The binding site for coat protein on bacteriophage Qbeta RNA.

The site of interaction of phage Qbeta coat protein with Qbeta RNA was determined by ribonuclease T1 degradation of complexes of coat protein and [32P]-RNA obtained by codialysis of the components from urea into buffer solutions. The degraded complexes were recovered by filtration through nitrocellulose filters, and bound [32P]RNA fragments were extracted and separated by polyacrylamide gel electrophoresis. Fingerprinting and further sequence analysis established that the three main fragments obtained (chain lengths 88, 71 and 27 nucleotides) all consist of sequences extending from the intercistronic region to the beginning of the replicase cistron. These results suggest that in the replication of Qbeta, as in the case of R17, coat protein acts as a translational repressor by binding to the ribosomal initiation site of the replicase cistron.     

Escherichia coli ribosomal protein S1 recognizes two sites in bacteriophage Qbeta RNA.

As a component of bacteriophage Qbeta replicase, S1 is required both for initiation of Qbeta minus strand RNA synthesis and for translational repression, which has been traced to the ability of the enzyme to bind to an internal site in the Qbeta RNA molecule. Previously, Senear and Steitz (Senear, A. W., and Steitz, J. A. (1976) J. Biol. Chem. 251, 1902-1912) found that isolated S1 protein binds specifically to an oligonucleotide spanning residues -38 to -63 from the 3' terminus of Qbeta RNA. Here we report that S1 also interacts strongly with a second oligonucleotide in Qbeta RNA, which is derived from the region recognized by replicase just 5' to the Qbeta coat protein cistron. Both sequences exhibit pyrimidine-rich regions.     

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.     

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.     

The complete nucleotide sequence of the group II RNA coliphage GA

The complete nucleotide sequence of the RNA coliphage GA, a group II phage, is presented. The entire genome comprises 3466 bases. Three large open reading frames were identified, which correspond to the maturation protein gene (390 amino acids), the coat protein gene (129 amino acids) and the replicase beta-subunit protein gene (531 amino acids). In addition, untranslated regions occur at the 5' (135 bases) and 3' (122 bases) ends of the molecule. Two intercistronic untranslated regions occur between the cistrons for the maturation and coat proteins, and between the coat and beta-subunit proteins. We have compared the nucleotide sequence of GA RNA with the published sequence of MS2 RNA, and show that they are related. The comparative structures of two important regulatory regions are presented; the coat protein binding site which is involved in translational repression of the replicase beta-subunit protein gene, and a hairpin in a region proximal to the lysis protein gene.     

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.     

Defective lysis of streptomycin-resistant escherichia coli cells infected with bacteriophage f2

A lysis defect was found to account for the failure of a streptomycin-resistant strain of Escherichia coli to form plaques when infected with the male-specific bacteriophage f2. The lysis defect was associated with the mutation to streptomycin resistance. Large amounts of apparently normal bacteriophage accumulated in these cells. Cell-free extracts from both the parental and mutant strains synthesized a potential lysis protein in considerable amounts in response to formaldehyde-treated f2 RNA but not in response to untreated RNA. As predicted from the nucleotide sequence of the analogous MS2 phage, the protein synthesized in vitro had the expected molecular weight and lacked glycine. The cistron for the lysis protein overlapped portions of the coat and replicase cistrons and was translated in the +1 reading frame. Initiation at the lysis protein cistron may be favored by translation errors that expose the normally masked initiation site, and streptomycin-resistant ribosomes, known to have more faithful translation properties, may be unable to efficiently synthesize the lysis protein.     

A hybridization procedure for the isolation of specific RNA segments applied to the analysis of bacteriophage Qbeta RNA.

A method for the isolation of RNA fragments originating from defined regions of bacteriophage Qbeta RNA minus strands is described. Large RNase T1 oligonucleotides were isolated on a preparative scale from Qbeta RNA. The nucleotide sequences (13 to 26 nucleotides) and map positions of these oligonucleotides were known from previous work (Billeter, M. A. (1978) J. Biol. Chem. 253, 8381-8389). After addition of AMP residues (50 in the average) using terminal adenylate transferase, these pure oligonucleotides were hybridized to 32P-labeled Qbeta RNA minus strands synthesized in vitro. Fragments in the size range of 100 to 500 nucleotides were then generated by partial digestion with RNase T1. Fragments hybridized to such oligonucleotides were recovered by chromatography on poly(U)-Sephadex and then resolved according to their size by polyacrylamide gel electrophoresis. The specificity and reproducibility of the method as well as its suitability for the sequence analysis of Qbeta RNA was verified by using in particular a linker oligonucleotide derived from a Qbeta RNA region near the 3' end. The sequence catalogues of the RNase T1 and RNase A oligonucleotides of two fragments isolated in this way, 202 and 310 nucleotides in length, were established and all fragments isolated were shown to contain a sequence complementary to the linker oligonucleotide.     

Overproduction of bacteriophage Qβ maturation (A2) protein leads to cell lysis

Double-stranded cDNA from the maturation (or A₂) protein gene of the RNA bacteriophage Qβ has been cloned such that its expression is regulated by the E. coli lac promoter/operator. Induction of the A₂ clone is lethal to the host. The basis of this lethality is cell lysis, which is correlated with synthesis of the A₂ protein. No other major proteins appear to be made from the A₂ gene when inducer is added. Plasmid-derived A₂ protein specifically complements infecting Qβ A₂ amber mutants. Lysis activity is abolished in clones that synthesize truncated or internally deleted A₂ polypeptides ranging from 10% to 95% of the length of wild-type protein. We conclude that host lysis is promoted by the maturation protein itself, rather than by a separate lysis protein. The A₂ protein probably allows for the release of progeny Qβ phage particles following normal infection.     

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.     

