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At least 19 recordsLinked to original sources
PubMed1985

Genetic analysis of subunit assembly of the tail fiber of bacteriophage T3.

Bacteriophage T3 virions have six tail fibers composed of the product of gene 17 (gp17). Each tail fiber is a trimer of gp17 polypeptide. To characterize the assembly process of the tail fiber, temperature-sensitive (ts) mutants of gene 17 (ts17) were analyzed by SDS-polyacrylamide gel electrophoresis and by extract complementation. Newly synthesized gp17 polypeptide chains matured to SDS-resistant native trimers with a half time of about 7.5 min at 30 degrees. Although all ts17 mutants had similar plating efficiencies at restrictive temperature (41.5 degrees or 42 degrees), they showed different phenotypes. tsNG75, whose mutation was located in the carboxyl-terminal region of gene 17, was defective in trimer assembly at 41.5 degrees. The ts tail fibers formed at 30 degrees lost the ability to attach to the tail upon treatment at 41.5 degrees. There was a change in temperature sensitivity of tsNG75 tail fibers upon attachment to the tail, suggesting that the tail fiber may change conformation after attachment to the tail. tsNG215 and tsNG169, whose mutation sites were located in the amino-terminal region of gene 17, were not defective in the trimer assembly and attachment to the tail at the restrictive temperature. tsNG215 tail fibers formed at 41.5 degrees appear to be aberrant because they were not active in extract complementation and their attachment to fiberless particles resulted in production of noninfectious phage. Tail fibers produced by cells infected with tsNG169 at the restrictive temperature were active in extract complementation. Phage particles were formed in tsNG169-infected cells at the restrictive temperature. These particles were infectious at the permissive temperature and the mutant was non-infectious only if infection was continued at the restrictive temperature. These phenotypic differences exhibited by different gene 17 mutants may indicate the regions within the gene 17 polypeptide that play a role(s) in the folding and assembly of gp17 and in the biological activity of the mature tail fiber.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

Substitution of a single bacteriophage T3 residue in bacteriophage T7 RNA polymerase at position 748 results in a switch in promoter specificity.

The bacteriophage T3 and T7 RNA polymerases (RNAP) are closely related, yet exhibit high specificity for their own promoter sequences. In this work the primary determinant of T7 versus T3 promoter specificity has been localized to a single amino acid residue at position 748 in the T7 RNAP. Substitution of this residue (Asn) with the corresponding residue found in T3 RNAP (Asp) results in a switch in promoter specificity, and specifically alters recognition of the base pairs (bp) at positions -11 and, possibly, -10 in the promoter. A complementary mutation in T3 RNAP (T3-D749N) results in a similar switch in promoter preference for that enzyme. The hierarchy of bp preference by the mutant and wild-type enzymes for bp at -10 and -11, and the results of previous experiments, lead to a model for specificity in which it is proposed that N748 in T7 RNAP (and D749 in T3 RNAP) make specific hydrogen bonds with bases at -11 and -10 on the non-template strand in the major groove. The specificity determining region of T7 RNAP does not appear to exhibit homology to any known sequence-dependent DNA binding motif.

Amino Acid Sequence↗
PubMed1989

Relative efficiency of utilization of promoter and termination sites by bacteriophage T3 RNA polymerase.

Bacteriophage T3 RNA polymerase promoters have been classified as class II and class III on the basis of their relative location in T3 DNA as well as on the function of the protein products encoded by the messages transcribed from them. In the present work, the efficiency of utilization of several class II and class III promoters by bacteriophage T3 RNA polymerase was compared with regard to (a) rate of initiation of transcription as determined by [32P]PPi exchange with GTP; (b) complex formation between polymerase and promoters in the presence of GTP; and (c) competition between different promoters for T3 RNA polymerase in a standard transcription assay. The results of these experiments indicated that the class II promoters at 1.05 and 22.8 T3 map units, whose promoter sequences are remarkably similar to the consensus class III promoter sequences, are nearly as strong as typical class III promoters. In contrast, the class II promoter at 14.3 T3 map units, whose promoter sequence differs from the consensus class III promoter sequence by having a C:G base pair instead of a usual A:T base pair at the -1 position, was considerably weaker than the class III promoter. When the C:G base pair at this position was changed to A:T using site-directed mutagenesis, the rate of initiation of RNA synthesis from the mutant promoter was similar to that of a typical class III promoter. In agreement with this observation, it was observed that changing the A:T base pair at the -1 position of a strong class II promoter, at 1.05 T3 map units, to C:G decreased the rate of RNA synthesis from this promoter by about 65%. These observations indicate that the nucleotide residues at the -1 position play a critical role in determining the efficiency of promoter utilization by T3 RNA polymerase. The two termination sites recognized in vitro by bacteriophage T3 RNA polymerase on the T3 genome have been cloned, sequenced, and mapped. Analysis of the DNA nucleotide sequence surrounding the termination site at 59.7 map units indicated that the putative RNA transcript arising from this region can be arranged into a GC-rich stem-loop structure followed by a U-rich 3' tail. However, a major fraction of T3 RNA polymerase molecules read through this terminator in vitro to transcribe regions of T3 DNA beyond this terminator. In contrast to termination at 59.7 map units, termination of transcription at 100 T3 map units does not occur in response to any putative terminator structure or sequence.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1988

