Bacteriophage f1 DNA replication genes. II. The roles of gene V protein and gene II protein in complementary strand synthesis

Filamentous phage gene V, which encodes a single-stranded DNA binding protein, has been cloned and placed under control of the lac promoter. Cells bearing the clone are refractory to filamentous phage infection if the expression of the gene is induced with isopropyl-1-thio-beta-D-galactoside. The inhibition of infection is shown to occur at an early stage, and can be reversed if the cells express gene II in addition to gene V protein. These observations support the hypothesis that gene II protein, in addition to its role in nicking and facilitating the synthesis of phage viral (+) strand DNA, functions to prevent the gene V-mediated inhibition of complementary (-) strand synthesis. We proposed a model in which the absolute and relative concentrations of the products of genes II, X and V determine whether a single strand is to be exported as phage or incorporated into double-stranded replicative form DNA.     

Regulation of bacteriophage f1 DNA replication. I. New functions for genes II and X

Gene II protein is required for all phases of filamentous phage DNA synthesis other than the conversion of the infecting single strand to the parental double-stranded molecule. It introduces a specific nick into the double-stranded replicative form DNA, is required for the initiation of (+) strand synthesis and is responsible for termination and ring closure of the (+) strand product. Here we show that the gene II protein also promotes minus strand synthesis later in infection. Over-expression of gene II protein can induce the conversion of all nascent single-stranded phage DNA to the double-stranded form, even in the presence of the single-stranded DNA-binding gene V protein that would normally sequester the newly synthesized single strands. We also present evidence that the gene X protein (separately translated from an initiator codon within gene II, and identical to the C-terminal one-third of the gene II protein) is a powerful inhibitor of phage-specific DNA synthesis in vivo.     

Interaction between gene II protein and the DNA replication origin of bacteriophage f1

The origin of DNA replication of the filamentous bacteriophage f1 binds its initiator protein (gene II protein) in vitro to form a complex that can be trapped on nitrocellulose filters. The binding occurs with both superhelical form DNA and linear DNA fragments. A number of defective mutants of the origin were tested for the ability to bind gene II protein. The region of DNA required for the binding is around a second palindrome downstream from the palindrome that contains the DNA replication initiation site. It overlaps, but is not identical to, the region required for the nicking reaction by the protein. The nicking site itself was dispensable for the binding. In vivo, a number of defective deletion mutants of the origin, when in a plasmid, inhibited growth of superinfecting phage if the intracellular level of gene II protein was low. In addition, these defective origins inhibited the activity of the functional phage origin located on the same replicon. The domain of the DNA sequence required for inhibition in vivo was consistent with that for the binding in vitro.     

Characterization of the DNA binding protein encoded by the N-specific filamentous Escherichia coli phage IKe. Binding properties of the protein and nucleotide sequence of the gene

A DNA binding protein encoded by the filamentous single-stranded DNA phage IKe has been isolated from IKe-infected Escherichia coli cells. Fluorescence and in vitro binding studies have shown that the protein binds co-operatively and with a high specificity to single-stranded but not to double-stranded DNA. From titration of the protein to poly(dA) it has been calculated that approximately four bases of the DNA are covered by one monomer of protein. These binding characteristics closely resemble those of gene V protein encoded by the F-specific filamentous phages M13 and fd. The nucleotide sequence of the gene specifying the IKe DNA binding protein has been established. When compared to the nucleotide sequence of gene V of phage M13 it shows an homology of 58%, indicating that these two phages are evolutionarily related. The IKe DNA binding protein is 88 amino acids long which is one amino acid residue larger than the gene V protein sequence. When the IKe DNA binding protein sequence is compared with that of gene V protein it was found that 39 amino acid residues have identical positions in both proteins. The positions of all five tyrosine residues, a number of which are known to be involved in DNA binding, are conserved. Secondary structure predictions indicate that the two proteins contain similar structural domains. It is proposed that the tyrosine residues which are involved in DNA binding are the ones in or next to a beta-turn, at positions 26, 41 and 56 in gene V protein and at positions 27, 42 and 57 in the IKe DNA binding protein.     

Selection and characterization of randomly produced mutants of gene V protein of bacteriophage M13.

