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• W2076427794 abstract "The gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain is responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding whereas the N-terminal primase domain is responsible for template-directed oligoribonucleotide synthesis. A 26 amino acid linker region (residues 246-271) connects the two domains and is essential for the formation of functional hexamers. In order to further dissect the role of the linker region, three residues (Ala257, Pro259, and Asp263) that was disordered in the crystal structure of the hexameric helicase fragment were substituted with all amino acids, and the altered proteins were analyzed for their ability to support growth of T7 phage lacking gene 4. The in vivo screening revealed Ala257 and Asp263 to be essential whereas Pro259 could be replaced with any amino acid without loss of function. Selected gene 4 proteins with substitution for Ala257 or Asp263 were purified and examined for their ability to unwind DNA, hydrolyze dTTP, translocate on ssDNA, and oligomerize. In the presence of Mg2+, all of the altered proteins oligomerize. However, in the absence of divalent ion, alterations at position 257 increase the extent of oligomerization whereas those at position 263 reduce oligomer formation. Although dTTP hydrolysis activity is reduced only 2-3-fold, none of the altered gene 4 proteins can translocate effectively on single-strand DNA, and they cannot mediate the unwinding of duplex DNA. Primer synthesis catalyzed by the altered proteins is relatively normal on a short DNA template but it is severely impaired on longer templates where translocation is required. The results suggest that the linker region not only connects the two domains of the gene 4 protein and participates in oligomerization, but also contributes to helicase activity by mediating conformations within the functional hexamer. The gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain is responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding whereas the N-terminal primase domain is responsible for template-directed oligoribonucleotide synthesis. A 26 amino acid linker region (residues 246-271) connects the two domains and is essential for the formation of functional hexamers. In order to further dissect the role of the linker region, three residues (Ala257, Pro259, and Asp263) that was disordered in the crystal structure of the hexameric helicase fragment were substituted with all amino acids, and the altered proteins were analyzed for their ability to support growth of T7 phage lacking gene 4. The in vivo screening revealed Ala257 and Asp263 to be essential whereas Pro259 could be replaced with any amino acid without loss of function. Selected gene 4 proteins with substitution for Ala257 or Asp263 were purified and examined for their ability to unwind DNA, hydrolyze dTTP, translocate on ssDNA, and oligomerize. In the presence of Mg2+, all of the altered proteins oligomerize. However, in the absence of divalent ion, alterations at position 257 increase the extent of oligomerization whereas those at position 263 reduce oligomer formation. Although dTTP hydrolysis activity is reduced only 2-3-fold, none of the altered gene 4 proteins can translocate effectively on single-strand DNA, and they cannot mediate the unwinding of duplex DNA. Primer synthesis catalyzed by the altered proteins is relatively normal on a short DNA template but it is severely impaired on longer templates where translocation is required. The results suggest that the linker region not only connects the two domains of the gene 4 protein and participates in oligomerization, but also contributes to helicase activity by mediating conformations within the functional hexamer. The replisomes of essentially all replication systems have five essential components, a DNA polymerase, a processivity factor for the polymerase, a DNA helicase, a DNA primase, and a single-stranded DNA (ssDNA) 1The abbreviations used are: ssDNA, single-stranded DNA; pfu, plaque-forming unit.1The abbreviations used are: ssDNA, single-stranded DNA; pfu, plaque-forming unit.-binding protein (1Kornberg A. Baker T.A. DNA Replication. 