Genetic mapping of regA mutants of bacteriophage T4D.

SP62, a mutant of bacteriophage T4 shown by Wiberg et al. (1973) to be defective in regulation of T4 protein synthesis, was shown by complementation tests to define a new gene, regA, and by intergenic mapping to lie between genes 43 and 62. The mapping involved crossing SP62 with a quadruple amber mutant defective in genes 42, 43, 62, and 44, selecting all six classes of amber-containing recombinants caused by single crossover events, and then scoring the presence or absence of SP62 in these recombinants. In addition, 15 new, spontaneous regA mutants were isolated, and 13 of these were mapped against each other; a total of eight different mutation sites were thus defined. Most of the new mutants were isolated as pseudorevertants of a leaky amber mutant in gene 62, according to Karam and Bowles (1974), whereas one was identified by virtue of the "white ring" around its plaque, a phenotype possessed by all the regA mutants at high temperature, SP62 was renamed regA1, and the new mutants were named regA2, regA3, etc.     

Bacteriophage T4 gene 44 DNA polymerase accessory protein. Sequences of gene 44 and its protein product

Bacteriophage T4 gene 44 protein is a DNA polymerase accessory protein which is required for T4 DNA replication. We have isolated the gene for 44 protein from a previously constructed lambda-T4 hybrid phage (Wilson, G. G., Tanyashin, V. I., and Murray, N. E. (1977) Mol. Gen. Genet. 156, 203-214). We report here the nucleotide sequence of gene 44 and about 60 nucleotides 5' upstream from its coding region, which is immediately adjacent to gene 45. We have also purified 44 protein from T4-infected cells and submitted it to extensive protein chemistry characterization. Thus, considerable portions of the protein sequence predicted from the DNA sequence were confirmed by direct protein sequencing of peptides or by matching amino acid compositions of purified peptides. A total of 84% of the predicted amino acids was confirmed by the protein data. These studies indicate that gene 44 codes for a polypeptide containing 319 amino acids, with a calculated Mr = 35,371. The coding region of gene 44 is preceded by a potential regulatory region containing sequences homologous to the Escherichia coli (-10) RNA polymerase binding region and to a conserved sequence at -25 to -30 found in other T4 middle genes. In addition, there are sequence similarities in the translation initiation regions of genes 44, 45, and rIIB, all of which are subject to regulation by regA protein.     

Characterization of bacteriophage T4 regA protein-nucleic acid interactions

The bacteriophage T4 regA protein is a translational repressor of a group of T4 early mRNAs. We have characterized the binding of regA protein to polynucleotides and to specific RNAs. Binding to nucleic acids was monitored by the quenching of the intrinsic tryptophan fluorescence of regA protein. regA protein exhibited differential affinities for the polynucleotides examined, with the order of affinity being poly(rU) greater than poly(dT) greater than poly(dU) = poly(rG) greater than poly(rC) = poly(rA). The binding site size calculated for regA protein binding to poly(rU) was n = 9 +/- 1 nucleotides. Cooperativity was observed in binding to multiple-site oligonucleotides, with a cooperativity parameter (omega) value of 10-22. To study the specific interaction between regA protein and T4 gene 44 mRNA, the affinity of regA protein for synthetic gene 44 RNA fragments was measured. The association constant (Ka) for regA protein binding to gene 44 RNA fragments was 100-fold higher than for binding to nontarget RNA. Study of variant gene 44 RNA fragments indicated that the nucleotides required for specific binding are contained within a 12-nucleotide sequence spanning -12 to -1, relative to the AUG codon. The bases of five nucleotides (indicated in upper case type) are critical for specific regA protein interaction with the gene 44 recognition element, 5'-aaUGAGgAaauu-3'. These studies further showed that formation of a regA protein-RNA complex involves a maximum of 2-3 ionic interactions and is primarily an enthalpy-driven process.     

Bacteriophage T4 regA protein binds RNA as a monomer, overcoming dimer interactions.

The stoichiometry of the complex formed between the T4 translational repressor protein regA and the 16 nt gene 44 recognition element (gene 44RE) RNA has been determined. Under quantitative binding conditions, the association of wild-type regA protein with gene 44RE RNA exhibits saturation at a 1:1 ratio of protein to RNA. It is known that regA protein exists as a dimer in protein crystals. Thus, the stoichiometry may be indicative of a regA dimer bound to two RNAs or a regA monomer bound to one RNA. Gel filtration through Sephadex G-75 revealed that wild-type and R91L regA proteins (14.6 kDa) elute at a mass of 29 kDa, consistent with the mass of a dimer. However, wild-type regA preincubated with gene 44RE (1:1) resulted in a complex that eluted at approximately 20 kDa, consistent with a regA monomer-RNA complex. Covalent crosslinking of surface lysines with glutaraldehyde confirmed that wild-type and R91L proteins exist as dimers and higher oligomers in solution. However, the addition of RNA to wild-type regA protein prior to crosslinking inhibited the formation of crosslinked dimers. Thus, the regA protein-protein interactions observed in solution are disrupted or blocked in the presence of gene 44RE RNA. Together, these studies demonstrate that regA protein binds RNA as a monomer, although free protein exists predominantly as a dimer.     