Gene V protein of bacteriophage Ff (M13, f1, fd) is a master regulator of phage DNA replication and phage mRNA translation. It exerts these two functions by binding to single-stranded viral DNA or to specific sequences in the 5' ends of its target mRNAs, respectively. To study the structure/function relationship of gene V protein, M13 gene V was inserted in a phagemid expression vector and a library of missense and nonsense mutants was constructed by random chemical mutagenesis. Phagemids encoding gene V proteins with decreased biological activities were selected and the nucleotide sequences of their gene V fragments were determined. Furthermore, the mutant proteins were characterized both with respect to their ability to inhibit the production of phagemid DNA transducing particles and their ability to repress the translation of a chimeric lacZ reporter gene whose expression is controlled by the promoter and translational initiation signals of M13 gene II. From the data obtained, it can be deduced that the mechanism by which gene V protein binds to single-stranded DNA differs from the mechanism by which it binds to its target sequence in the gene II mRNA.     

Assignment of the 1H NMR spectrum and secondary structure elucidation of the single-stranded DNA binding protein encoded by the filamentous bacteriophage IKe.

By means of 2D NMR techniques, all backbone resonances in the 1H NMR spectrum of the single-stranded DNA binding protein encoded by gene V of the filamentous phage IKe have been assigned sequence specifically (at pH 4.6, T = 298 K). In addition, a major part of the side chain resonances could be assigned as well. Analysis of NOESY data permitted the elucidation of the secondary structure of IKe gene V protein. The major part of this secondary structure is present as an antiparallel beta-sheet, i.e., as two beta-loops which partly combine into a triple-stranded beta-sheet structure, one beta-loop and one triple-stranded beta-sheet structure. It is shown that a high degree of homology exists with the secondary structure of the single-stranded DNA binding protein encoded by gene V of the distantly related filamentous phage M13.     

Translational control of bacteriophage f1 gene II and gene X proteins by gene V protein

The gene II region of bacteriophage f1 DNA codes for two proteins, the 46 kd gene II protein and the 13 kd gene X protein, which results from an in-phase start at codon 300 of gene II. Using antigens II protein IgG, we show that the intracellular concentration of both proteins is controlled by the phage gene V protein. In wild-type f1-infected cells, the amount of gene II protein reaches a plateau of about 1500 molecules per cell at 20 min after infection, as measured by blot immunoassay. Similarly, the amount of gene X protein reaches a peak of about 500 molecules per cell around 10 min after infection. In contrast, when the gene V protein is inactive, both gene II and gene X proteins continue to accumulate at a high rate for at least 40 min after infection. This difference is caused by decreased synthesis of gene II and gene X proteins in the presence of gene V protein, which represses the translation of these two proteins.     

Gene X of bacteriophage f1 is required for phage DNA synthesis. Mutagenesis of in-frame overlapping genes

The gene II protein of bacteriophage f1 is a site-specific endonuclease required for initiation of phage viral strand DNA synthesis. Within gene II is another gene, X, encoding a protein of unknown function identical to the C-terminal 27% of the gene II protein, and separately translated from codon 300 (AUG) of gene II. By oligonucleotide mutagenesis, we constructed phage mutants in which this codon has been changed to UAG (amber) or UUG (leucine), and propagated them on cells carrying a cloned copy of gene X on a plasmid. The amber mutant makes no gene X protein, and cannot grow in the absence of the complementing plasmid; the leucine-inserting mutant can make gene X protein, and grows normally without the plasmid. Without gene X protein, phage DNA synthesis (particularly viral strand synthesis) is impaired. We discuss this finding in the context of other known in-frame overlapping genes (particularly genes A and A* of phage phi X174), many of which are also involved in the specific initiation of DNA synthesis, and suggest applications for the mutagenic strategy we employed.     

In-frame overlapping genes: the challenges for regulating gene expression

In-frame overlapping genes in phage, plasmid and bacterial genomes permit synthesis of more than one form of protein from the same gene. Having one gene entirely within another rather than two separate genes presumably precludes recombination events between the identical sequences. However, studies of such gene pairs indicate that the overlapping arrangement can make regulation of the genes more difficult. Here, we extend studies of in-frame overlapping genes II and X from filamentous phage f1 to determine if translational controls are required to regulate the gene properly. These genes encode proteins (pII and pX) with essential but opposing roles in phage DNA replication. They must be tightly regulated to maintain production of the proteins at relative steady state levels that permit continuous replication without killing the host. To determine why little or no pX appears to be made on the gene II/X mRNA, gene II translation was lowered by progressively deleting into the gene II initiator region. Increased pX translation resulted, suggesting that elongating ribosomes on the gene II mRNA interfere with internal initiation on the gene X ribosome binding site and limit gene X translation. As judged from systematically lowering the efficiency of suppression at a gene II amber codon upstream from the gene X start, the already modest level of gene II translation would have to be reduced by more than twofold to relieve all interference with internal initiation. Further downregulation of gene X expression proved to be required to maintain pX at levels relative to pII that are tolerated by the cell. Site-directed mutagenesis and nuclease mapping revealed that the gene X initiation site is sequestered in an extended RNA secondary structure that lowers gene X translation on the two mRNAs encoding it. The more general implications of the results for expression of in-frame overlapping genes are discussed.     

