Phage Coat Research Articles

Translation of MS2 RNA in vitro in the absence of initiation factor IF-3.

An Escherichia coli cell-free translational system, deprived of initiation factor IF-3, has been used to study the role of the factor in protein synthesis. In this system, 30-S ribosomal subunits are preincubated together with MS2 phage RNA in a small volume in the presence of 10 mM Mg(Ac)2; the missing components required for protein synthesis are then added and assembly of elongating ribosomes is allowed to occur. This stepwise assembly process permits formation of functional complexes which can carry out protein synthesis in the complete absence of IF-3. The translational products, obtained in the absence of IF-3, have been analysed and shown to be similar to those synthesized in the presence of the factor. The main product observed is the phage coat protein.

Detection of prokaryotic signal peptidase in an Escherichia coli membrane fraction: endoproteolytic cleavage of nascent f1 pre-coat protein.

An inverted membrane vesicle fraction isolated from uninfected Escherichia coli and largely derived from the inner membrane has been shown to contain an endoproteolytic activity that cleaves nascent bacteriophage f1 pre-coat protein into two identifiable products. The electrophoretic mobility on sodium dodecyl sulfate/urea/polyacrylamide gels and the partial amino-terminal sequence of the larger fragment were indistinguishable from those of the mature phage coat protein. Partial amino-terminal sequence analysis showed that the smaller fragment corresponds to the amino-terminal "signal peptide" of f1 pre-coat protein. Cleavage occurred only if the membrane fraction was present during in vitro synthesis, and was not observed if it was added after completion of pre-coat protein synthesis. The cleavage reaction was strongly stimulated when the membrane fraction was present together with the nonionic detergent Nikkol. These results are consistent with and discussed in terms of the signal hyothesis.

Xf phage invading the host cells with their protein coats.

The Xf phage coat protein associated with infected cells could not be removed by washing with antiserum and tris-EDTA buffer. Although the infected cells were consecutively washed 6 times with tris-EDTA buffer, the ratios of parental phage 6H-DNA to 14C-protein were not changed. A considerable amount of the parental 14C-protein and 3H-DNA in the original ratio were detected in the membrane and the soluble cytoplasmic fractions of infected cells. The studies of the change in Xf 14C-protein and 3H-DNA incorporation into the host cells and their release showed that DNA and protein penetrate together into the host cells during the first 60 min after infection (p.i.). While virtually all parental DNA was conserved, re-utilized and released from the infected cell 60 min p.i., no apparent release of parental protein was observed. Approx. 40% of the parental protein became degraded and could be washed from the infected cell after 90 min; the rest of the parental protein remained and probably was re-utilized by the host.

Asymmetric orientation of a phage coat protein in cytoplasmic membrane of Escherichia coli.

The coat protein of a filamentous phage (M13) enters the cytoplasmic membrane from two directions: from the outside upon infection and from the cell interior late in the viral life cycle prior to phage assembly and extrusion. Binding of 125I-labeled anti-coat protein antibody to spheroplasts or to inverted vesicles was used to assay the orientation of coat protein in the membrane. Both parental and newly synthesized coat protein were found to be exposed on the outer surface of the cytoplasmic membrane. Coat protein in intact infected cells is also accessible to external antibody. Thus two different processes of assembling a protein into membrane, each starting from a different membrane surface, appear to produce similar surface orientations.

Deviations from compositional randomness in eukaryotic and prokaryotic proteins: The hypothesis of selective-stochastic stability and a principle of charge conservation

