Binding of mammalian ribosomes to MS2 phage RNA reveals an overlapping gene encoding a lysis function.

The main binding site for mammalian ribosomes on the single-stranded RNA of bacteriophage MS2 is located nine tenths of the way through the coat protein gene. Translation initiated at an AUG triplet in the +1 frame yields a 75 amino acid polypeptide which terminates within the synthetase gene at a UAA codon, also in the +1 frame. Partial amino acid sequence analysis of the product synthesized in relatively large amounts by mammalian ribosomes confirms this assignment of the overlapping cistron. The same protein is made in an E. coli cell-free system, but only in very small amounts. Analysis of the translation products directed by RNA from op3, a UGA nonsense mutant of phage f2, identifies the overlapping cistron as a lysis gene. In this paper we show that the op3 mutation is a C yield U transition occurring in the second codon of the synthetase cistron, which explains the lowered production of phage replicase (as well as lack of lysis) upon op3 infection of nonpermissive cells. We discuss the properties of the overlapping gene in relation to its lysis function, recognition of the lysis initiator region by E. coli versus eucaryotic ribosomes and op3 as a ribosome binding site mutant for the f2 synthetase cistron.     

Leakage induced in Escherichia coli cells by A protein-RNA complexes from bacteriophage f2

Complexes of f2 phage RNA and its A protein, or maturation protein, transfect Escherichia coli cells much better than does protein-free RNA. We used these complexes to introduce the bacteriophage f2 lysis gene into cells. The A protein-RNA complex was found to kill cells, probably by causing them to leak large macromolecules. Previously induced beta-galactosidase leaked from cells treated either with the A protein-RNA complex or with lethal but noninfectious complexes that had been treated with formaldehyde. This observation was consistent with an earlier finding that formaldehyde-treated f2 RNA stimulates the in vitro synthesis of a lysis protein. The complexes did not stimulate the rate of leakage of beta-galactosidase from a streptomycin-resistant mutant known to be lysis defective. On the other hand, the rate of leakage was increased in a double mutant resistant to both streptomycin and rifampin and which is lysed normally by f2 bacteriophage.     

The lysis function of RNA bacteriophage Qbeta is mediated by the maturation (A2) protein

Complete or partial cDNA sequences of the RNA bacteriophage Qbeta were cloned in plasmids under the control of the lambdaP(L) promoter to allow regulated expression in Escherichia coli harbouring the gene for the temperature-sensitive lambdaCI857 repressor. Induction of the complete Qbeta sequence leads to a 100-fold increase in phage production, accompanied by cell lysis. Induction of the 5'-terminal sequence containing the intact maturation protein (A2) cistron also causes cell lysis. Alterations of the A2 cistron, leading to proteins either devoid of approximately 20% of the C-terminal region or of six internal amino acids, abolish the lysis function. Expression of other cistrons in addition to the A2 cistron does not enhance host lysis. Thus, in Qbeta, the A2 protein, in addition to its functions as maturation protein, appears to trigger cell lysis. This contrasts with the situation in the distantly related group I RNA phages such as f2 and MS2 where a small lysis polypeptide is coded for by a region overlapping the end of the coat gene and the beginning of the replicase gene.     

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.     

Binding of wheat germ ribosomes to fragmented viral mRNA.

The specificity of binding of wheat germ ribosomes to mRNA was greatly altered by cleavage of the message. Fragmentation of reovirus mRNA allowed wheat germ ribosomes to bind and protect a variety of internal sequences which were not accessible to ribosomes in the intact message. In experiments using the polycistronic mRNA from bacteriophage R17, wheat germ ribosomes bound preferentially at the beginning of the lysis peptide and synthetase cistrons, and at a third site which may be derived from the C-terminal region of the A protein cistron. This result is similar to that reported previously in a mammalian translational system (J.F. Atkins et al., Cell 18:246-256, 1979) except that, in the present study, limited cleavage of the phage RNA was necessary to activate these sites. More extensive fragmentation of R17 RNA permitted wheat germ ribosomes to bind and protect a great many additional sites. Thus, presence of an (exposed) 5'-terminus on an RNA molecule appears to be necessary and sufficient for attachment of eucaryotic ribosomes.     

