Bacteriophage P4 DNA replication. Nucleotide sequence of the P4 replication gene and the cis replication region.

A 3100 base piece of DNA from the 11,500 base genome of bacteriophage P4 was analyzed for its nucleotide sequence. This segment of DNA contains two open reading frames of 106 and 777 amino acid residues; the latter of which is the coding sequence for the Mr 84,841 alpha protein, which is necessary for P4 DNA replication and is thought to act as a P4-specific DNA primase. A region of about 300 base-pairs localized just beyond the alpha gene and about 4500 bases from the origin of replication (ori), was defined as the locus for P4's cis replication region (crr). This region is required for replication both in vivo and in vitro, and consists of two directly repeated sequences of 120 base-pairs that match one another at 98 positions. These directly repeated sequences are separated by 60 base-pairs, which are not necessary for replication. Each repeat in crr contains three copies of the octamer TGTTCACC that is found six times in ori. Either of the 120 base-pair repeat sequences in crr is sufficient for replication, and the entire crr can function in an inverted orientation. crr is also active at a distance of 1800 bases from the P4 origin of replication.

The replication of bacteriophage P4 DNA in vitro. Partial purification of the P4 alpha gene product.

A soluble enzyme system has been prepared from a phage P4-infected Escherichia coli strain that supports the replication of exogenous, supercoiled P4 DNA. This DNA synthesis in vitro depends upon the four deoxyribonucleotides and ATP, but is enhanced about four- to fivefold by the presence of other ribonucleotides. E. coli DNA polymerase III holoenzyme, the E. coli single-strand DNA binding protein, and the partially purified P4 alpha gene product are required for replication in vitro. Rifamycin does not inhibit P4 replication in vitro. Since the P4 alpha gene codes for a rifamycin-resistant RNA polymerase (Barrett et al., 1983), and since P4 DNA replication is independent of the host primase (Bowden et al., 1975), we believe the alpha gene product is functioning as a P4-specific DNA primase.

Bacteriophage P2 late promoters. II. Comparison of the four late promoter sequences.

The late genes of bacteriophage P2 are clustered into four transcription units. We have reported the transcription initiation sites for two of the late messenger RNAs, encoding genes QP and ONMLKRS. We have now located the 5' ends of the two remaining late mRNAs. The first gene in the VJHG transcription unit has been located by DNA sequence determination of the single nucleotide change in a V amber mutant. Location of the first gene in the FETUD transcription unit has been inferred from the DNA sequence. The 5' ends of the mRNAs for these two transcription units were located by protection of end-labeled restriction fragments in RNA-DNA hybrids from digestion with nuclease S1. Similar protection of hybrids using RNA that had been 5' end-labeled with [alpha-32P]GTP and guanylyl transferase confirmed that these 5' termini resulted from initiation of transcription. The DNA sequences preceding the P2 late transcription starts are different from the Escherichia coli promoter consensus sequences at -10 and -35, consistent with the apparent requirement for phage-encoded proteins in the regulation of P2 late gene expression. The four P2 late promoters do share sequence homologies in the -10 and -35 regions, however, and several additional homologies further upstream. P2 late gene expression also appears to involve negative regulation by a product of the ONMLKRS gene cluster. When cells are infected with P2 polar O amber mutants, a marked increase in the levels of proteins encoded by the other three gene clusters is observed. This increase is reflected in the amounts of late mRNAs, suggesting that RNA synthesis is normally repressed or that late mRNAs are more labile in the presence of a gene product from the ONMLKRS transcription unit. Satellite phage P4 induced P2 late gene expression without the usual requirement for P2 DNA replication. The 5' ends of the P2 late mRNAs are the same during P4 transactivation as during normal P2 late gene expression. Thus, the regulation of P2 late gene expression by P4 does not involve altered promoter selection.

Bacteriophage P2 late promoters. Transcription initiation sites for two late mRNAs.