Efficiency and Frequency of Translational Coupling between the Bacteriophage T4 Clamp Loader Genes

The bacteriophage T4 DNA polymerase holoenzyme is composed of the core polymerase, gene product 43 (gp43), in association with the “sliding clamp” of the T4 system, gp45. Sliding clamps are the processivity factors of DNA replication systems. The T4 sliding clamp comes to encircle DNA via the “clamp loader” activity inherent in two other T4 proteins: 44 and 62. These proteins assemble into a pentameric complex with a precise 4:1 stoichiometry of proteins 44 and 62. Previous work established that T4 genes 44 and 62, which are directly adjacent on polycistronic mRNA molecules, are—to some degree—translationally coupled. In the present study, measurement of the levels (monomers/cell) of the clamp loader subunits during the course of various T4 infections in different host cell backgrounds was accomplished by quantitative immunoblotting. The efficiency of translational coupling was obtained by determining the in vivo levels of gp62 that were synthesized when its translation was either coupled to or uncoupled from the upstream translation of gene 44. Levels of gp44 were also measured to determine the relative stoichiometry of synthesis and the percentage of gp44 translation that was transmitted across the intercistronic junction (coupling frequency). The results indicated a coupling efficiency of ~85% and a coupling frequency of ~25% between the 44-62 gene pair during the course of infection. Thus, translational coupling is the major factor in maintaining the 4:1 stoichiometry of synthesis of the clamp loader subunits. However, coupling does not appear to be an absolute requirement for the synthesis of gp62.     

Translational repression: biological activity of plasmid-encoded bacteriophage T4 RegA protein

The RegA protein of bacteriophage T4 is a translational repressor that regulates expression of several phage early mRNAs. We have cloned wild-type and mutant alleles of the T4 regA gene under control of the heat-inducible, plasmid-borne leftward promoter (PL) of phage lambda. Expression of the cloned regA+ gene resulted in the synthesis of a protein that closely resembled phage-encoded RegA protein in biological properties. It repressed its own synthesis (autogenous translational control) as well as the synthesis of specific T4-encoded proteins that are known from other studies to be under RegA-mediated translational control. Cloned mutant alleles of regA exhibited derepressed synthesis of the mutant regA gene products and were ineffective in trans against RegA-sensitive mRNA targets. The effects of plasmid-encoded RegA proteins were also demonstrated in experiments using two compatible plasmids in uninfected Escherichia coli. The two-plasmid assays confirm the sensitivities of several cloned T4 genes to RegA-mediated translational repression and are well-suited for genetic analysis of RegA target sites. Repression specificity in this system was demonstrated by using wild-type and operator-constitutive translational initiation sites of T4 rIIB fused to lacZ. The results show that no additional T4 products are required for RegA-mediated translational repression. Additional evidence is provided for the proposal that uridine-rich mRNA sequences are preferred targets for the repressor. Surprisingly, plasmid-generated RegA protein represses the synthesis of some E. coli proteins and appears to enhance selectively the synthesis of others. The RegA protein may have multiple functions, and its binding sites are not restricted to phage mRNAs.     

The complex of T4 bacteriophage gene 44 and 62 replication proteins forms an ATPase that is stimulated by DNA and by T4 gene 45 protein

The bacteriophage T4 genome is believed to encode all of the proteins needed for the replication of its own DNA. Included among these proteins are the "polymerase accessory proteins", the products of T4 genes 44, 62 and 45. The first two of these genes specify the synthesis of the 44/62 protein complex, which is here shown to be a DNA-dependent ATPase, hydrolyzing either ATP or dATP to the corresponding nucleoside diphosphate and releasing inorganic phosphate. This nucleotide hydrolysis is greatly stimulated by addition of the gene 45 protein and by single-stranded DNA termini. A rapid micro DNA-cellulose assay is introduced and used to measure accessory protein binding to the complex of T4 gene 32 protein and single-stranded DNA. In the presence of ATP, the 44/62 protein binds to this complex but not to naked DNA, while the 45 protein requires both the 32 protein and the 44/62 protein for detectable binding.     