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.     

Preparation and properties of 5,6-monoepoxyvitamin A acetate, 5,6-monoepoxyvitamin A alcohol, 5,6-monoepoxyvitamin A aldehyde and their corresponding 5,8-monoepoxy (furanoid) compounds

1. A method is described for the preparation and purification of the RNA from the RNA coliphage ZIK/1. 2. Some of the physical characteristics and infective properties of coliphage-ZIK/1 RNA were examined. 3. A method is also described for examining the type and quantity of RNA synthesized after bacteriophage infection. 4. Ribosome synthesis was decreased 15min. after bacteriophage adsorption, bacteriophage RNA was synthesized from 15min. to 120min. after adsorption and intracellular bacteriophages appeared 40min. after adsorption. Cell lysis commenced 60min. after adsorption, and was half complete 20min. later and 90-95% complete 120min. after adsorption. 5. Cell division continued until 40min. after bacteriophage adsorption. 6. Bacterial ribosomes were conserved during the infective process. 7. Intracellular bacteriophage RNA has sedimentation coefficient 28s but after cell lysis it has sedimentation coefficient 10-5s.     

Determination of the first half of the coat protein cistron of bacteriophage Qbeta as an application of a mapping procedure for RNA fragments.

A method is described to classify, in regard to their location within the genome, fragments obtained by partial cleavage of 32P-labeled bacteriophage Qbeta RNA. The location of many fragments suitable for sequence analysis could be established using as markers 29 large RNase T1-resistant oligonucleotides with known map positions. Applying this method four fragments originating from the coat protein cistron were isolated and analyzed. The sequence of a segment of 239 nucleotides located immediately adjacent to the initiation triplet was determined to be G-C-A-A-A-A-U-U-A-G-A-G-A-C-U-G-U-U-A-C-U-U-U-A-G-G-U-A-A-C-A-U-C-G-G-G-A-A-A-G-A-U-G-G-A-A-A-A-C-A-A-A-C-U-C-U-G-G-U-C-C-U-C-A-A-U-C-C-G-C-G-U-G-G-G-G-U-A-A-A-U-C-C-C-A-C-U-A-A-C-G-G-C-G-U-U-G-C-C-U-C-G-C-U-U-U-C-A-C-A-A-G-C-G-G-G-U-G-C-A-G-U-U-C-C-U-G-C-G-C-U-G-G-A-G-A-A-G-C-G-U-G-U-U-A-C-C-G-U-U-U-C-G-G-U-A-U-C-U-C-A-G-C-C-U-U-C-U-C-G-C-A-A-U-C-G-U-A-A-G-A-A-C-U-A-C-A-A-G-G-U-C-C-A-G-G-U-U-A-A-G-A-U-C-C-A-G-A-A-C-C-C-G-A-C-C-G-C-U-U-G-C-A-C-U-G-C-A-A-A-C-G-G-U-U-C-U-U-Gp. The primary structure and the secondary structure model derived from it did not provide any evidence of homology with the corresponding RNA region of bacteriophage MS2.     

Process of Infection with Bacteriophage φX174: XXXVI. Measurement of Virus-Specific Proteins During a Normal Cycle of Infection

Double-labeling techniques in which (14)C-labeled, phiX174-infected cells and (3)H-labeled, uninfected cells were used permitted the identification of the virus-specific proteins after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis without prior inhibition of host-cell protein synthesis by ultraviolet irradiation. It was also possible to detect previously undescribed components of high molecular weight which may represent induced host proteins. The gel regions specifically corresponding to cistron II protein and the chloramphenicol-resistant VI protein were identified, and a third new, small peak of unknown origin was detected. Studies of the rate of synthesis of virus-specific proteins at various times after infection indicated that the product of cistron I (lysis) is made only late in infection, but the other proteins seemed to be synthesized at the same relative rates throughout infection (although in different amounts). Studies of the proteins obtained from uniformly labeled phiX virus particles indicated that all of the spikes are identical and allowed a formulation of the structure of the phage capsid.     

