The bacteriophage T7 gene 4 protein, like a number of helicases, is believed to function as a hexamer. The amino acid sequence of the T7 gene 4 protein from residue 475 to 491 is conserved in the homologous proteins of the related phages T3 and SP6. In addition, part of this region is conserved in DNA helicases such as Escherichia coli DnaB protein and phage T4 gp41. Mutations within this region of the T7 gene 4 protein can reduce the ability of the protein to form hexamers. The His475-->Ala and Asp485-->Gly mutant proteins show decreases in nucleotide hydrolysis, single-stranded DNA binding, double-stranded DNA unwinding, and primer synthesis in proportion to their ability to form hexamers. The mutation Arg487-->Ala has little effect on oligomerization, but nucleotide hydrolysis by this mutant protein is inhibited by single-stranded DNA, and it has a higher affinity for dTTP, suggesting that this protein is defective in the protein-protein interactions required for efficient nucleotide hydrolysis and translocation on single-stranded DNA. Gene 4 protein can form hexamers in the absence of a nucleotide, but dTTP increases hexamer formation, as does dTDP, to a lesser extent, demonstrating that the protein self-association affinity is influenced by the nucleotide bound. Together, the data demonstrate that this region of the gene 4 protein is important for the protein-protein contacts necessary for both hexamer formation and the interactions between the subunits of the hexamer required for coordinated nucleotide hydrolysis, translocation on single-stranded DNA, and unwinding of double-stranded DNA. The fact that the gene 4 proteins form dimers, but not monomers, even while hexamer formation is severely diminished by some of the mutations, suggests that the proteins associate in a manner with two separate and distinct protein-protein interfaces.

The gene 4 proteins of bacteriophage T7 provide both primase and helicase activities at the replication fork. Efficient DNA replication requires that the functions of the gene 4 protein be coordinated with the movement of the T7 DNA polymerase. We show that a carboxyl-terminal domain of the gene 4 protein is required for interaction with T7 DNA polymerase during leading strand DNA synthesis. The carboxyl terminus of the gene 4 protein is highly acidic: of the 17 carboxyl-terminal amino acids 7 are negatively charged. Deletion of the coding region for these 17 residues results in a gene 4 protein that cannot support the growth of T7 phage. The purified mutant gene 4 protein has wild-type levels of both helicase and primase activities; however, DNA synthesis catalyzed by T7 DNA polymerase on a duplex DNA substrate is stimulated by this mutant protein to only about 5% of the level of synthesis obtained with wildtype protein. The mutant gene 4 protein can form hexamers and bind single-stranded DNA, but as determined by native PAGE analysis, the protein cannot form a stable complex with the DNA polymerase. The mutant gene 4 protein can prime DNA synthesis normally, indicating that for lagging strand synthesis a different set of helicase/primase-DNA polymerase interactions are involved. These findings have implications for the mechanisms coupling leading and lagging strand DNA synthesis at the T7 replication fork.

The helicase and primase activities of bacteriophage T7 are distributed between the 56-and 63-kDa gene 4 proteins. The 56-kDa gene 4 protein lacks 63 amino acids found at the N terminus of the colinear 63-kDa protein and catalyzes helicase activity. The 63-kDa gene 4 protein catalyzes both primase and helicase activities. A bacteriophage deleted for gene 4, T7 A4-1, has been tested for growth by complementation on Escherichia coli strains that contain plasmids expressing either one or both of the gene 4 proteins. 17 A4-1 cannot grow (efficiency of plating, 10-7) on E. coil cells that express only 56-kDa gene 4 protein. In contrast, 17 A4-1 has an efficiency of plating of 0.1 on an E. coil strain that expresses only 63-kDa gene 4 protein in which glycine is substituted for methionine at position 64. A bacteriophage, T7 4B-, in which methionine at residue 64 is replaced by glycine, expresses only 63-kDa gene 4 protein. The burst sizes, latency periods, and Okazaki fragment sizes of 17 4B-are similar in the presence and absence of the 56-kDa gene 4 protein; however, 17 4B-has a reduced rate of DNA synthesis when compared with a phage that synthesizes both gene 4 proteins.

We analyzed protein expression from a cloned Mycoplasma hyorhinis genomic fragment that produces in Escherichia coli a set of related polypeptides of 110, 100, 65, and 55 kilodaltons from a coding region of just over 3.0 kilobases. Expression of these multiple products resulted from a mechanism operatng at the translational level but not from tun tion at UGA termination codons, which are known to encode tryptophan in several mycoplasma species. The structural relatedness of the proteins was demonstrated by two-diensional tryptic peptic mapping, but their generation by posttranslational processing was ruled out by puls-chase labeling analysis. Emiaon of proteins expressed from plasmid constructs and tryptic peptide analysis of these polypeptides and the original set of proteins revealed that they share carboxy-terminal regios, an observation inconsistent with runcation at UGA codons. Expression of proteins from this cloned fragment was not dependent on vector sequences and was observed when the coding region was placed under control of a T7 promoter, suggesting that all products were translated from a single message. Expression of related products in mycoplasmas was examined by immunoblot analysis of M. hyorhinis proteins with antiserum against overexpressed recombinant proteins. A single 115-klodalton mycoplasma protein was detected, which is larger than any of the related proteins expressed in E. coli. Our analysis indicated that translation initiation sites are used in E. coli that are not active in mycoplasmas, thereby defining differences between the translational regulatory signals of mycoplasmas and eubacteria.

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