2nd Ed. W. H. Freeman, New York1992Google Scholar). The replisome of bacteriophage T7 is unique in that the proteins that account for these five functions mediate coordinated leading and lagging strand DNA synthesis without the aid of other accessory proteins such as clamp-loading proteins and helicase-loading proteins (2Lee J. Chastain II, P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). All of these activities with the exception of the processivity factor are encoded by the phage. The economy of proteins in the phage T7 system is also manifest in the residence of the helicase and primase within a single polypeptide, the T7-encoded gene 4 protein (3Richardson C.C. Cell. 1983; 33: 315-317Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 4Dunn J.J. Studier F.W. J. Mol. Biol. 1983; 166: 477-535Crossref PubMed Scopus (971) Google Scholar). There is a requirement for an association of DNA primase with its cognate helicase in all replication systems, but the two proteins are usually encoded by distinct and separate genes, thus necessitating an association at the replication fork. As shown in Fig. 1, the helicase and primase activities of the gene 4 protein each reside in separate domains located in the C-terminal and N-terminal halves of the 63-kDa gene 4 protein, respectively (5Ilyina T.V. Gorbalenya A.E. Koonin E.V. J. Mol. Evol. 1992; 34: 351-357Crossref PubMed Scopus (170) Google Scholar). The DNA sequence encoding each domain has been cloned and the resulting helicase and primase fragments have full helicase and primase activities, respectively (6Bird L.E. Hakansson K. Pan H. Wigley D.B. Nucleic Acids Res. 1997; 25: 2620-2626Crossref PubMed Scopus (53) Google Scholar, 7Frick D.N. Baradaran K. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7957-7962Crossref PubMed Scopus (66) Google Scholar, 8Guo S. Tabor S. Richardson C.C. J. Biol. Chem. 1999; 274: 30303-30309Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). However, each domain, particularly the primase domain, is impaired in specific reactions when it is not coupled to the other domain. The crystal structures of both the helicase (9Sawaya M.R. Guo S. Tabor S. Richardson C.C. Ellenberger T. Cell. 1999; 99: 167-177Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 10Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar) and primase (11Kato M. Ito T. Wagner G. Richardson C.C. Ellenberger T. Mol. Cell. 2003; 11: 1349-1360Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) domains have been solved. Like other DNA helicases of the DnaB family, the gene 4 protein functions as a hexamer (12Patel S.S. Picha K.M. Annu. Rev. Biochem. 2000; 69: 651-697Crossref PubMed Scopus (455) Google Scholar). The hexameric gene 4 protein binds tightly to single-stranded DNA in the presence of dTTP and uses the energy of hydrolysis of dTTP to translocate unidirectionally 5′ to 3′ on the DNA strand to which it is bound (13Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 205-209Crossref PubMed Scopus (107) Google Scholar, 14Matson S.W. Richardson C.C. J. Biol. Chem. 1983; 258: 14009-14016Abstract Full Text PDF PubMed Google Scholar). Biochemical analysis of C-terminal helicase fragments of the gene 4 protein demonstrated that the region of the gene 4 protein linking the primase and helicase domains (residues 246-271) is critical for oligomerization (8Guo S. Tabor S. Richardson C.C. J. Biol. Chem. 1999; 274: 30303-30309Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The primase and helicase domains of gene 4 protein share extensive homology with primases and helicases from other organisms (5Ilyina T.V. Gorbalenya A.E. Koonin E.V. J. Mol. Evol. 1992; 34: 351-357Crossref PubMed Scopus (170) Google Scholar). However, this small portion of residues 246 through 271 does not share amino acid sequence with other members of either the helicase or primase families, and thus was defined as the linker region. Helicase fragments lacking this linker region failed to oligomerize except at extremely high protein concentration and were severely impaired for all helicase activities (8Guo S. Tabor S. Richardson C.C. J. Biol. Chem. 1999; 274: 30303-30309Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The crystal structure of the hexameric helicase fragment