Packaging and transduction of non-T3 DNA by bacteriophage T3.

A defined in vitro system for packaging T3 DNA also packaged other linear DNAs, including T4, lambda, and plasmid DNAs. The packaging capacity was determined to be 40 kb (kilobase pairs) by measuring the packaged length of T4 DNA. Packaged lambda and plasmid DNAs were injected into host cells to form plaques and transductants, respectively. The yield of transducers increased by using artificially ligated plasmid oligomers. The T3 mutant in gene 3 endonuclease (T3 3-) packaged plasmid DNA during abortive infection and transduced it into the recipient. Transduction of recombinant plasmids was not affected by the presence of the terminally redundant sequence (TR sequence) but increased by 4 orders of magnitudes when the genetic right-end 2.7-kb sequences, containing gene 19 (E1) but lacking TR, were present and by 7 orders when both E1 and TR sequences were present. However, these sequences did not increase transduction of these plasmids by T7 3-. Analysis of the structure of transduced plasmid DNAs indicates that transducing particles carry head-to-tail oligomers of plasmid DNA with the same termini as those of T3 genomic DNA. The mechanism of formation of transducing particles is discussed.

Bacteriophage lambda↗
PubMed1989

Terminator-distal sequences determine the in vitro efficiency of the early terminators of bacteriophages T3 and T7.

Bacteriophages T3 and T7 contain homologous terminators for Escherichia coli RNA polymerase that restrict early phage transcription to the leftmost 20% of the linear phage genomes. These two terminators serve equally well as p-independent terminators in vivo, but their in vitro efficiencies and sensitivity to salt and nucleotide concentrations differ dramatically. Sequence analysis shows that the T7 and T3 terminators differ at only two sites in the region normally accepted as defining terminator function. In order to determine which structural features of these two terminators are responsible for their functional differences, a series of hybrid terminators were constructed in which structural features of the two terminators were systematically interchanged. Transcription of hybrid terminator templates revealed that sequences downstream of the termination release sites are responsible for the differences in efficiency of in vitro termination. These sequences also determine the sensitivity of these terminators to elevated salt concentrations and to alterations of substrate concentrations. Alteration of the sequences in the region between three and seven nucleotides downstream of the final T7Te release site is sufficient to reduce termination efficiency to that of T3Te, and point mutations in this region yield terminators with intermediate efficiency. Hence, the determinants of p-independent terminator efficiency in vitro must include elements of the transcription complex other than the structure of the 3' end of the transcript. The termination differences between T7Te, T3Te, and their hybrid derivatives are overcome in vivo; all of these sites become very efficient. This finding further supports the hypothesis that protein factors or other cellular features enhance the efficiency and specificity of p-independent terminators in vivo.

DNA↗
PubMed2004

Characterization of bacteriophage T3 DNA ligase.

DNA ligases of bacteriophage T4 and T7 have been widely used in molecular biology for decades, but little is known about bacteriophage T3 DNA ligase. Here is the first report on the cloning, expression and biochemical characterization of bacteriophage T3 DNA ligase. The polyhistidine-tagged recombinant T3 DNA ligase was shown to be an ATP-dependent enzyme. The enzymatic activity was not affected by high concentration of monovalent cations up to 1 M, whereas 2 mM ATP could inhibit its activity by 50%. Under optimal conditions (pH 8.0, 0.5 mM ATP, 5 mM DTT, 1 mM Mg(2+) and 300 mM Na(+)), 1 fmol of T3 DNA ligase could achieve 90% ligation of 450 fmol of cohesive dsDNA fragments in 30 min. T3 DNA ligase was shown to be over 5-fold more efficient than T4 DNA ligase for ligation of cohesive DNA fragments, but less active for blunt-ended DNA fragments. Phylogenetic analysis showed that T3 DNA ligase is more closely related to T7 DNA ligase than to T4 DNA ligase.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence and complementation studies of the gene 10 region of bacteriophage T3.

