weight 41,000 (refs. 4, 5)), (2) that for the major coat protein of the virus (molecular weight 14,000 (ref. 9)) and (3) the gene for the phage-specific subunit of the Qβ replicase (molecular weight 64,000 (ref. 10) or 69,000 (ref. 24)), listed in the probable order^7,8 that they occur on the Qβ RNA. The fourth Qβ-specific protein, A₁ or IIb (molecular weight 36,000 (refs. 4–6, 10)), has recently been shown by Weiner and Weber to have an N-terminal sequence which is identical (for eight amino-acids) to that of the coat protein⁷. Because increased amounts of A₁ appear in virus particles grown in cells containing a UGA suppressor, Weiner and Weber postulate⁷ that this protein is the product of natural read-through at the UGA termination signal of the Qβ coat cistron. Such read-through (involving about 600 nucleotides) could occur entirely within a large “intercistronic” region between the coat and replicase genes, or could involve translation, either in or out of phase, of the replicase cistron. In hopes of distinguishing between these alternatives, I have isolated and examined the nucleotide sequence of the region surrounding the initiator codon of the Qβ replicase gene.     

Identification of an Escherichia coli inner membrane polypeptide specified by a lambda-tonB transducing.

Analysis by polyacrylamide gel electrophoresis of the proteins coded by a λ$\text{ton}$B transducing phage, after infection of UV-irradiated bacteria, revealed the presence of at least 7 new polypeptides. Three of these were identified as proteins of the $\text{trp}$ operon whilst three others were deleted by a spontaneous mutation in the $\text{ton}$B region carried by the phage. A single polypeptide, molecular weight 40,000 was absent from a phage carrying a proflavine induced mutation in $\text{ton}$B. We conclude that this protein, which was localised in the inner membrane by sarkosyl fractionation of the envelope, is the $\text{tonB}$ product.     

Viral Genomic Single-Stranded RNA Directs the Pathway Toward a T = 3 Capsid

The molecular mechanisms controlling genome packaging by single-stranded RNA viruses are still largely unknown. It is necessary in most cases for the protein to adopt different conformations at different positions on the capsid lattice in order to form a viral capsid from multiple copies of a single protein. We showed previously that such quasi-equivalent conformers of RNA bacteriophage MS2 coat protein dimers (CP₂) can be switched by sequence-specific interaction with a short RNA stem-loop (TR) that occurs only once in the wild-type phage genome. In principle, multiple switching events are required to generate the phage T = 3 capsid. We have therefore investigated the sequence dependency of this event using two RNA aptamer sequences selected to bind the phage coat protein and an analogous packaging signal from phage Qβ known to be discriminated against by MS2 coat protein both in vivo and in vitro. All three non-cognate stem-loops support T = 3 shell formation, but none shows the kinetic-trapping effect seen when TR is mixed with equimolar CP₂. We show that this reflects the fact that they are poor ligands compared with TR, failing to saturate the coat protein under the assay conditions, ensuring that sufficient amounts of both types of dimer required for efficient assembly are present in these reactions. Increasing the non-cognate RNA concentration restores the kinetic trap, confirming this interpretation. We have also assessed the effects of extending the TR stem-loop at the 5′ or 3′ end with short genomic sequences. These longer RNAs all show evidence of the kinetic trap, reflecting the fact that they all contain the TR sequence and are more efficient at promoting capsid formation than TR. Mass spectrometry has shown that at least two pathways toward the T = 3 shell occur in TR-induced assembly reactions: one via formation of a 3-fold axis and another that creates an extended 5-fold complex. The longer genomic RNAs suppress the 5-fold pathway, presumably as a consequence of steric clashes between multiply bound RNAs. Reversing the orientation of the extension sequences with respect to the TR stem-loop produces RNAs that are poor assembly initiators. The data support the idea that RNA-induced protein conformer switching occurs throughout assembly of the T = 3 shell and show that both positional and sequence-specific effects outside the TR stem-loop can have significant impacts on the precise assembly pathway followed.