subsequently revealed that the subunit interface of the helicase ring is stabilized by interactions between an N-terminal region (residues 264-284 or helix A) of one subunit and a pocket on the adjacent subunit (residues 364-395 or helices D2 to D3) (10Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar) (Fig. 1). Thus, a portion of the linker region identified by biochemical studies (residues 246-271) is found in this interaction. However, residues 241-260 in the structure were poorly ordered. In addition to these interactions at the interface, a series of loops near the nucleotide binding site also make contact between adjacent subunits with potential contacts of adjacent subunits with the bound nucleotide. While it is clear from the biochemical and structural data that the linker region is essential for oligomerization of the gene 4 protein, the identity of the key residues critical for protein-protein interaction are unknown since previous studies have made use of truncated helicase domains. Furthermore, it is not known if the linker region via its contact with adjacent subunits assists in coordinating helicase and primase activities at the replication fork. One study (15Rosenberg A.H. Griffin M. Washington M.T. Patel S.S. Studier F.W. J. Biol. Chem. 1996; 271: 26819-26824Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) involving a random mutagenesis of the entire T7 gene 4 protein did find that substitutions of residues within this region such as A257T, A257V, and G258D give rise to proteins that cannot support T7 phage lacking gene 4. One of these altered proteins, a gene 4 protein with the substitution of threonine for alanine at position 257 (gp4-A257T), had reduced dTTPase activity and no DNA unwinding activity. In order to define more precisely the role of the linker, we have carried out an extensive study of three amino acid residues (Ala257, Pro259, and Asp263) all located in the linker region. Three libraries of gene 4 were generated such that each of the three amino acids was replaced with all other amino acids, and the resulting proteins were analyzed for their ability to support T7 phage lacking gene 4. We find that two of these residues, Ala257 and Asp263, play a critical role in oligomerization and translocation of the gene 4 protein. Materials—Oligonucleotides were obtained from Invitrogen. Plasmid DNA purification kits were from Qiagen. Restriction endonucleases, alkaline phosphatase, and Deep Vent® polymerase were purchased from New England Biolabs. T4 polynucleotide kinase, T4 DNA ligase, radiolabeled nucleotides, high molecular weight protein markers, and desalting column S-400HR were purchased from Amersham Biosciences. Agarose and β,γ-methylene dTTP were from USB Corp. Polyethyleneimine cellulose thin layer chromatography (TLC) plates were from J. T. Baker. T7 DNA polymerase (T7 gene 5 protein-Escherichia coli thioredoxin complex), M13mp18 ssDNA, and a primer used for strand displacement DNA synthesis assay were kindly provided by Donald Johnson (Harvard Medical School). Random Mutagenesis of Selected Codons—Random mutagenesis of the codons specifying Ala257, Pro259, and Asp263 in gene 4 protein was carried out using primers containing mixed nucleotides at these positions. The codon site was flanked by 10-12 nucleotides of the wild-type sequence. For example, a set of primers for Ala257 was 5′-CAAGTGTGGAATNNNGGTCCTTGGATT-3′ and 5′-AATCCAAGGACCNNNATTCCACACTTG-3′ (N, any base). Using these mutagenic primers and outside primers in a two-step overlap PCR procedure (16Lee S.J. Richardson C.C. J. Biol. Chem. 2001; 276: 49419-49426Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), a pool of DNA containing mutated codons was generated. After digestion with BstBI and Bsu36I, the DNA (0.62 pmol) was ligated into plasmid pET24gp4-63 (0.049 pmol) previously cut with the same restriction enzymes. One-third of the ligation reaction mixture was transformed into 0.1 ml of E. coli strain DH5α (2.6 × 108 cells) by heat shock at 42 °C for 1 min followed by the addition of 0.9 ml of SOC medium. The cells were incubated at 37 °C for 1 h, and 0.1 ml of the culture was spread onto agar plates layered with either T7Δ4 phage or no phage. After overnight growth, bacterial