Assembly of the Bacteriophage T4 Replication Machine Requires the Acidic Carboxy Terminus of Gene 32 Protein
Identifieur interne : 004685 ( Main/Exploration ); précédent : 004684; suivant : 004686Assembly of the Bacteriophage T4 Replication Machine Requires the Acidic Carboxy Terminus of Gene 32 Protein
Auteurs : J. Michael Hurley [États-Unis] ; Stephen A. Chervitz [États-Unis] ; Thale C. Jarvis [États-Unis] ; Britta S. Singer [États-Unis] ; Larry Gold [États-Unis]Source :
- Journal of Molecular Biology [ 0022-2836 ] ; 1993.
English descriptors
Abstract
Abstract: The acidic carboxy-terminal 89-amino acid fragment of bacteriophage T4 gene 32 protein was expressed in Escherichia coli to high levels from an inducible plasmid construct. Infection of induced cells by wild-type T4 phage results in impaired phage DNA synthesis. The time at which DNA synthesis begins and the diminution in DNA synthesis rates correlate with the amount of carboxy-terminal peptide that accumulates intracellularly prior to infection. Correspondingly, when induced cells are induced cells are infected with viable phage containing a small deletion near the carboxy-terminus of 32 protein (ΔPR201), the inhibition of phage DNA synthesis was much more severe. The mutant 32 protein competes less well against overproduced wild-type acid peptide than does wild-type 32 protein. The purified acid peptide, when used as the attached ligand for affinity chromatography, binds several T4 proteins from phage-infected cells, including 43 protein (T4 DNA polymerase), Dda protein (a DNA helicase), and UvsX protein (a Rec-like recombination protein). Furthermore, at 50- to 100-fold molar excess of acid peptide over intact 32 protein, phage DNA synthesis was specifically inhibited at the initiation step in an in vitro 5-protein DNA replication experiment. We propose that one or more phage replication proteins are titrated as non-productive protein-protein complexes at a site away from the DNA template. This implies that the carboxy-terminal domain of 32 protein is involved in an obligate step of replication machine assembly when the protein is properly attached to ssDNA in the vicinity of a primer-template junction. The assembly defect we observe is strikingly similar to the repression, or "squelching", of the activity of certain eukaryotic transcriptional activators.
Url:
DOI: 10.1006/jmbi.1993.1042
Affiliations:
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Jarvis</name><affiliation wicri:level="1"><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Molecular, Cellular and Developmental Biology University of Colorado, Boulder, CO 80309</wicri:regionArea><wicri:noRegion>CO 80309</wicri:noRegion></affiliation></author><author><name sortKey="Singer, Britta S" sort="Singer, Britta S" uniqKey="Singer B" first="Britta S." last="Singer">Britta S. 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Infection of induced cells by wild-type T4 phage results in impaired phage DNA synthesis. The time at which DNA synthesis begins and the diminution in DNA synthesis rates correlate with the amount of carboxy-terminal peptide that accumulates intracellularly prior to infection. Correspondingly, when induced cells are induced cells are infected with viable phage containing a small deletion near the carboxy-terminus of 32 protein (ΔPR201), the inhibition of phage DNA synthesis was much more severe. The mutant 32 protein competes less well against overproduced wild-type acid peptide than does wild-type 32 protein. The purified acid peptide, when used as the attached ligand for affinity chromatography, binds several T4 proteins from phage-infected cells, including 43 protein (T4 DNA polymerase), Dda protein (a DNA helicase), and UvsX protein (a Rec-like recombination protein). Furthermore, at 50- to 100-fold molar excess of acid peptide over intact 32 protein, phage DNA synthesis was specifically inhibited at the initiation step in an in vitro 5-protein DNA replication experiment. We propose that one or more phage replication proteins are titrated as non-productive protein-protein complexes at a site away from the DNA template. This implies that the carboxy-terminal domain of 32 protein is involved in an obligate step of replication machine assembly when the protein is properly attached to ssDNA in the vicinity of a primer-template junction. The assembly defect we observe is strikingly similar to the repression, or "squelching", of the activity of certain eukaryotic transcriptional activators.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country></list><tree><country name="États-Unis"><noRegion><name sortKey="Hurley, J Michael" sort="Hurley, J Michael" uniqKey="Hurley J" first="J. Michael" last="Hurley">J. Michael Hurley</name></noRegion><name sortKey="Chervitz, Stephen A" sort="Chervitz, Stephen A" uniqKey="Chervitz S" first="Stephen A." last="Chervitz">Stephen A. Chervitz</name><name sortKey="Gold, Larry" sort="Gold, Larry" uniqKey="Gold L" first="Larry" last="Gold">Larry Gold</name><name sortKey="Jarvis, Thale C" sort="Jarvis, Thale C" uniqKey="Jarvis T" first="Thale C." last="Jarvis">Thale C. Jarvis</name><name sortKey="Singer, Britta S" sort="Singer, Britta S" uniqKey="Singer B" first="Britta S." last="Singer">Britta S. Singer</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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