Suppression of Amber Mutations of Bacteriophage T4 Gene 43 (DNA Polymerase) by Translational Ambiguity

The growth properties of twelve different amber (am) mutants of bacteriophage T4 gene 43 (DNA polymerase) were examined by using nonpermissive (su(-)) as well as permissive (su(+)) Escherichia coli hosts. It was found that most of these mutants were measurably suppressed in su(-) hosts by translational ambiguity (misreading of codons during protein synthesis). The ability of these mutants to grow in response to this form of weak suppression probably means that the T4 gene 43 DNA polymerase can be effective in supporting productive DNA replication when it is supplied in small amounts. By similar criteria, studies with other phage mutants suggested that the products of T4 genes 62 (uncharacterized), 44 (uncharacterized), 42 (dCMP-hydroxymethylase), and 56 (dCTPase) are also effective in small amounts. Some T4 gene products, such as the product of gene 41 (uncharacterized), seem to be partially dispensable for phage growth since am mutants of such genes do propagate, although weakly, in streptomycin-resistant su(-) hosts which appear to have lost the capacity to suppress am mutations by ambiguity.     

The Riia Gene of Bacteriophage T4. I. Its DNA Sequence and Discovery of a New Open Reading Frame between Genes 60 and Riia

We have determined the DNA sequence of the rIIA gene and have discovered a small open reading frame, rIIA.1, between genes 60 and rIIA. The predicted molecular weights of these proteins are 82,840 for rIIA and 8,124 for rIIA.1. The rIIA protein has a repeated motif which suggests that the gene has evolved by duplication. It also has a motif which suggests that it belongs to a group of ompR-like proteins that control regulation of gene expression in response to changes in the external environment. We have sequenced three different missense mutants whose mutations lie in the Ala segment of the rIIA genetic map. All three changes are found within the first 35 bp of the rIIA coding sequence. The region of control of protein synthesis is identical in the rIIA gene and in gene 44 of T4. We relate this finding to the high sensitivity of both RNAs to translational repression by the T4 regA gene product.     

An ATP stimulation of T4 DNA polymerase mediated via T4 gene 44/62 and 45 proteins. The requirement for ATP hydrolysis.

An in vitro replication system reconstituted from six purified T4 bacteriophage proteins, each of which is essential for T4 DNA replication in vivo, requires ATP. Because of the complexity of the complete system, we examine in this report the involvement of ATP in two subsystems of the overall DNA synthesis reaction. One subsystem consists of the T4 DNA polymerase (gene 43 protein) and its "accessory proteins," the gene 44/62 and 45 products. An even simpler subsystem consists of the gene 44/62 and 45 proteins alone, which together have a DNA-dependent ATPase activity. The combination of the 44/62 and 45 proteins hydrolyze ATP to ADP and inorganic phosphate in the presence of DNA. These essential accessory proteins have been previously shown to increase T4 DNA polymerase activity on primed, single-stranded DNA templates. In this report we use nucleotide analogues to demonstrate that this polymerase stimulation requires hydrolysis of the beta,gamma-phosphate bond of ATP. However, our data suggest that the mechanism of accessory protein stimulation is such that less than 1 ATP molecule need be hydrolyzed per 10 deoxyribonucleotides incorporated by the DNA polymerase into DNA.     

Autogenous Regulation of the Rega Gene of Bacteriophage T4: Derepression of Translation

The regA gene of phage T4 encodes a translational repressor that inhibits utilization of its own mRNA as well as the translation of a number of other phage-induced mRNAs. In recombinant plasmids, autogenous translational repression limits production of the RegA protein when the cloned structural gene is expressed under control of a strong, plasmid-borne promoter (lambda PL). We have found that a genetic fusion which places the regA ribosome binding domain in proximity to active translation leads to partial derepression of wild-type RegA protein synthesis. The derepression is not due to increased synthesis of regA RNA, suggesting that it occurs at the translational level. Derepressed clones of the wild-type regA gene were used to overproduce and purify the repressor. In an in vitro assay the wild-type target was sensitive and a mutant target was resistant to inhibition by the added protein. The results suggest that the sensitivity of a regA-regulated cistron to translational repression may depend on the competition between ribosomes and RegA protein for overlapping recognition sequences in the translation initiation domain of the mRNA.     

The T4 DNA polymerase accessory proteins form an ATP-dependent complex on a primer-template junction

The DNA polymerase holoenzyme of bacteriophage T4 contains, besides the DNA polymerase itself (the gene 43 protein), a complex of the protein products of T4 genes 44 and 62 (a DNA-dependent ATPase) and of gene 45. Together, the 44/62 and 45 proteins form an ATP-dependent "sliding clamp" that holds a moving DNA polymerase molecule at the 3' terminus of a growing DNA chain. We have used a unique DNA fragment that forms a short hairpin helix with a single-stranded 5' tail (a "primer-template junction") to map the binding sites for these polymerase accessory proteins by DNA footprinting techniques. In the absence of the DNA polymerase, the accessory proteins protect from DNase I cleavage 19-20 nucleotides just behind the 3' end of the primer strand and 27-28 nucleotides on the complementary portion of the template strand. Detection of this DNA-protein complex requires the 44/62 and 45 proteins plus the nonhydrolyzable ATP analogue adenosine 5'-O-(thiotriphosphate). The complex is not detected in the presence of ATP. We suggest that ATP hydrolysis by the 44/62 protein normally activates the accessory proteins at a primer-template junction, permitting the DNA polymerase to bind and thus form the complete holoenzyme. However, when the polymerase is missing, as in these experiments, ATP hydrolysis is instead followed by a release (or loosening) of the accessory protein complex.     

regA protein of bacteriophage T4D: identification, schedule of synthesis, and autogenous regulation.