Divergent transcription of two of the bacteriophage P2 late mRNAs, encoding genes QP and ONMLKRS, is initiated from opposite strands of the DNA in a region near the left end of the P2 genome. The first gene in each of these transcription units (P and O) has been located in the nucleotide sequence by amino-terminal sequence analysis of the P gene product and by DNA sequence determination of the single nucleotide changes in two O amber mutants. The 5' ends of the P and O gene mRNAs are separated by 109 nucleotide pairs in the DNA template. The locations of these 5' termini were determined by protection of end-labeled restriction fragments in RNA-DNA hybrids from digestion with nuclease S1. Sequence analysis of mRNA that had been labeled at the 5' end with [alpha-32P]GTP and guanylyl transferase confirmed that these termini resulted from initiation of transcription. The DNA sequences preceding the O and P transcription starts have poor homologies to the bacterial promoter consensus sequences at -10 and -35, consistent with the apparent requirement for phage-encoded proteins in the regulation of P2 late gene expression. The O and P promoter regions also have no detectable homology to each other in the -10 or -35 regions, and are unusually G + C-rich. There are, however, blocks of sequence homology within the transcribed region of each of these two late operons near the 5' end. Satellite phage P4 induces P2 late gene expression without the usual requirement for P2 DNA replication. The 5' ends of the P2 P and O gene transcripts are the same during P4 "transactivation" as during normal P2 late gene expression. Thus the regulation of P2 late gene expression by P4 does not involve a change in the site for initiation of transcription.

Organization and expression of the satellite bacteriophage P4 late gene cluster.

The satellite bacteriophage P4 genes for capsid size determination (sid), transactivation (delta), and polarity suppression (psu) are cotranscribed at late times after infection from a single P4 late promoter (Psid) that lies to the left of the sid gene. While the -10 region of this promoter is similar to the consensus sequence for Escherichia coli RNA polymerase, the -35 region shares no homology with known classes of E. coli promoters. The -10 and -35 regions of Psid share no homology with the late gene promoters of helper phage P2. Nonetheless, P4 late transcription is stimulated by coinfecting P2, as well as by P2 prophage. This stimulation depends on the P2 encoded transcription factor ogr; transcription from Psid is stimulated following the induction of the P2 ogr gene carried on a plasmid. P4 late transcription in the absence of P2 requires the P4 delta product, which is partially homologous to the P2 ogr gene product. DNA sequence analysis shows that the psu gene codes for a protein of Mr = 21,314 that is unrelated to the antitermination gene products of the lambdoid phages.

Bacteriophage P4 DNA replication. Location of the P4 origin.

An electron microscopic examination of replicating bacteriophage P4 DNA molecules has revealed theta-type structures that replicate bidirectionally from a single origin. Many replicating P4 DNA molecules also contain long (2000 bases) single-strand DNA regions at the growing fork that are deployed in a trans configuration, which supports the concept of continuous leading strand and discontinuous lagging strand syntheses. The position of the P4 origin was localized by the use of a plasmid complementation test for replication in vivo, as well as by labeling of DNA replicating in vitro in the presence of a chain-terminating inhibitor. During this study we discovered a second site on the P4 genome which is essential for replication, and we have named it crr (cis region required for replication). The site is located at least 3300 bases from the origin but appears to be required for the initiation of DNA replication in vivo as well as in vitro.

Characterization of the cos sites of bacteriophages P2 and P4.

A 641-bp cos-containing P2 DNA fragment was sequenced and compared to the P4 cos region. Alignment of the P2 and P4 cos regions shows a homologous region of 55 bp that has only three mismatches and contains a completely conserved region of dyad symmetry. A number of P4- and P2-derived cosmids were tested in an in vivo transduction assay in order to determine the minimal cos region required for packaging. These experiments show that the common region of 55 bp is sufficient for transduction with low frequency, but that a 125-bp cos-containing fragment contains all the information for transduction with optimal frequency.

Selection of lambda Spi- transducing phages using the P2 old gene cloned onto a plasmid.

The old gene product of the P2 prophage interferes with plaque formation by lambda wild type phage but allows lambda phages whose red and gam genes have been deleted to form small, visible plaques (the lambda Spi- phenotype). The old gene product also kills Escherichia coli recB or recC mutants. We have cloned the old gene into the high-copy-number plasmid pBR322, where it prevents plaque formation by both lambda Spi+ and lambda Spi- phages. We transferred a DNA fragment that carries the old gene to the low-copy-number plasmid pSC101 and found that lambda Spi- phages can be selected on strains that carry this plasmid. The plasmid-borne old gene kills E. coli recB mutants, providing a selection for old- mutants.