Eight proteins of diverse lengths, functions, and origin, are examined for compositional non-randomness amino acid by amino acid. The proteins investigated are human fibrinopeptide A, guinea pig Insulin, rattlesnake cytochrome c, MS2 phage coat protein, rabbit triosephosphate isomerase, bovine pancreatic deoxyribonuclease A, bovine glutamate dehydrogenase, and Bacillus thermoproteolyticus thermolysin. As a result of this study the experimentally testable hypothesis is put forth that for a large class of proteins the ratio of that fraction of the molecule which exhibits compositional non-randomness to that fraction which does not is on the average, stable about a mean value (estimated as 0.32 plus or minus 0.17) and (nearly) independent of protein length. Stochastic and selective evolutionary forces are viewed as interacting rather than independent phenomena. With respect to amino acid composition, this coupling ameliorates the current controversy over Darwinian vs. non-Darwinian evolution, selectionist vs. neutralist, in favor of neither: Within the context of the quantitative data, the evolution of real proteins is seen as a compromise between the two viewpoints, both important. The compositional fluctuations of the electrically charged amino acids glutamic and aspartic acid, lysine and arginine, are examined in depth for over eighty protein families, both prokaryotic and eukaryotic. For both taxa, each of the acidic amino acids is present in amounts roughly twice that predicted from the genetic code. The presence of an excess of glutamic acid is independent of the presence of an excess of aspartic acid and vice versa.

Hexamer of bacteriophage f2 coat protein as a repressor of bacteriophage RNA polymerase synthesis.

Formation of complex I between phage f2 RNA and coat protein, leading to repression of phage RNA polymerase synthesis, depends nonlinearly upon the concentration of the coat protein. Maximum formation of complex I was observed when six molecules of coat protein were bound to one molecule of RNA. RNase digestion of a glutaraldehyde-fixed complex left, as the products, coat protein oligomers. The heaviest, hexamers, predominated in the mixture. It was also shown that, in an ionic environment required for phage protein synthesis, coat protein at a concentration optimum for complex I formation exists in solution as a dimer. The results indicate that the translational repression of the RNA polymerase cistron is due to a cooperative attachment to phage template of three dimers of coat protein, forming a hexameric cluster on an RNA strand.

Role of ribosomal protein S1 in portein synthesis: effects of its addition to Bacillus stearothermophilus cell-free system.

Products of the f2 phage RNA-directed protein synthesizing systems in vitro, derived from Escherichia coli and Bacillus stearothermophilus, were analyzed. In contrast to other reports, it was found that B. stearothermophilus ribosomes synthesized phage coat protein when the high-speed (100 000 X g) supernatant (S100) and ribosomal wash (crude initiation factors) of homologous origin were used. Furthermore, a marked stimulation of coat protein synthesis was observed with B. stearothermophilus ribosomes when either S100 or ribosomal wash or both from E. coli cells were used instead of the respective homologous components. The principle responsible for this stimulation was identified as the 30-S ribosomal protein S1 present in S100 and ribosomal wash. Purified S1 from E. coli at roughly one-to-one molar ratio to ribosomes resulted in about 10-fold stimulation of the incorporation of [14C]valine when added to the B. stearothermophilus system. This stimulation was observed mainly in the synthesis of coat protein and replicase.

Translational repression of a viral messenger RNA by a host protein.

It is shown that factor i, a bacterial protein, specifically inhibits that step in the initiation of R17 bacteriophage RNA translation that involves the attachment of native R17 RNA to 30 S ribosomal subunits carrying fMet-tRNA. This inhibition by factor i is relieved by the addition of excess R17 RNA, but not by the addition of excess 30 S subunits. That R17 RNA is the only target of the inhibition is demonstrated further by the fact that in a cell-free extract containing all components for protein synthesis, factor i-mediated inhibition of exogenous R17 RNA translation can be overcome only by the addition of excess R17 RNA and not by excess cell-free extract. Upon relief of inhibition, phage coat protein synthesis is restored; enhancement of formation of other cistron products is not seen. While initiation of R17 RNA translation is blocked by factor i, chain elongation is not affected. Although foactor i inhibits the IF-3-dependent binding of R17 RNA to fMet-tRNA-30 S complexes, under conditions of initiation of protein synthesis formation of stable complexes between factor i and IF-3 could not be detected, and factor i did not interfere with the binding of IF-3 to free, native R17 RNA. Instead of affecting the function of IF-3 or ribosomes, factor i exerts its inhibition by binding to R17 RNA and acting as a translational repressor. Factor i prefers intact R17 RNA to fragments generated by autoradiolysis; its binding to R17 RNA is specific in that little competition is observed by transfer RNA, ribosomal RNA or poly(A). However, factor i has a high affinity for poly(U) sequences.