colonies that emerged from plates infected with T7Δ4 phage (1 × 107 pfu) were picked and re-streaked on a new plate. Inability of the isolated colonies to support T7Δ4 phage was confirmed by infecting separate bacterial cultures with the phage. Plasmid DNA from the surviving bacteria was prepared and the integrity of the gene 4 coding region near the substitution site was examined by restriction enzyme analysis with SnaBI and AflII. Finally, the DNA sequence of the gene 4 coding from Gly179 through Ser312 of the protein was determined by the DNA sequencing facility at The Dana-Farber/Harvard Cancer Center. Protein Overproduction and Purification—The entire gene 4 coding region of selected gene 4 proteins was confirmed by DNA sequence analysis. The plasmids were transformed into E. coli HMS 174(DE3), and gene 4 proteins were overproduced by isopropyl-1-thio-β-d-galactopyranoside induction. Genetically altered gene 4 proteins were purified following procedures described previously (16Lee S.J. Richardson C.C. J. Biol. Chem. 2001; 276: 49419-49426Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The purified proteins were all greater than 95% pure as determined by SDS-PAGE analysis and staining with Coomassie Blue. Biochemical Assays of Gene 4 Protein—Most of the assays used in this study have been described previously in detail (16Lee S.J. Richardson C.C. J. Biol. Chem. 2001; 276: 49419-49426Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). All assays (DNA unwinding, DNA binding, dTTP hydrolysis, oligomerization of gene 4 protein, primer synthesis, and DNA synthesis assays) used a reaction buffer containing 40 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 10 mm dithiothreitol, and 50 mm potassium glutamate plus additional components described in each assay. All reactions were carried out at 37 °C for the indicated time period. DNA Unwinding Assay—The DNA substrate for DNA unwinding assay was prepared by annealing a 5′-32P radiolabeled 45-mer (5′-ATAAC TCTAT GCACA TTGAC CATGC TTCAG ATTCG TATTG TTACT-3′) to a 65-mer (5′-TTTTT TTTTT TTTTT TTTTT ATTCG TAATC CGACC TCGAG GCATG GTCAA TGTGC ATAGA GTTAT-3′) in 50 mm NaCl. The DNA substrate (100 nm) was incubated with the indicated amounts of gene 4 protein for 5 min in the presence of 0.5 mm dTTP. After termination of the reaction by the addition of EDTA to a final concentration of 25 mm, the reaction mixture was loaded onto a 10% non-denaturing gel. Oligonucleotides separated from the partial duplex substrate by the helicase were measured using a Fuji BAS 1000 Bioimaging analyzer. In a control experiment in which 100 nm each of ssDNA 45- and 65-mer were incubated, we found that there is significant annealing (∼60%) of the DNA strands. Therefore, it is likely that some reannealing of the ssDNA strands arising during the helicase reaction occurs thus leading to an underestimation of DNA unwinding. However, lowering the DNA concentration 10-fold to reduce the annealing does not alter the results. DNA Binding Assay—DNA binding affinity of gene 4 protein was measured by nitrocellulose (NC) filter binding. The reaction (10 μl) containing 1.3 nm of 5′-32P-radiolabeled DNA (5′-GGGTCA10-3′) was incubated with various amounts of gene 4 protein in the presence of 1 mm non-hydrolyzable β,γ-methylene dTTP for 30 min. The reaction mixture was loaded onto two layers of filters, a NC membrane (0.45 μm, Bio-Rad) laid atop a Zeta-Probe® membrane (Bio-Rad) fixed on a Dot microfiltration apparatus (Bio-Rad). The protein-DNA complex bound to the NC membrane and free DNA on the Zeta-Probe® membrane was measured using a Fuji BAS 1000 Bioimaging analyzer. DNA-dependent Hydrolysis of dTTP—ssDNA-dependent hydrolysis of dTTP by gene 4 protein was determined by incubating 0.25 mm [α-32P]dTTP (0.1 μCi), 8 nm M13 ssDNA with the indicated concentration of the protein for 20 min. After termination of the reaction by the addition of EDTA to a final concentration of 25 mm, the reaction mixture was spotted onto a polyethyleneimine cellulose TLC plate. The TLC plate was developed with a solution containing 1 m formic acid and 0.8 m LiCl. The amount of [α-32P]dTDP formed in the reaction was measured using a Fuji BAS 1000 Bioimaging analyzer. Oligomerization of Gene 