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.     

Analysis of six new genes encoding lysis proteins and coat proteins in Escherichia coli group A RNA phages

Group A RNA phages consist of four genes-maturation protein, coat protein, lysis protein and replicase genes. We analyzed six plasmids containing lysis protein genes and coat protein genes of Escherichia coli group A RNA phages and compared their amino acid sequences with the known proteins of E. coli(group A), Pseudomonas aeruginosa(PP7) RNA phages and Rg-lysis protein from Qbeta phage. The size of lysis proteins was different by the groups but the coat proteins were almost the same size among phages. The phylogenetic analysis shows that the sub-groups A-I and A-II of E. coli RNA phages were clearly dispersed into two clusters.     

Escherichia coliSkp Chaperone Coexpression Improves Solubility and Phage Display of Single-Chain Antibody Fragments

Expression of single-chain antibody fragments (scAb)in the periplasm ofEscherichia colioften results in low soluble product yield and cell lysis. We have increased scAb solubility and prevented cell culture lysis by coexpressing theE. coliSkp chaperone gene. A mutant Skp cistron was linked to a bacteriophage T7 gene 10 translational initiation region and placed either downstream of a scAb gene within an isopropyl β- -thiogalactopyranoside-inducible expression cassette or on a separate colE1-compatible arabinose-inducible vector. Increases in scAb solubility reflected the amount of coexpressed Skp. A bacteriophage display vector that was also engineered to coexpress Skp permitted display of a virtually undisplayable scAb and should prove useful in expanding library sizes.     

Sequence and location of large RNase T1 oligonucleotides in bacteriophage Qbeta RNA.

Twenty-nine oligonucleotides, 11 to 26 nucleotides in length, arising by complete RNase T1 digestion of bacteriophage Qbeta RNA and isolated by two-dimensional polyacrylamide gel electrophoresis, were sequenced. Their location within the genome was established with two methods. (a) In vitro synthesis of Qbeta RNA plus strands was started synchronously, using minus strands as template and nucleoside [alpha-32P]triphosphates as substrate; after various times, the reaction was stopped and the length of the products formed was correlated with their content of T1 oligonucleotides. (b) Qbeta [32P]RNA was elongated with poly(A) using terminal riboadenylate transferase; after mild treatment with alkali the fragments were fractionated by size and the poly(A)-containing molecules of each size class were isolated by chromatography on poly(U)-Sephadex and assayed for T1 oligonucleotides. The oligonucleotides in the 5' region were localized more precisely with method a, those near the 3' end with method b; in the middle region, the results of the two sets of analyses confirmed each other. The use of these oligonucleotides in the sequence determination of Qbeta RNA is discussed.     

Resynchronization of RNA synthesis by coliphage Qbeta replicase at an internal site of the RNA template

In previous work Qβ replicase has been used to synthesize labelled 5′ terminal segments of Qβ plus or minus strands of defined length. A procedure has now been developed which allows resynchronization of Qβ replicase at an internal position and synthesis of a labelled minus-strand segment complementary to the coat cistron ribosome binding site and the intercistronic region between the A₂ (maturation) and the coat cistron. Resynchronization is accomplished by binding a ribosome to Qβ RNA and allowing Qβ replicase to initiate and elongate up to the ribosome, using unlabelled ribonucleoside triphosphates. The ribosome is dissociated by EDTA treatment and the EDTA is removed. The replicating complex remains functional after this treatment, and addition of labelled substrates leads to synchronized elongation. The radioactive part of the product recovered after a short elongation period with labelled substrates was shown to be complementary to the coat protein ribosome binding site.     

Escherichia coli skp chaperone coexpression improves solubility and phage display of single-chain antibody fragments

Expression of single-chain antibody fragments (scAb)in the periplasm of Escherichia coli often results in low soluble product yield and cell lysis. We have increased scAb solubility and prevented cell culture lysis by coexpressing the E. coli Skp chaperone gene. A mutant Skp cistron was linked to a bacteriophage T7 gene 10 translational initiation region and placed either downstream of a scAb gene within an isopropyl beta-d-thiogalactopyranoside-inducible expression cassette or on a separate colE1-compatible arabinose-inducible vector. Increases in scAb solubility reflected the amount of coexpressed Skp. A bacteriophage display vector that was also engineered to coexpress Skp permitted display of a virtually undisplayable scAb and should prove useful in expanding library sizes.     

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.     

Translational interference at overlapping reading frames in prokaryotic messenger RNA

In overlapping reading frames of prokaryotic mRNA, the ribosome-binding site (RBS) of the downstream cistron is part of the coding sequence of the upstream message. We have examined whether the rate of translation in Escherichia coli can be sufficiently high to preclude the use of an RBS in initiation of protein synthesis when it is part of an actively decoded reading frame. The two sets of gene overlap present in the RNA phage MS2 are used as a model system. We find that translation of an upstream cistron can fully block initiation of protein synthesis at the overlapping RBS of the downstream cistron. Nonsense mutations in the upstream gene restore the translation of the downstream gene.     

The regulatory region of phage fr replicase cistron. III. Initiation activity of specific fr RNA fragments.

RNA fragments from phage fr covering the complete or part of the replicase cistron initiation region have been used as templates in the formation of a ribosomal initiation complex in vitro. The results so obtained together with our earlier findings in a similar approach applied to fragments of the structurally related RNA from phage MS2 have allowed us to pinpoint the boundaries of the replicase cistron initiation region on phage RNA. A structural model of the above initiation region has been provided which shows that besides the minimal initiation region (comprises the Shine-Dalgarno sequence and initiator AUG), the flanking regions are also involved and are responsible for additional interactions with the ribosome. The flanking regions possibly contribute to the stability of specific contact between the ribosome and template realized by the minimal initiation region.