Mutational analysis of a satellite phage activator.

The late gene activator, Delta, of satellite phage P4 is more efficient than the Delta of satellite phage phiR73 in utilizing a P2 helper prophage that lacks an activator (ogr) gene. Analysis of P4 Delta is complicated by the fact that this protein contains two tandem phiR73 Delta-like domains. We performed a mutational analysis of phiR73 Delta, in order to select mutations that might not be found using P4 Delta. The host RNA polymerase alpha subunit mutation rpoA155 (L289F) blocks the growth of P2, P4, and P4 carrying the delta gene of phiR73. A mutant of this latter phage that can grow in the presence of rpoA155 carries a V19M mutation in phiR73 Delta. This suggests that aa 19 contacts RNA polymerase, in addition to the aa residues 13, 42 and 44, that have been implicated in interactions with RNA polymerase by previous mutational analyses of P2 ogr and P4 delta. In corroboration of the proposed role of the regions at aa residues 19, 42, and 44, we found phiR73 Delta mutations in these regions that showed a reduced activation of late gene expression, but a normal ability to bind to late gene promoters. All activators in the Delta class contain four Cys residues that bind Zn2+. Mutation of these aa residues in phiR73 Delta eliminated late gene activation. Spectroscopic analysis of these mutant proteins revealed that they were unable to bind Zn2+. Histidine residues were substituted for two of the Cys residues (C30 and C35), changing a C2C2 type Zn-binding motif to a C2H2 motif. Although His residues are used in coordinating Zn2+ in other proteins, these His substitutions resulted in complete loss of activity and the inability to bind Zn2+.

The P2 phage old gene: sequence, transcription and translational control.

The old (overcoming lysogenization defect) gene product of bacteriophage P2 kills Escherichia coli recB and recC mutants and interferes with phage lambda growth [Sironi et al., Virology 46 (1971) 387-396; Lindahl et al., Proc. Natl. Acad. Sci. USA 66 (1970) 587-594]. Specialized transducing lambda phages, which lack the recombination region, can be selected by plating lambda stocks on E. coli that carry the old gene on a prophage or plasmid [Finkel et al., Gene 46 (1986) 65-69]. Deletion and sequence analyses indicate that the old-encoded protein has an Mr of 65,373 and that its transcription is leftward. Primer extension analyses locate the transcription start point near the right end of the virion DNA. A bacterial mutant, named pin3 and able to suppress the effects of the old gene, has been isolated [Ghisotti et al., J. Virol. 48 (1983) 616-626]. In a pin3 mutant strain, carrying the old gene on a prophage or plasmid, the amount of old transcript is greatly reduced. The effect of the pin3 mutation is abolished by the wild-type allele of argU, an arginine tRNA that reads the rare Arg codons AGA and AGG, which are used for eight of the 14 Arg codons in the old gene. Thus the pin3 allele probably stalls translation of the old mRNA, causing this mRNA to be degraded. Isoelectric focusing and electrophoretic analysis identify the old gene product as a basic protein of approx. 65 kDa.

Evidence for an active role of the DnaK chaperone system in the degradation of sigma(32).

Under non-stressed conditions in Escherichia coli, the heat shock transcription factor sigma(32) is rapidly degraded by the AAA protease FtsH. The DnaK chaperone system is also required for the rapid turnover of sigma(32) in the cell. It has been hypothesized that the DnaK chaperone system facilitates the degradation of sigma(32) by sequestering it from RNA polymerase core. This hypothesis predicts that mutant sigma(32) proteins, which are deficient in binding to RNA polymerase core, will be degraded independently of the DnaK chaperone system. We examined the in vivo stability of such mutant sigma(32) proteins. Results indicated that the mutant sigma(32) proteins as similar as authentic sigma(32) were stabilized in DeltadnaK and DeltadnaJ/DeltacbpA cells. The interaction between sigma(32) and DnaK/DnaJ/GrpE was not affected by these mutations. These results strongly suggest that the degradation of sigma(32) requires an unidentified active role of the DnaK chaperone system.