4 Proteins—The ability of gene 4 protein to oligomerize was determined by analyzing the oligomerized protein by electrophoresis on a non-denaturing polyacrylamide gel. The complete reaction (20 μl) contained 1 μm gene 4 protein, 1 mm β,γ-methylene dTTP, and 0.1 μm 45-mer oligonucleotide (5′-AGAGC GTCAC TCTTG TGACT ACCAG TGGTC GCAAA GTTCT TATCT-3′). After incubation for 20 min at 37 °C, the reaction mixture was loaded onto a 10% non-denaturing polyacrylamide gel and electrophoresed at 4 °C for 5 h. Gel running buffer (25 mm Tris-HCl, pH 7.0, 190 mm glycine) contained 10 mm Mg(OAc)2 or did not. The protein was stained with Coomassie Blue in order to visualize the oligomerized protein. Primase Assay—Template-directed oligoribonucleotide synthesis was determined by measuring the incorporation of [α-32P]CMP into oligoribonucleotides using a synthetic DNA template containing a primase recognition site. The reaction (10 μl) included the indicated template, 0.1 mm each of ATP and [α-32P]CTP (0.1 μCi), and the indicated amount of gene 4 protein. The DNA template was 100 μm 6-mer (5′-GGGTCA-3′) or 1 μm 65-mer used for DNA unwinding substrate. In the case of the 65-mer, the indicated amounts of dTTP or β,γ-methylene dTTP were added in the reaction. After incubation at 37 °C for 20 min, the reaction was terminated by the addition of 3 μl of sequencing dye and loaded onto a 25% denaturing polyacrylamide sequencing gel containing 3 m urea. Electrophoresis was carried out at 1800 V for 3 h, and the gel was dried. Radioactive oligoribonucleotide products were analyzed using a Fuji BAS 1000 Bioimaging analyzer. Strand Displacement DNA Synthesis by T7 DNA Polymerase—A DNA template used to measure DNA strand displacement DNA synthesis was prepared by annealing M13 ssDNA in 10 mm NaCl to a 5′-32P radiolabeled 66-mer primer (5′-T36AATTC GTAAT CATGG TCATA GCTGT TTCCT-3′) to create a non-complementary T36 tail at the 5′-end of a 30-bp duplex region. Excess primers in the annealing mixture were removed using a desalting column S-400HR. The reaction containing 5 nm template DNA, 0.3 mm of all four dNTPs, and 10 nm T7 DNA polymerase was initiated for 2 min before the indicated amounts of gene 4 protein were added. After incubation for 30 min, the reaction was terminated by the addition of EDTA to a final concentration of 20 mm and loaded on a 1% agarose gel containing 0.5 mg/ml ethidium bromide. The gel was run in a buffer (45 mm Tris borate, pH 8.3, 1 mm EDTA) and dried for autoradiography. RNA-primed DNA Synthesis by T7 DNA Polymerase—The ability of gene 4 protein to prime DNA synthesis catalyzed by T7 DNA polymerase on M13 ssDNA was measured by incubating 9.8 nm M13 ssDNA, 0.3 mm each of dTTP, dCTP, dATP, and [α-32P]dGTP (0.1 μCi), 0.1 mm each of ATP and CTP, 100 nm gene 4 protein, and 20 nm T7 DNA polymerase. After incubation for 5 min, the reaction was terminated by the addition of EDTA to a final concentration of 20 mm and spotted onto a DE-81 membrane (Whatman). The amount of radioactively synthesized DNA was determined by measuring the radioactive products retained on the membrane after washing the membrane three times with 10 ml of 0.3 m ammonium formate (pH 8.0). A short segment of 26 amino acids lacking homology to members of either the helicase or primase families has been termed the “linker region” (5Ilyina T.V. Gorbalenya A.E. Koonin E.V. J. Mol. Evol. 1992; 34: 351-357Crossref PubMed Scopus (170) Google Scholar). Biochemical data suggested that this linker region is critical for the functioning of the gene 4 protein (8Guo S. Tabor S. Richardson C.C. J. Biol. Chem. 1999; 274: 30303-30309Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). As anticipated from the biochemical data, crystal structures of the helicase domains revealed residues in the linker of one subunit of the hexamer contacting residues in the adjacent subunit, thus contributing to the stability of the hexamer (9Sawaya M.R. Guo S. Tabor S. Richardson C.C. Ellenberger T. Cell. 1999; 99: 167-177Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 10Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). Although the structure showed clearly the participation of the linker in subunit interaction, 9 of 26 residues were disordered