Gene V protein-mediated translational regulation of the synthesis of gene II protein of the filamentous bacteriophage M13: a dispensable function of the filamentous-phage genome.

Introduction of a deletion in the genome of wild-type M13 bacteriophage that eliminates translational repression of M13 gene II by its cognate gene V protein had no effect on phage viability. Furthermore, it was noted that gene V protein of phage IKe, a distant relative of M13, does not function as a translational repressor of its cognate gene II protein. The data strongly indicate that the gene V protein-mediated control of gene II expression in bacteriophage M13 is an evolutionary relic of the ancestral filamentous-phage genome and thus dispensable for proper filamentous-phage replication.     

Orientation of the DNA in the filamentous bacteriophage f1

The filamentous bacteriophage f1 consists of a molecule of circular single-stranded DNA coated along its length by about 2700 molecules of the B protein. Five molecules of the A protein and five molecules of the D protein are located near or at one end of the virion, while ten molecules of the C protein are located near or at the opposite end. The two ends of the phage can be separated by reacting phage fragments, which have been generated by passage of intact phage through a French press, with antibody directed against the A protein (Grant et al., 1981a). By hybridizing the DNA isolated from either end of ³²P-labeled phage to specific restriction fragments of fl replicative form I DNA, we have determined that the single-stranded DNA of the filamentous bacteriophage f1 is oriented within the virion. For wild-type phage, the DNA that codes for the gene III protein is located at the A and D protein end and that which corresponds to the intergenic region is located close to the C protein end of the particle. The intergenic region codes for no protein but contains the origins for both viral and complementary strand DNA synthesis. Analysis of the DNA orientation in phage in which the plasmid pBR322 has been inserted into different positions within the intergenic region of fl shows that the C protein end of all sizes of filamentous phage particles appears to contain a common sequence of phage DNA. This sequence is located near the junction of gene IV and the intergenic region, and probably is important for normal packaging of phage DNA into infectious particles. There appears to be no specific requirement for the origins of viral and complementary strand DNA synthesis to be at the end of a phage particle.     

Filamentous phage replication initiator protein gpII forms a covalent complex with the 5' end of the nick it introduced

Rolling circle type DNA replication is initiated by introduction of a nick in the leading strand of the origin by the initiator protein, which in most cases binds covalently to the 5' end of the nick. In filamentous phage, however, such a covalent complex has not been detected. Using a suitable substrate and short reaction time, we show that filamentous phage initiator gpII forms a covalent complex with nicked DNA, which rapidly dissociates unless gpII is inactivated. A peptide-DNA complex was isolated from trypsin digest of the complex by ion-exchange column chromatography and gel filtration, and its peptide sequence was determined. The result indicated that gpII was linked to DNA by the tyrosine residue at position 197 from the N-terminus. The mutant protein in which this tyrosine was replaced by phenylalanine did not show any detectable activity to complement gene II amber mutant phage in vivo. In vitro, the mutant protein recognized the origin and bent DNA as well as the wild-type does, but failed to introduce a nick and to relax the superhelicity of cognate DNA.     

A bacterial gene, fip, required for filamentous bacteriophage fl assembly.

An Escherichia coli mutant which does not support the growth of filamentous bacteriophage fl allows phage fl DNA synthesis and gene expression in mutant cells, but progeny particles are not assembled. The mutant cells have no other obvious phenotype. On the basis of experiments with phage containing nonlethal gene I mutations and with mutant fl selected for the ability to grow on mutant bacteria, we propose an interaction between the morphogenetic function encoded by gene I of the phage and the bacterial function altered in this mutant. The bacterial mutation defines a new gene, fip (for filamentous phage production), located near 84.2 min on the E coli chromosome.     