Strand separation of DNA induced by ultraviolet irradiation in vitro

1. Ultraviolet irradiation of double stranded DNA in vitro caused strand separation, as judged by the susceptibility of the DNA to degradation by an endonuclease from Neurospora crassa that is specific for single strands, by its behavior on elution from hydroxylapatite and by its sedimentation properties. The extent of degradation by the endonuclease was almost linearly proportional to the ultraviolet dose up to 2.8 · 10 5 J/m 2, when 45% of the DNA was degraded. Increased DNA concentration and high concentrations of Na + and Mg 2+ were inhibitory to degradation. The pH of the DNA solution had no significant effect. 2. The ultraviolet-irradiated DNA was resistant to single strand-specific exonuclease I from Escherichia coli even after removal of 5′-phosphate termini or limited endonucleolytic cleavage. Ultraviolet-induced denaturation was not reversible even under optimum conditions. 3. The elution profile of irradiated DNA from hydroxylapatite was gradually shifted, depending on the ultraviolet dose, from the position of double stranded DNA toward that of single stranded DNA, although there was no significant overlap with the profile of single stranded DNA. The elution profile also indicated that the denaturation was not an all-or-none phenomenon and that all the DNA molecules were similarly affected. Unlike X-irradiation, ultraviolet light did not generate single stranded fragments. 4. The sedimentation coefficient of T7 phage DNA increased from 31 to 67 S after ultraviolet irradiation (1.4 · 10 5 J/m 2 incident dose), presumably due to localized collapse of the double stranded structure and formation of interstrand cross-links. 50% of the total irradiated DNA was rapidly renaturable after denaturation by alkali. 5. Localized strand separation in DNA after ultraviolet irradiation was related to pyrimidine dimer formation; proflavine reduced both. The denatured regions contained almost all of the pyrimidine dimers. When the irradiated DNA was treated with N. crassa endonuclease, thymine-containing dimers were released as acid-soluble products more readily than was thymine. 6. DNA inside T7 phage was also denatured after ultraviolet irradiation of the whole phage. Such DNA was not denatured by alkali, and was covalently cross-linked with the phage coat.

Recognition of Protein Synthesis Initiation Signals on Bacteriophage Ribonucleic Acid by Mammalian Ribosomes

Previous work has shown that bacteriophage Qβ RNA is translated by cell-free extracts of Krebs II mouse ascites cells. The major product of the reaction is the phage coat protein (M orrison , T. G. & L odish , H. F. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 315–319; A viv , H., B oime , I., L oyd , B. & L eder , P. (1972) Science 178, 1293–1295). Here we show that ascites cell extracts directed by Qβ RNA also synthesize Qβ replicase and, in addition, a polypeptide which results from initiation of protein synthesis in the interior of the replicase gene. This polypeptide is made in amounts equivalent to that of the replicase. Extracts of ascites cells directed by bacteriophage f2 RNA synthesize the phage coat and replicase, but the major products of the reaction do not co-migrate with authentic f2 protein. These major products are also made in reactions directed by f2 RNA which contain nonsense mutations in any of the three f2 genes. f2 RNA directs incorporation of initiator formyl[35S]methionine into authentic chymotryptic and tryptic peptides of the f2 coat and replicase, but formyl-[35S]methionine is also incorporated into several initiator sequences not found in authentic f2 proteins. These results indicate that ascites ribosomes initiate protein synthesis at both authentic initiation regions and at spurious regions. These incorrect initiations on bacteriophage RNAs by mammalian ribosomes are in marked contrast to the correct initiation by mammalian ribosomes on mammalian viral and cellular RNAs in cell-free systems.

P22 morphogenesis. I: Catalytic scaffolding protein in capsid assembly.

AbstractAbout 250 molecules of the 42,000 molecular weight gene 8 product catalyze the polymerization of the major phage coat protein into a precursor shell temporarily containing both proteins. The resulting prohead appears to be a shell structure with the P8, or scaffolding protein, on the inside, and the coat protein on the outside. In concert with DNA condensation inside the shell, all 250 scaffolding molecules exit from the prohead, without proteolytic cleavage. These molecules then recycle and catalyze the formation of more proheads from newly synthesized coat protein. Such proteins, which catalyze assembly by temporarily associating with an intermediate stage, may represent a general mechanism of macromolecular assembly.