in the structure. In order to examine the role of these 9 amino acids in helicase-primase function, we selected three for detailed analysis. Residues Pro259 and Asp263 were chosen since they are conserved in gene 4 protein of phage T3 where the primase is also fused to the helicase. Residue Ala257 was included in this study since alteration at this position had been shown previously to affect DNA-dependent dTTP hydrolysis and DNA unwinding (17Washington M.T. Rosenberg A.H. Griffin K. Studier F.W. Patel S.S. J. Biol. Chem. 1996; 271: 26825-26834Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The codons for each of the three selected amino acid residues were mutated randomly in gene 4 and cloned into a plasmid so as to give rise to all possible amino acid substitutions at these three sites (see “Experimental Procedure”). In order to select for altered gene 4 proteins that were defective in primase and/or helicase activity, we first selected for gene 4 proteins that could not support the growth of T7Δ4 lacking gene 4. In this selection, E. coli cells transformed with the pools of plasmids harboring the mutated gene 4 are plated onto plates previously layered with T7Δ4 phage. If the appropriate phage concentration is present on the plate, E. coli cells expressing gene 4 proteins that are defective in supporting T7Δ4 phage will survive. Cells that have functional gene 4 protein expressed will allow T7 phage to propagate, resulting in lysis of the colony. Since too high a concentration of phage can kill all of the cells lacking a functional gene 4 protein, it was first necessary to select an appropriate number of T7Δ4 phage to layer on each plate. As shown in Table I, infection with 1.3 × 108 pfu/plate resulted in lysis of ∼90% of cells regardless of the position mutated, suggesting that lysis of cells occurred even in the absence of a functional gene 4 protein. However, at 1.3 × 107 pfu/plate, a clear distinction in survival among the three libraries was apparent (42% for Ala257, 4% for Pro257, and 66% for Asp263). Since further reduction (1.3 × 106 pfu/plate) of infecting phage did not significantly change the number of surviving cells, we chose 1.3 × 107 pfu/plate for the screening procedure.Table ISelection of gene 4 clones that do not support growth of T7Δ4 The codons for each of the selected amino acids (Ala257, Pro259, and Asp263) were randomly mutated in a cloned gene 4 so as to give three libraries, each containing all possible amino acid replacements in the respective codons. After transformation of plasmids from the library into E. coli DH5α, bacterial cells were spread on plates containing the indicated number of T7Δ4. The numbers of colonies were determined by plating 1/10 of the transformation reactions.Position of random mutationAla257Pro259Asp263No phage7412365T7Δ4 (1.3 × 106 pfu)431046T7Δ4 (1.3 × 107 pfu)31 ± 35 ± 143 ± 2T7Δ4 (1.3 × 108 pfu)725 Open table in a new tab Using the screen described above, individual colonies that survived phage infection were isolated. Lack of ability to support T7Δ4 phage was confirmed by infecting cell cultures derived from individual colonies with T7Δ4. Plasmid DNAs from surviving colonies were purified and subjected to restriction enzyme analysis with SnaBI and AflII and only those that contained the intact coding segment for gene 4 were retained. Initially 151, 20, and 136 colonies survived from plating on the libraries of gene 4 with altered amino acids at Ala257, Pro259, and Asp263, respectively. After restriction enzyme analysis, 124 (Ala257), 8 (Pro259), and 105 (Asp263) plasmid DNAs remained. In order to identify the amino acid replacements that led to gene 4 proteins that could not support T7Δ4 phage growth, the sequence of each plasmid DNA selected in the screen was determined. The results of the DNA sequence analysis are presented in Table II where the amino acid substitutions are tabulated. The striking observation is that only 8 plasmids were identified in the Pro259 library, and none of these had single amino acid substitution at position 259; they consisted of stop codons, deletions, an insertion, or multiple amino acid replacements (Table II). In order to assure that amino acid substitutions were occurring at position 