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N- and C-terminal regions of the quorum-sensing activator TraR cooperate in interactions with the alpha and sigma-70 components of RNA polymerase.

ABSTRACT: Positive control (PC) mutants defining 20 residues of the quorum-sensing activator TraR were isolated that bind DNA but show defects in activating transcription from class I, class II or both types of promoters. These PC residues, located in both the N- and C-terminal regions, combine to form three patches, one on the top (II) and two near the DNA binding domain on both lateral faces of the dimer (I and III). Patches I and II, but not patch III, involve residues from both protomers and are essential for activation. TraR-mediated activation in Escherichia coli requires expression of the alpha-subunit of Agrobacterium (alpha(At)). We report that TraR also activates a class II promoter in E. coli when coexpressed with sigma(70)(At). Analyses in E. coli expressing alpha(At), sigma(70)(At) or both subunits indicate that most of the PC residues are important for interactions with alpha(At) and that these interactions are predominant for activation of class II promoters. Using the E. coli system we identified nine residues in the C-terminal domain of alpha(At) that are required for stimulating TraR-mediated activation. We conclude that N- and C-terminal residues of TraR from both protomers cooperate to define regions of the protein important for interactions with RNAP.

SUBMITTER: Qin Y

PROVIDER: S-EPMC2765545 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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N- and C-terminal regions of the quorum-sensing activator TraR cooperate in interactions with the alpha and sigma-70 components of RNA polymerase.

Qin Yinping Y Keenan Carrie C Farrand Stephen K SK

Molecular microbiology 20090902 2

Positive control (PC) mutants defining 20 residues of the quorum-sensing activator TraR were isolated that bind DNA but show defects in activating transcription from class I, class II or both types of promoters. These PC residues, located in both the N- and C-terminal regions, combine to form three patches, one on the top (II) and two near the DNA binding domain on both lateral faces of the dimer (I and III). Patches I and II, but not patch III, involve residues from both protomers and are essen ...[more]

PMID: 19732344

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Project description:Conjugal transfer of Ti plasmids from Agrobacterium spp. is controlled by a hierarchical regulatory system designed to sense two environmental cues. One signal, a subset of the opines produced by crown gall tumors initiated on plants by the pathogen, serves to induce production of the second, an acyl-homoserine lactone quorum-sensing signal, the quormone, produced by the bacterium itself. This second signal activates TraR, and this transcriptional activator induces expression of the tra regulon. Opines control transfer because the traR gene is a member of an operon the expression of which is regulated by the conjugal opine. Among the Ti plasmid systems studied to date, only one of the two or more opine families produced by the associated tumor induces transfer. However, two chemically dissimilar opines, nopaline and agrocinopines A and B, induce transfer of the opine catabolic plasmid pAtK84b found in the nonpathogenic Agrobacterium radiobacter isolate K84. In this study we showed that this plasmid contains two copies of traR, and each is associated with a different opine-regulated operon. One copy, traR(noc), is the last gene of the nox operon and was induced by nopaline but not by agrocinopines A and B. Mutating traR(noc) abolished induction of transfer by nopaline but not by the agrocinopines. A mutation in ocd, an upstream gene of the nox operon, abolished utilization of nopaline and also induction of transfer by this opine. The second copy, traR(acc), is located in an operon of four genes and was induced by agrocinopines A and B but not by nopaline. Genetic analysis indicated that this gene is required for induction of transfer by agrocinopines A and B but not by nopaline. pAtK84b with mutations in both traR genes was not induced for transfer by either opine. However, expression of a traR gene in trans to this plasmid resulted in opine-independent transfer. The association of traR(noc) with nox is unique, but the operon containing traR(acc) is related to the arc operons of pTiC58 and pTiChry5, two Ti plasmids inducible for transfer by agrocinopines A-B and C-D, respectively. We conclude that pAtK84b codes for two independently functioning copies of traR, each regulated by a different opine, thus accounting for the activation of the transfer system of this plasmid by the two opine types.

Project description:BACKGROUND:Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been optimized to work with bacteriophage RNA polymerases, which put significant restrictions to engineer processes related to biological information. The development of efficient cell-free systems with broader transcription capabilities is required to study complex informational processes in vitro. RESULTS:In this work, an efficient cell-free expression system that uses the endogenous E. coli RNA polymerase only and sigma factor 70 for transcription was prepared. Approximately 0.75 mg/ml of Firefly luciferase and enhanced green fluorescent protein were produced in batch mode. A plasmid was optimized with different regulatory parts to increase the expression. In addition, a new eGFP was engineered that is more translatable in cell-free systems than the original eGFP. The protein production was characterized with three different adenosine triphosphate (ATP) regeneration systems: creatine phosphate (CP), phosphoenolpyruvate (PEP), and 3-phosphoglyceric acid (3-PGA). The maximum protein production was obtained with 3-PGA. Preparation of the crude extract was streamlined to a simple routine procedure that takes 12 hours including cell culture. CONCLUSIONS:Although it uses the endogenous E. coli transcription machinery, this cell-free system can produce active proteins in quantities comparable to bacteriophage systems. The E. coli transcription provides much more possibilities to engineer informational processes in vitro. Many E. coli promoters/operators specific to sigma factor 70 are available that form a broad library of regulatory parts. In this work, cell-free expression is developed as a toolbox to design and to study synthetic gene circuits in vitro.

Dataset Information

N- and C-terminal regions of the quorum-sensing activator TraR cooperate in interactions with the alpha and sigma-70 components of RNA polymerase.

Publications

N- and C-terminal regions of the quorum-sensing activator TraR cooperate in interactions with the alpha and sigma-70 components of RNA polymerase.

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