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The Bacteriophage T4 Transcription Activator MotA Interacts with the Far-C-Terminal Region of the σ70Subunit ofEscherichia coliRNA Polymerase
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| Zeitschriftentitel: | Journal of Bacteriology |
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| Personen und Körperschaften: | , , , , , |
| In: | Journal of Bacteriology, 184, 2002, 14, S. 3957-3964 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
American Society for Microbiology
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| Schlagwörter: |
| Zusammenfassung: | <jats:title>ABSTRACT</jats:title><jats:p>Transcription from bacteriophage T4 middle promoters uses<jats:italic>Escherichia coli</jats:italic>RNA polymerase together with the T4 transcriptional activator MotA and the T4 coactivator AsiA. AsiA binds tightly within the C-terminal portion of the σ<jats:sup>70</jats:sup>subunit of RNA polymerase, while MotA binds to the 9-bp MotA box motif, which is centered at −30, and also interacts with σ<jats:sup>70</jats:sup>. We show here that the N-terminal half of MotA (MotA<jats:sup>NTD</jats:sup>), which is thought to include the activation domain, interacts with the C-terminal region of σ<jats:sup>70</jats:sup>in an<jats:italic>E. coli</jats:italic>two-hybrid assay. Replacement of the C-terminal 17 residues of σ<jats:sup>70</jats:sup>with comparable σ<jats:sup>38</jats:sup>residues abolishes the interaction with MotA<jats:sup>NTD</jats:sup>in this assay, as does the introduction of the amino acid substitution R608C. Furthermore, in vitro transcription experiments indicate that a polymerase reconstituted with a σ<jats:sup>70</jats:sup>that lacks C-terminal amino acids 604 to 613 or 608 to 613 is defective for MotA-dependent activation. We also show that a proteolyzed fragment of MotA that contains the C-terminal half (MotA<jats:sup>CTD</jats:sup>) binds DNA with a<jats:italic>K</jats:italic><jats:sub>D(app)</jats:sub>that is similar to that of full-length MotA. Our results support a model for MotA-dependent activation in which protein-protein contact between DNA-bound MotA and the far-C-terminal region of σ<jats:sup>70</jats:sup>helps to substitute functionally for an interaction between σ<jats:sup>70</jats:sup>and a promoter −35 element.</jats:p> |
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| Umfang: | 3957-3964 |
| ISSN: |
0021-9193
1098-5530 |
| DOI: | 10.1128/jb.184.14.3957-3964.2002 |