Sequence of 1000 nucleotides at the 3'' end of tobacco mosaic virus RNA.

The sequence of 1000 nucleotides at the 3' end of tobacco mosaic virus RNA has been determined. The sequence contains the entire coat protein cistron as well as regions to its left and right. Sequence characterization was by conventional methods for use with uniformly 32P labeled RNA complemented by newer methods for in vitro 5' and 3' 32P end-labeling of RNA and its subsequent rapid analysis. The noncoding region separating the coat protein cistron from the 3' terminus is 204 residues long and may be folded into a clover-leaf-type secondary structure. The distribution of termination codons to the left of the coat protein cistron suggests that the end of the adjacent cistron is separated from the beginning of the coat protein cistron by only two nucleotides. The subgenomic viral coat protein mRNA was isolated from infected tissue and shown to be capped. The nontranslated sequence separating the cap from the AUG initiation codon is 9 residues long and thus overlaps a portion of the adjacent cistron on the genome RNA.     

Single stranded DNA SP6 promoter plasmids for engineering mutant RNAs and proteins: synthesis of a ''stretched'' preproparathyroid hormone.

The intergenic region of bacteriophage f1 has been subcloned into the bacteriophage SP6 promoter plasmids, pSP64 and pSP65, in both orientations. Coinfection of E. coli with these SP6 promoter/phage f1 chimeric plasmids and the interference resistance phage, IR1, results in the replication and secretion of the pSP6.f1 plasmids as single stranded DNA. Bovine preProPTH cDNAs in both the native form and a form containing an insertion of 117 base pairs in the protein coding region have been inserted in these plasmids. The RNA transcribed from the SP6.f1/preProPTH cDNA constructs was efficiently translated in the wheat germ or reticulocyte cell free systems without addition of a 7-methylguanosine cap to the RNA. In the presence of dog pancreatic or chicken oviduct microsomal membranes, conversion of the resultant pre-proteins to pro-proteins was observed. Confirmation of the "mutated" preProPTH cDNA was determined by dideoxyribonucleotide DNA sequencing of single stranded plasmid DNA. These vectors are suitable for the efficient biosynthesis of large amounts of single or double stranded DNA, and translationally active RNA. The combined properties of single stranded DNA replication and the SP6 promoter simplify the engineering of mutant RNAs and their corresponding proteins. In addition, single stranded DNA or RNA corresponding to either complementary strand may be synthesized as nucleic acid hybridization probes.     

Competition between bacteriophage f2 RNA and bacteriophage T4 messenger RNA

In an $\text{Escherichia coli}$ cell-free protein synthesis assay, mRNA isolated from cells late after infection by phage T4 out-competes bacteriophage f2 RNA. Addition of a saturating or subsaturating amount of T4 mRNA inhibits translation of f2 RNA, while even an excess of f2 RNA has no effect on translation of T4 mRNA. Peptide mapping of reaction products labeled with formyl-[³⁵S]-methionyl-tRNA was used to quantitate f2 and T4 protein products synthesized in the same reaction. We suggest that messenger RNA competition might be one mechanism by which T4 superinfection of cells infected with phage f2 blocks translation of f2 RNA and possibly host mRNA.     

Studies on the bacteriophage MS2. XVI. The termination signal of the A protein cistron

The RNA of bacteriophage MS2 codes for three viral proteins: the coat protein, the A protein and the replicase. Upon infection of various amber suppressor strains of Escherichia coli, we found a fourth viral protein, the synthesis of which was specifically dependent on the presence of an amber suppressor gene. It is shown that this polypeptide is formed by reading through the natural termination signal of the A protein cistron. This cistron therefore terminates with the nonsense codon UAG. The observed prolongation accounts for the addition of some 30 amino acids. Unlike the normal A protein, the longer polypeptide is probably not incorporated into mature phage particles.     