Proteins labeled with 14C-amino acids after infection of Escherichia coli B by T4 phage were examined by electrophoresis in the presence of sodium dodecyl sulfate. Four regA mutants (regA1, regA8, regA11, and regA15) failed to make a protein having a molecular weight of about 12,000, whereas mutant regA9 did make such a protein; regA15 produced a new, apparently smaller protein that was presumably a nonsense fragment, whereas regA11 produced a new, apparently larger protein. We conclude that the 12,000-dalton protein was the product of the regA gene. The molecular weight assignment rested primarily on our finding that the regA protein had the same mobility as the T4 gene 33 protein, which we identified by electrophoresis of whole-cell extracts of E. coli B infected with a gene 33 mutant, amE1120. Synthesis of wild-type regA protein occurred from about 3 to 11 min after infection at 37 degrees C in the DNA+ state and extended to about 20 min in the DNA- state. However, synthesis of the altered regA proteins of regA9, regA11, and regA15 occurred at a higher rate and for a much longer period in both the DNA+ and DNA- states; thus, the regA gene is autogenously regulated. At 30 degrees C, both regA9 and regA11 exhibited partial regA function by eventually shutting off the synthesis of many T4 early proteins; the specificity of this shutoff differed between these two mutants. We also obtained evidence that the regA protein is not Stevens's "polypeptide 3." As a technical point, we found that, when quantitating acid-precipitable radioactivity in protein samples containing sodium dodecyl sulfate, it was necessary to use 15 to 20% trichloroacetic acid; use of 5% acid, e.g., resulted in loss of over half of the labeled protein.     

Sequence analysis of conserved regA and variable orf43.1 genes in T4-like bacteriophages.

Bacteriophage T4 RegA protein is a translational repressor of several phage mRNAs. In the T4-related phages examined, regA nucleotide sequences are highly conserved and the inferred amino acid sequences are identical. The exceptional phage, RB69, did not produce a RegA protein reproducibly identifiable by Western blots (immunoblots) nor did it produce mRNA that hybridized to T4 regA primers. Nucleotide sequences of either 223 or 250 base pairs were identified immediately 3' to regA in RB18 and RB51 that were absent in T-even phages. Open reading frames in these regions, designated orf43.1RB18 and orf43.1RB51, potentially encode related proteins of 8.5 and 9.2 kilodaltons, respectively. orf43.1 sequences, detected in 13 of 27 RB bacteriophage chromosomes analyzed by polymerase chain reaction, are either RB18- or RB51-like and have flanking repeat sequences that may promote orf43.1 deletion.     

Purification of the gene 43, 44, 45, and 62 proteins of the bacteriophage T4 DNA replication apparatus.

A procedure has been developed which allows the T4 bacteriophage proteins corresponding to the products of genes 43, 44, 45, and 62 to be purified to near homogeneity from a single T4-infected cell lysate (greater than 90% single species as judged by sodium dodecyl sulfate polyacrylamide elctrophoresis). In these preparations, the major problem of removing all contaminating nucleases has been overcome. Each of the above proteins is known from genetic analysis to be essential for phage DNA replication. The protein product of gene 43 is T4 DNA polymerase, and its recovery can be monitored using a standard DNA polymerase assay. The other three gene products have been designated as "polymerase accessory proteins," since they directly enhance polymerase function on both single- and double-stranded DNA templates. Their activities were monitored by an "in vitro complementation assay," which measures the stimulation of DNA synthesis observed in a concentrated lysate of T4 mutant-infected Escherichia coli cells when the missing T4 wild type protein is added. Starting from 300 g of infected cell paste, we obtained 9.3 mg of gene 43 protein, 21 mg of gene 45 protein, and 70 mg of a tight complex made up of 44 and 62 proteins; final yields were estimated at 30%, 14%, and 28%, respectively, of the initial activity present in the lysate. When the above purified proteins are incubated with preparations of two other T4 DNA replication proteins (gene 41 and gene 32 proteins) plus deoxyribonucleoside and ribonucleoside triphosphates, extensive DNA synthesis occurs on both single- and double-stranded DNA templates. As reported elsewhere, this synthesis mimicks that catalyzed by the T4 DNA replication apparatus in vivo.