Bacteriophage T4 gene 41 protein, required for the synthesis of RNA primers, is also a DNA helicase

Bacteriophage T4 gene 41 protein is one of the two phage proteins previously shown to be required for the synthesis of the pentaribonucleotide primers which initiate the synthesis of new chains in the T4 DNA replication system. We now show that a DNA helicase activity which can unwind short fragments annealed to complementary single-stranded DNA copurifies with the gene 41 priming protein. T4 gene 41 is essential for both the priming and helicase activities, since both are absent after infection by T4 phage with an amber mutation in gene 41. A complete gene 41 product is also required for two other activities previously found in purified preparations of the priming activity: a single-stranded DNA-dependent GTPase (ATPase) and an activity which stimulates strand displacement synthesis catalyzed by T4 DNA polymerase, the T4 gene 44/62 and 45 polymerase accessory proteins, and the T4 gene 32 helix-destabilizing protein (five-protein reaction). The 41 protein helicase requires a single-stranded DNA region adjoining the duplex region and begins unwinding at the 3' terminus of the fragment. There is a sigmoidal dependence on both nucleotide (rGTP, rATP) and protein concentration for this reaction. 41 Protein helicase activity is stimulated by our purest preparation of the T4 gene 61 priming protein, and by the T4 gene 44/62 and 45 polymerase accessory proteins. The direction of unwinding is consistent with the idea that 41 protein facilitates DNA synthesis on duplex templates by destabilizing the helix as it moves 5' to 3' on the displaced strand.     

The J gene of bacteriophage phi X174: in vitro analysis of J protein function.

The J protein of phi X174 is a small, highly basic protein and is a component of the phage capsid. We have investigated the role of J protein during single-stranded viral DNA synthesis and phage morphogenesis by using an in vitro system composed of purified viral and host components (Aoyama et al., Proc. Natl. Acad. Sci. U.S.A. 80:4195-4199, 1983). The characterization of the products made in the presence and absence of J protein shows that J protein is not required for viral DNA synthesis, but is required for the packaging of DNA into infectious phage. The ability of J protein to bind to double-stranded DNA as well as single-stranded DNA and other interactions with DNA suggest a model in which J protein binds to double-stranded, replicative form DNA and enters the phage prohead by remaining bound to viral DNA as it is encapsidated.     

The putative single-stranded DNA-binding protein of the filamentous bacteriophage, Ifl. Amino acid sequence of the protein and structure of the gene.

The protein product corresponding to the gene located in the region of the coliphage Ifl genome shown to contain the code for the single-stranded DNA (ssDNA)-binding proteins of all filamentous phages so far studied has been isolated from infected bacterial cells and its amino acid sequence determined. The mature protein contains 95 amino acids (calculated molecular mass 10553 Da). Its sequence corresponds to that predicted from the DNA sequence but lacks the initiating methionine residue. Although there is little direct sequence homology between the phage Ifl protein and the ssDNA-binding proteins of the other filamentous phages that have been studied, computer-based comparisons of various physical and structural parameters showed that the phage Ifl protein contains a domain that is closely related to domains in the coliphage T4 gene 32 protein and the Pseudomonas phage Pfl ssDNA-binding protein and suggest that the Ifl protein does have a ssDNA-binding function although we were unable to show this directly.     

Translation at higher than an optimal level interferes with coupling at an intercistronic junction

In pairs of adjacent genes co-transcribed on bacterial polycistronic mRNAs, translation of the first coding region frequently functions as a positive factor to couple translation to the distal coding region. Coupling efficiencies vary over a wide range, but synthesis of both gene products at similar levels is common. We report the results of characterizing an unusual gene pair, in which only about 1% of the translational activity from the upstream gene is transmitted to the distal gene. The inefficient coupling was unexpected because the upstream gene is highly translated, the distal initiation site has weak but intrinsic ability to bind ribosomes, and the AUG is only two nucleotides beyond the stop codon for the upstream gene. The genes are those in the filamentous phage IKe genome, which encode the abundant single-stranded DNA binding protein (gene V) and the minor coat protein that caps one tip of the phage (gene VII). Here, we have used chimeras between the related phage IKe and f1 sequences to localize the region responsible for inefficient coupling. It mapped upstream from the intercistronic region containing the gene V stop codon and the gene VII initiation site, indicating that low coupling efficiency is associated with gene V. The basis for inefficient coupling emerged when coupling efficiency was found to increase as gene V translation was decreased below the high wild-type level. This was achieved by lowering the rate of elongation and by decreasing the efficiency of suppression at an amber codon within the gene. Increasing the strength of the Shine-Dalgarno interaction with 16S rRNA at the gene VII start also increased coupling efficiency substantially. In this gene pair, upstream translation thus functions in an unprecedented way as a negative factor to limit downstream expression. We interpret the results as evidence that translation in excess of an optimal level in an upstream gene interferes with coupling in the intercistronic junction.