The ratio of coat protein to bacteriophage f2 RNA in the translational repressor complex]

One of the mechanisms underlying the regulation of the bacteriophage f2 RNA translation is the repression of the phage RNA-replicase formation by coat protein. This repression is due to the formation of a complex between f2 RNA and coat protein (complex I). In this work the mechanism of complex I formation as well as the effect of this complex on the f2 RNA-replicase formation was followed by inhibition of alanine incorporation into RNA-replicase polypeptide which was separated by polyacrylamide gel electrophoresis. The molar ratios of protein to f2 RNA in complex I were analyzed by sucrose gradient sedimentation. It was been found that complex I consists of six molecules of coat protein bound per one molecule of RNA. Ribonuclease digestion of the glutaraldehyde-fixed complex resulted in a mixture of products in which the hexamers of coat protein molecules were predominant. This indicates that the six molecules of coat protein bound to f2 RNA are neighbouring. It has been also shown that under conditions required for phage protein synthesis, coat protein occurs in solution is dimer. The results show that the translational repression of the RNA-replicase cistron is due to the cooperative attachment of three dimers of coat protein to phage template, forming a hexameric cluster on the RNA strand. The proposed mechanism of the complex I formation seems to be in good agreement with the sequence of events in the phage F2 life cycle. It is known that shortly after infection of the host cell the coat protein and phage RNA-replicase begin to be synthesised. According to our findings, the first portions of coat protein do not affect the translation of the RNA-replicase gene since at low concentration the coat protein occure in the form of monomers. At a later period of phage development, when the concentration of coat protein is sufficiently high to promote the formation of protein dimers, the translational repressor complex is formed and the RNA-replicase gene becomes inoperative.     

Template activity of complexes formed between bacteriophage f2 RNA and coat protein.

Formation of complexes between f2 RNA polymerase cistron was partially inhibited, some RNA and coat protein was studied using salt conditions which are optimum for phage protein synthesis. In this ionic environment, coat protein precipitation can be prevented by sulfhydryl group-protecting agents. Complexes formed at different protein-RNA input molar ratios were isolated and tested for template activity in an in vitro protein synthesizing system. Simultaneously, the number of protein molecules bound per RNA strand in such complexes was measured by the membrane (Millipore) filtration technique. Under conditions in which translation of the RNA strands were complexed with six molecules of coat protein, whereas some remained unbound. Strong inhibition of the translation of the RNA polymerase cistron was observed when each of the RNA strands present in the mixture was associated with six molecules of coat protein.     

Initial velocity kinetic analysis of 30 S initiation complex formation in an in vitro translation system derived from Escherichia coli

The analysis of initial velocity kinetic data was used to examine the order in which fMet-tRNA and the coat cistron of genomic bacteriophage R17 or Q beta RNA bind to the 30 S ribosome subunit. These data were obtained using a quantitative assay for protein synthesis in Escherichia coli extracts where the rate of accumulation of protein product is dependent on the concentration of mRNA and is partially dependent on fMet-tRNA. Under the conditions of this assay, the amount of protein synthesized was proportional to the formation of ternary complexes between the mRNA, fMet-tRNA, and the 30 S ribosomal subunit. The results from the initial velocity and alternative substrate experiments are consistent with a rapid equilibrium ordered mechanism as opposed to a rapid equilibrium random mechanism. Analysis of the rate of coat protein synthesis at varied concentrations of mRNA and fixed concentrations of fMet-tRNA indicated that fMet-tRNA was the first substrate to bind to the 30 S subunit when either coat cistron was used as the mRNA. This scheme assumes the existence of a relatively slow step in protein synthesis that occurs after both the initiating tRNA and mRNA are bound to the ribosome and which allows substrate addition to reach thermodynamic equilibrium.     