The nucleotide sequence of bacteriophage T3 gene 10 and surrounding regulatory elements has been determined and compared to the analogous region of bacteriophage T7. T3 genes 9, 10 and 11 have been shown to complement T7 mutants. The DNA sequences of T3 and T7 gene 10A are homologous, as are the amino acid sequences of the respective products. The translational shift to the -1 frame is predicted to occur at the same position in gene 10 of T3 and T7, though different nucleotide sequences are probably responsible. The resulting gp10B products have completely different C termini.

Base Sequence↗
PubMed1980

Derivation of a restriction map of bacteriophage T3 DNA and comparison with the map of bacteriophage T7 DNA.

The DNA of bacteriophage T3 was characterized by cleavage with seven restriction endonucleases. AvaI, XbaI, BglII, and HindIII each cut T3 DNA at 1 site, KpnI cleaved it at 2 sites, MboI cleaved it at 9 sites, and HpaI cleaved it at 17 sites. The sizes of the fragments produced by digestion with these enzymes were determined by using restriction fragments of T7 DNA as molecular weight standards. As a result of this analysis, the size of T3 DNA was estimated to be 38.74 kilobases. The fragments were ordered with respect to each other and to the genetic map to produce a restriction map of T3 DNA. The location and occurrence of the restriction sites in T3 DNA are compared with those in the DNA of the closely related bacteriophage T7.

Base Sequence↗
PubMed1985

Purification and characterization of gene 17 product of bacteriophage T3.

Tail fiber proteins of bacteriophage T3 are encoded by gene 17. By using in vitro complementation for phage assembly as an assay, the product of gene 17 (gp17) was purified to near homogeneity from cells infected with a double mutant of T3 defective in DNA synthesis and head assembly. The purified gp17 consists of a single polypeptide having a molecular weight of 67,000. Electron microscopy of the purified gp17 showed a fiber structure similar to the tail fiber in a virion. The subunit structure of the purified, native gp17 was analyzed by using a crosslinking agent, dimethyl suberimidate. The results indicate that native gp17 is a trimer of gp17 monomer.

Macromolecular Substances↗
PubMed1978

[Effect of temperature on formation of lysozyme in E. coli CRT 266 (dnaB ts) after infection with bacteriophage T3].

Lysozyme formation induced by bacteriophage T3 was studied in the ts-mutant E. coli CRT 266 (dnaBts) and in the wild-type E. coli CR 34--45 (dnaB+) at different temperatures. It was found that lysozyme was formed in E. coli CRT 266, however, no lysozyme synthesis took place at 41.5 degrees C. These results indicate that the expression of the lysozyme gene is disturbed in the ts-mutant at 41.5 degrees C.

Coliphages↗
PubMed1976

Bacteriophage T3 and T7 early RNAs are translated by eukaryotic 80S ribosomes: active phage T3 coded S-adenosylmethionine cleaving enzyme is synthesized.

RNA transcribed in vitro from the early region of bacteriophage T3 or T7 was translated by cytoplasmic ribosomes which synthesized protein in cell-free systems prepared from mammalian cells and wheat germ. The proteins synthesized in vitro and their counterparts prepared from infected Escherichia coli comigrate by polyacrylamide gel electrophoresis with sodium dodecyl sulfate and are similarly affected by deletion or amber bacteriophage mutations. Bacteriophage T3 codes for an enzyme that cleaves S-adenosylmethionine and this activity was detected among the products of the mammalian cell-free system. Bacteriophage T3 or T7 RNA, after endoribonuclease III (EC 3.1.4.24) cleavage, gave higher levels of incorporation into phage T3 or T7 polypeptides than when an equivalent amount of the uncleaved RNA was added to the eukaryotic cell-free systems. Methylation of phage T3 or T7 RNAs is apparently not required for translation in either the wheat germ or mammalian cell-free system. The ability of T3 and T7 RNA to be translated in the presence of saturating amounts of natural eukaryotic mRNAs suggests that many prokaryotic genes introduced into mammalian cells might be expressed if they were transcribed in an appropriate form.

Cell-Free System↗