Mechanism of head assembly and DNA encapsulation in Salmonella phage P22 : II. Morphogenetic pathway

We have identified and characterized structural intermediates in phage P22 assembly. Three classes of particles can be isolated from P22-infected cells: 500 S full heads or phage, 170 S empty heads, and 240 S “proheads”. One or more of these classes are missing from cells infected with mutants defective in the genes for phage head assembly. By determining the protein composition of all classes of particles from wild type and mutant-infected cells, and examining the time-course of particle assembly, we have been able to define many steps in the pathway of P22 morphogenesis. In pulse-chase experiments, the earliest structural intermediate we find is a 240 S prohead; it contains two major protein species, the products of genes 5 and 8. Gene 5 protein (p5) is the major phage coat protein. Gene 8 protein is not found in mature phage. The proheads contain, in addition, four minor protein species, PI, P16, P20 and PX. Similar prohead structures accumulate in lysates made with mutants of three genes, 1, 2 and 3, which accumulate uncut DNA. The second intermediate, which we identify indirectly, is a newly filled (with DNA) head that breaks down on isolation to 170 S empty heads. This form contains no P8, but does contain five of the six protein species of complete heads. Such structures accumulate in lysates made with mutants of two genes, 10 and 26. Experiments with a temperature-sensitive mutant in gene 3 show that proheads from such 3 − infected cells are convertible to mature phage in vivo, with concomitant loss of P8. The molecules of P8 are not cleaved during this process and the data suggest that they may be re-used to form further proheads. Detailed examination of 8 − lysates revealed aberrant aggregates of P5. Since P8 is required for phage morphogenesis, but is removed from proheads during DNA encapsulation, we have termed it a scaffolding protein, though it may have DNA encapsulation functions as well. All the experimental observations of this and the accompanying paper can be accounted for by an assembly pathway, in which the scaffolding protein P8 complexes with the major coat protein P5 to form a properly dimensioned prohead. With the function of the products of genes 1, 2 and 3, the prohead encapsulates and cuts a headful of DNA from the concatemer. Coupled with this process is the exit of the P8 molecules, which may then recycle to form further proheads. The newly filled heads are then stabilized by the action of P26 and gene 10 product to give complete phage heads.

Controlled alterations in the physical and biological properties of R17 bacteriophage induced by guanidine hydrochloride

The effects of guanidine hydrochloride on the physical and biological properties of R17 phage were studied to ascertain how these effects relate to the structure of the phage particle. The dialysis of phage against 3.0 M guanidine at 4 C resulted in a loss of infectivity and subtle changes in the sedimentation properties of the resulting non-infectious particles. Upon increasing the temperature of the reaction from 4 C to 37 C, additional physical modifications were found to occur in a sequential manner, giving rise to three unique particle forms followed by the eventual breakdown of phage structure. These three modified phage forms were found to have S values of 78, 58, and 44, respectively, to contain all three macromolecules characteristic of R17 phage, and to have the buoyant density of normal phage. The RNA of these particles were sensitive to RNase, and negative stains readily penetrated their interior, suggesting that changes had occurred in the capsid. The 44S particles in particular were found to be highly unstable as judged by electron microscopic and analytical ultracentrifugational studies. Various stages in the disassembly of these particles were identified. Protein fragments directly released from 44S particles were found to be morphologically similar to 11S subunits produced during the breakdown of phage in higher concentrations of guanidine. The morphological identify of the subunits released from 44S particles and the 11S protein subunits was supported by the similarity of the two types of subunits in forming branched chains, in the arrangement of subunits within the chains, and by their cosedimentation in sucrose gradients. Further analysis of the 11S subunits showed the following characteristics: (1) three morphological configurations with the most common form having dimensions of 106 × 50 Å; (2) a sedimentation rate of 10.5–11.4S; (3) molecular weight of 240,000 daltons. On the basis of these properties and on the known properties of phage coat protein, a model is proposed to explain the structure of these subunits and their possible relalationship to the structure of the phage particle. Additional support for the proposed model of phage structure is provided by electron microscopic observations and the recently discovered functions of the A (maturation) protein.