Studies on the ribosomal binding sites of natural messenger RNA

Synthetic RNAs (poly AUG, poly UG, poly AUC, and poly AG) were observed to inhibit initiation of translation of maturation protein and coat protein cistrons on f2 phage RNA. The former was affected more significantly than the latter by poly AUG and poly UG, whereas the latter by poly AUC. Poly AG markedly blocked initiation of both cistrons at the same level. The synthetic RNAs interfered with the binding of f2 RNA to ribosomes. The results suggested that each cistron of mRNA may have a specific initiation signal.     

Polysomal Localization of R17 Bacteriophage-Specific Protein Synthesis

Synthesis of viral ribonucleic acid (RNA) polymerase, maturation protein, and coat protein in Escherichia coli infected with bacteriophage R17 occurs mainly on polysomes containing four or more ribosomes. The 30S ribosomal subunits through trimer-size polysomes, which are associated with all of the R17-specific proteins and are predominant in the infected cell, synthesize only coat protein. These structures may accumulate as products derived from larger polysomes as a result of failure in the release of nascent polypeptides after termination of chain growth. Appreciable amounts of viral coat protein remain attached to ribosomes and polysomes during R17 bacteriophage replication, supporting the hypothesis of the repressor role of this protein. The time course of synthesis of virus-specific proteins obtained from the polysomes of infected cells demonstrated regulated R17 messenger RNA translation consistent with the idea that coat protein is preferentially synthesized whereas the synthesis of noncoat proteins is suppressed.     

Bacteriophage SPO1 development: defects in a gene 31 mutant.

SPO1 temperature-sensitive mutant ts14-1, located in cistron 31, has a DD (DNA synthesis-delayed) phenotype at 37 degrees C and produces progeny in a stretched program. At 44 degrees C it behaves as a DO (DNA synthesis-defective) mutant and shuts off the viral RNA synthesis about 10 min after infection. The thermal sensitivity of this mutant is due to the inactivity of gp-31 (the product of gene 31) at 44 degrees C. However, gp-31 is synthesized at that temperature and partly recovers its activity at 37 degrees C. Only 5 min at the permissive temperature is enough to trigger the continuation of the phage program and to produce progeny. The partial defect at 37 degrees C and the expansion of the middle program together with the pleiotropic defects at the nonpermissive temperature could be suitable for the study of the controls involved in bacteriophage development.     

Regulation of gene expression at the translational level. The rescue factor reverses thermosensitive protein synthesis in N4316, a conditionally-lethal mutant of Escherichia coli defective in translation

Extracts of the conditionally-lethal mutant Escherichia coli N4316 are defective in a newly described translation factor, the rescue protein. We have analyzed the in vitro translation products of this mutant by gel electrophoresis during normal and arrested synthesis at the permissive and non-permissive temperatures. Translation programmed with MS2 bacteriophage RNA at the non-permissive temperature results in highly reduced synthesis of the coat protein with no detectable levels of the maturation and replicase products. Thus the relative number of copies of proteins synthesized by the ribosomes is altered in this mutant. In addition, there is mistranslation of the coat gene which results in the overproduction of the phage encoded no. 7 protein. Aberrant synthesis is also reflected in the increased read-through of termination codons during synthesis directed by phage RNAs harbouring amber mutations in the coat cistron. The rescue protein, purified from the parental strain, is able to complement the thermosensitive defect and restore proper synthesis. Biochemical characterization of the defect in the absence of rescue shows no detectable deficiency in the extent of initiation complex formation in reactions inhibited with sparsomycin. Peptidyltransferase is fully active as judged by the kinetics of formylmethionine-puromycin formation. However, rescue does exert an effect at the level of termination. In addition, the thermolability of the mutant can be reversed by dissociating 70S ribosomes into 30S and 50S subunits. Based on these and other observations, we propose tht rescue mediates a novel function in the association/dissociation of ribosomal subunits which is essential to the accuracy and efficiency of translation.