A Family of Anti-σ⁷⁰ Proteins in T4-type Phages and Bacteria that are Similar to AsiA, a Transcription Inhibitor and Co-activator of Bacteriophage T4

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• 作者：Pineda ; Melissa ; Gregory ; Brian D. ; Szczypinski ; Bridget ; Baxter ; Kimberly R. ; Hochschild ; Ann ; et. al.
• 关键词：phage ; AsiA ; Rsd ; AlgQ ; transcription ; PCR ; polymerase chain reaction ; ITPG ; isopropyl-beta-d-thiogalactopyranoside
• 刊名：Journal of Molecular Biology
• 出版年：2004
• 出版时间：December 10, 2004
• 年：2004
• 卷：344
• 期：5
• 页码：1183-1197
• 全文大小：643 K

文摘

Anti-σ⁷⁰ factors interact with σ⁷⁰ proteins, the specificity subunits of prokaryotic RNA polymerase. The bacteriophage T4 anti-σ⁷⁰ protein, AsiA, binds tightly to regions 4.1 and 4.2 of the σ⁷⁰ subunit of Escherichia coli RNA polymerase and inhibits transcription from σ⁷⁰ promoters that require recognition of the canonical σ⁷⁰ −35 DNA sequence. In the presence of the T4 transcription activator MotA, AsiA also functions as a co-activator of transcription from T4 middle promoters, which retain the canonical σ⁷⁰ −10 consensus sequence but have a MotA box sequence centered at −30 rather than the σ⁷⁰ −35 sequence. The E.coli anti-σ⁷⁰ protein Rsd also interacts with region 4.2 of σ⁷⁰ and inhibits transcription from σ⁷⁰ promoters. Our sequence comparisons of T4 AsiA with Rsd, with the predicted AsiA orthologs of the T4-type phages RB69, 44RR, KVP40, and Aeh1, and with AlgQ, a regulator of alginate production in Pseudomonas aeruginosa indicate that these proteins share conserved amino acid residues at positions known to be important for the binding of T4 AsiA to σ⁷⁰ region 4. We show that, like T4 AsiA, Rsd binds to σ⁷⁰ in a native protein gel and, as with T4 AsiA, a L18S substitution in Rsd disrupts this complex. Previous work has assigned σ⁷⁰ amino acid F563, within region 4.1, as a critical determinant for AsiA binding. This residue is also involved in the binding of σ⁷⁰ to the β-flap of core, suggesting that AsiA inhibits transcription by disrupting the interaction between σ⁷⁰ region 4.1 and the β-flap. We find that as with T4 AsiA, the interaction of KVP40 AsiA, Rsd, or AlgQ with σ⁷⁰ region 4 is diminished by the substitution F563Y. We also demonstrate that like T4 AsiA and Rsd, KVP40 AsiA inhibits transcription from σ⁷⁰-dependent promoters. We speculate that the phage AsiA orthologs, Rsd, and AlgQ are members of a related family in T4-type phage and bacteria, which interact similarly with primary σ factors. In addition, we show that even though a clear MotA ortholog has not been identified in the KVP40 genome and the phage genome appears to lack typical middle promoter sequences, KVP40 AsiA activates transcription from T4 middle promoters in the presence of T4 MotA. We speculate that KVP40 encodes a protein that is dissimilar in sequence, but functionally equivalent, to T4 MotA.