A coat protein of the bacteriophage M13 virion participates in membrane-oriented synthesis of DNA.

Several molecules of a protein specified by gene 3 of M13 comprise a minor fraction of the phage coat and have been assigned a role in adsorption to the bacterial cell. We find that the gene-3 protein molecules of the virion are fully conserved in phage that have attached irreversibly to the host cell, and they form a complex with the phage DNA when it has been converted to a duplex replicative form. In cells infected at a restrictive temperature with a thermosensitive mutant in gene 3, there is no conversion of the phage DNA to the replicative form. Both the adsorbed phage and the complex of replicative form DNA with the gene-3 protein were isolated with the inner membrane fraction of the cell. We suggest that the gene-3 protein may perform an essential function in the synthesis of replicative form by linking the phage DNA to a cellular replicative system in or on the inner cell membrane.

Relationship among temperate Agrobacterium phage genomes and coat proteins.

SUMMARY Five temperate Agrobacterium phages, two of which are defective, and one presumed temperate, phage PS8, isolated from crown gall tissue, were prepared, purified and compared. The four phages, omega, PS8, PB2A and LV-1, were indistinguishable. They possess a hexagonal head of about 70 nm. with a flexible tail about 200 nm. long. Their DNA had a T m of 92.7° ± 0.1°, a % GC of 56.7 ± 0.2, a mol. wt of 34.1 × 106 and comprised the four normal bases A, T, G and C. No evidence was found for repetitive units in PB2A. These four genome DNA types gave 100% hybridization (limit of error 6%). The electrophoretic protein profiles on SDS polyacrylamide gels are essentially identical: four major bands with similar mol. wt. About half of the phage protein has a mol. wt of 48,000 (perhaps a major head protein), about one-third has a mol. wt of 30,000, one sixth has a mol. wt of 15,500 and 4% has a mol. wt of 69,000. No omega-type prophage DNA was detectable in the cured bacteria v-ic by DNA:DNA hybridization. The latter bacteria were as pathogenic as the prophage-containing parent strain v-i, which throws some doubt on the role of the omega phage group in crown gall induction. The phage-like particle P0362, isolated from the pathogenic Agrobacterium tumefaciens 0362 had a 60 to 70 nm. head and a straight 130 nm. tail. Its DNA was double-stranded with a slightly lower T m of 92.4°, a mol. wt of 25 × 106. The electrophoretic profiles of coat proteins differed from those of the other phages. Its DNA is less than 17% homologous with the omega group. The eventual role of this phage in crown gall induction remains to be established. The phage-like particle P8149, isolated from the non-pathogenic Agrobacterium radiobacter ncib 8149, was quite different. The bipyramidal head was much smaller (40 nm. diameter) and had a very short bud-like tail. The DNA was double-stranded, with a T m of 93.9°, a mol. wt of 10 × 106. The electrophoretic profile of the coat proteins differed from those of the other phages. The DNA does not hybridize with the omega group. As this phage was isolated from a non-pathogenic A. radiobacter, it probably has no function in crown gall. A. radiobacter 8149 is bilysogenic.

Extent of variation in three related bacteriophage RNA molecules

The isolation and sequence analysis of a series of oligonucleotides from complete ribonuclease T 1 digests of bacteriophage R17 RNA have been reported (Jeppesen, 1971). We have obtained the homologous sets of large T 1-resistant oligonucleotides from the related phages f2 and MS2. As expected from earlier findings (Nichols & Robertson, 1971), each RNA can be uniquely characterized by its ribonuclease T 1 fingerprint. Comparison of nucleotide sequences from the sets of RNA fragments from R17, f2 and MS2 allows an estimate of the over-all degree of variation among the three RNA's (2.8 to 3.4%), and yields additional evidence that non-coding regions show significantly lower variation. R17, f2 and MS2 are apparently not linear descendants, but could have diverged from a common ancestor. Base substitutions involving U and C transitions occur at a higher frequency than would be expected on a random basis. It appears probable that a total of 15 to 20 base replacements in the phage coat protein cistron may accompany the single amino-acid difference between f2 coat protein and that of either R17 or MS2. We conclude that analysis of this sort should prove useful in determining the extent of variation among members of any set of closely related nucleic acid molecules.