259, we isolated several colonies prior to phage infection and sequenced the DNA to find that Val, Gln, His, Cys, and Gly had replaced Pro. None of these substitutions change the ability to support T7Δ4 (data not shown). We conclude that Pro259 is not a critical amino acid for gene 4 function, because it can be replaced with any amino acid. Although it is difficult to make generalizations regarding the substitutions for Ala257 and Asp263, it does appear that aliphatic residues such as valine, leucine, and isoleucine cannot replace the alanine at position 257, suggesting that the size of the residue at this position is important. Arginine, leucine, valine, and proline were found at a relatively high frequency at position 257. This finding is not unexpected since these amino acids have multiple codons (6, 6, 4, and 4, respectively). Interestingly, serine and glycine, having 6 and 4 codons, respectively, were not substituted at position 257. Since the side chains of these residues are relatively small, it appears likely that the size of the residue at position 257 is critical. Similarly, amino acids coded by multiple codons (alanine, arginine, and proline) were abundant at position 263. In this position, it is noteworthy that asparagine, lacking a negative charge, cannot substitute for aspartic acid. Some amino acid substitutions were not observed presumably because the numbers in the screen are not sufficiently large to be statistically significant.Table IINon-viable amino acid substitutions in linker region of gene 4 protein Plasmids encoding genetically altered gene 4 proteins at residues Ala257, Pro259, and Asp263 that could not support T7Δ4 phage were analyzed by DNA sequence analysis. The DNA sequence encoding the gene 4 sequence from Gly179 to Ser312 was determined, and the identity of the amino acid replacement at positions 257, 259, and 263 was deduced from the codon for those residues. The amino acids identified are listed in decreasing order of their frequency.Position of random mutationAla257Pro259Asp263ReplacementOccurrenceReplacementOccurrenceReplacementOccurrenceArg24Stop2Ala25Val21Deletion2Arg18Leu19Insertion1Pro16Pro18Cys8Glu11Tyr0Thr6Asp4Trp0Ser6Gln4Val0Val5Trp4Thr0His4Stop4Ser0Leu4Deletion4Arg0Met3Ile3Gln0Gln3Thr3Asn0Phe2His2Met0Deletion1Tyr1Leu0Lys1Ile0Asn1Cys0His0Phe0Gly0Glu0Gly0Phe0Stop0Lys0Glu0Ile0Met0Asp0Trp0Asn0Cys0Gly0Ser0Ala0Tyr0Multiple mutations2Multiple mutations3Multiple mutations2Total124Total8Total105 Open table in a new tab From the data in Table II, we identified the least drastic amino acid substitutions at positions 257 and 263. We chose three gene 4 proteins with the substitutions of leucine, proline, and valine for Ala257 and three with the substitutions of alanine, asparagine, and serine for Asp263. Prior to purification, the ability of these altered gene 4 proteins were examined for their ability to support the growth of T7Δ4 phage and for their effect on the growth of wild-type T7 phage (Table III). None of the altered proteins with single amino acid changes at positions 257 and 263 could support the growth of T7Δ4 phage, and none of the proteins demonstrated a dominant negative effect on the growth of wild-type T7 phage.Table IIIAbility of recombinant gene 4 protein to complement growth of T7 phage Gene 4 proteins containing the indicated amino acid substitution were expressed in E. coli DH5α. After infection with either T7Δ4 or wild-type T7 phage, the number of plaques were counted and normalized to the value obtained with the wild-type gene 4 protein.Alteration in gene 4 proteinT7 phageΔ4WTaWild-typeWT1.0bRelative efficiency of plating1.0A257L1.5 × 10-80.39A257P1.5 × 10-80.45A257V5.0 × 10-80.42D263A< 10-90.4" @default.
• W2076427794 created "2016-06-24" @default.
• W2076427794 creator A5011038929 @default.
• W2076427794 creator A5082077458 @default.
• W2076427794 date "2004-05-01" @default.
• W2076427794 modified "2023-09-28" @default.
• W2076427794 title "The Linker Region between the Helicase and Primase Domains of the Gene 4 Protein of Bacteriophage T7" @default.
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• W2076427794 doi "https://doi.org/10.1074/jbc.m400857200" @default.
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