Termination of messenger RNA translation in a temperature-sensitive mutant of Escherichia coli

A temperature-sensitive mutant of Escherichia, coli K12 is defective in a previously uncharacterized component essential for protein synthesis. The lesion in the mutant is expressed in vivo at 36 °C as the appearance of the capacity to suppress UAA and UGA nonsense mutations, and in cell-free extracts as the temperature-sensitive capacity to release newly formed R17 phage coat protein from the ribosome. 70 s ribosomes from extracts of the mutant are unable to participate in any R17 RNA-directed protein synthesis whereas the native subunits have limited polymerization activity at 43 °C. A non-ribosomal component (designated Z factor) which rescues the protein-synthesizing activity of the mutant crude extract has been partially purified from a parental strain. Z factor is shown to be distinct from G and T translocation factors. Evidence is also presented which indicates that Z factor is not one of the initiation factors, aminoacyl-tRNA synthetases, the release factor R 2, or the S factor. The inference presented is that the mutant is defective in a previously uncharacterized activity which is essential for completion of messenger RNA translation during or subsequent to recognition of the signal for polypeptide chain termination.

Virginiamycin M-A specific inhibitor of the acceptor site of ribosomes

Virginiamycin M (0.5 μg/ml) inhibits polyuridylic acid (poly U) dependent polyphenylalanine formation as well as MS2 phage coat protein synthesis directed by MS2 phage RNA. It does not inhibit aminoacyl tRNA synthetase reaction. It causes 50 p. cent inhibition of non-enzymatic specific binding of phenylalanyl tRNA and total suppression of the T-factor dependent binding of phenylalanyl tRNA to the poly U ribosome complex. The ribosomal complex of poly U and phenylalanyl tRNA which is obtained in the presence of Virginiamycin M does not form phenylalanyl puromycin nor does it get incorporated into polyphenylalanine. The complex of labeled Virginiamycin M, ribosomes and poly U has been isolated by column chromatography. These results suggest that Virginiamycin M binds to the acceptor site of the ribosomes resulting in the steric inhibition of peptide synthetase at the donor site.

R17 coat protein interaction with multi-stranded R17 RNA.

Phage coat protein reacted with preparations of multi‐stranded RNA at high protein inputs to yield complexes containing phage‐like particles. RNA complexes containing predominantly double‐stranded RNA (replicative form, “RI”) were considerably less efficient in promoting particle formation than those rich in single strands (replicative intermediate, “RF”). Similarly, binding of coat protein at low input multiplicities was less efficient with “RF” than with “RI” preparations.

Bacteriophage tail components. II. Dihydrofolate reductase in T4D bacteriophage.

The protein component of the T-even bacteriophage coat which binds the phage-specific dihydropteroyl polyglutamate has been identified as the phage-induced dihydrofolate reductase. Dihydrofolate reductase activity has been found in highly purified preparations of T-even phage ghosts and phage substructures after partial denaturation. The highest specific enzymatic activity was found in purified tail plate preparations, and it was concluded that this enzyme was a structural component of the phage tail plate. Phage viability was directly correlated with the enzymological properties of the phage tail plate dihydrofolate reductase. All reactions catalyzed by this enzyme which changed the oxidation state of the phage dihydrofolate also inactivated the phage. Properties of two T4D dihydrofolate reductase-negative mutants, wh1 and wh11, have been examined. Various lines of evidence support the view that the product of the wh locus of the phage genome is normally incorporated into the phage tail structure. The effects of various dihydrofolate reductase inhibitors on phage assembly in in vitro complementation experiments with various extracts of conditional lethal T4D mutants have been examined. These inhibitors were found to specifically block complementation when added to extracts which did not contain preformed tail plates. If tail plates were present, inhibitors such as aminopterin, did not affect further phage assembly. This specific inhibition of tail plate formation in vitro confirms the analytical and genetic evidence that this phage-induced "early" enzyme is a component of the phage coat.