Abstract
In vivo titration experiments have demonstrated a direct interaction between the Pseudomonas aeruginosa transcription antiterminator, AmiR, and the mRNA leader sequence of the amidase operon. A region of 39 nucleotides has been identified which is sufficient to partially titrate out the AmiR available for antitermination. Site-directed mutagenesis has shown that the leader open reading frame has no role in the antitermination reaction, and has identified two critical elements at the 5' and 3' ends of the proposed AmiR binding site which are independently essential for antitermination. A T7 promoter/RNA polymerase-driven system shows AmiR-mediated antitermination, demonstrating a lack of promoter/polymerase specificity. Using the operon negative regulator, AmiC, immobilized on a solid support and gel filtration chromatography, an AmiC-AmiR complex has been identified and isolated. Complex stability and molecular weight assayed by gel filtration alter depending on the type of amide bound to AmiC. AmiC-AmiR-anti-inducer is a stable dimer-dimer complex and the addition of the inducer, acetamide, causes a conformational change which alters the complex stability and either this new configuration or dissociated AmiR interacts with the leader mRNA to cause antitermination.
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Selected References
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- Amster-Choder O., Wright A. Modulation of the dimerization of a transcriptional antiterminator protein by phosphorylation. Science. 1992 Sep 4;257(5075):1395–1398. doi: 10.1126/science.1382312. [DOI] [PubMed] [Google Scholar]
- Amster-Choder O., Wright A. Transcriptional regulation of the bgl operon of Escherichia coli involves phosphotransferase system-mediated phosphorylation of a transcriptional antiterminator. J Cell Biochem. 1993 Jan;51(1):83–90. doi: 10.1002/jcb.240510115. [DOI] [PubMed] [Google Scholar]
- Aymerich S., Steinmetz M. Specificity determinants and structural features in the RNA target of the bacterial antiterminator proteins of the BglG/SacY family. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10410–10414. doi: 10.1073/pnas.89.21.10410. [DOI] [PMC free article] [PubMed] [Google Scholar]
- BRAMMAR W. J., CLARKE P. H. INDUCTION AND REPRESSION OF PSEUDOMONAS AERUGINOSA AMIDASE. J Gen Microbiol. 1964 Dec;37:307–319. doi: 10.1099/00221287-37-3-307. [DOI] [PubMed] [Google Scholar]
- Clarke P. H., Drew R. E., Turberville C., Brammar W. J., Ambler R. P., Auffret A. D. Alignment of cloned amiE gene of Pseudomonas aeruginosa with the N-terminal sequence of amidase. Biosci Rep. 1981 Apr;1(4):299–307. doi: 10.1007/BF01114869. [DOI] [PubMed] [Google Scholar]
- Cousens D. J., Clarke P. H., Drew R. The amidase regulatory gene (amiR) of Pseudomonas aeruginosa. J Gen Microbiol. 1987 Aug;133(8):2041–2052. doi: 10.1099/00221287-133-8-2041. [DOI] [PubMed] [Google Scholar]
- Drew R. Complementation analysis of the aliphatic amidase genes of Pseudomonas aeruginosa. J Gen Microbiol. 1984 Dec;130(12):3101–3111. doi: 10.1099/00221287-130-12-3101. [DOI] [PubMed] [Google Scholar]
- Drew R., Lowe N. Positive control of Pseudomonas aeruginosa amidase synthesis is mediated by a transcription anti-termination mechanism. J Gen Microbiol. 1989 Apr;135(4):817–823. doi: 10.1099/00221287-135-4-817. [DOI] [PubMed] [Google Scholar]
- Friedman A. M., Fischmann T. O., Steitz T. A. Crystal structure of lac repressor core tetramer and its implications for DNA looping. Science. 1995 Jun 23;268(5218):1721–1727. doi: 10.1126/science.7792597. [DOI] [PubMed] [Google Scholar]
- Fürste J. P., Pansegrau W., Frank R., Blöcker H., Scholz P., Bagdasarian M., Lanka E. Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene. 1986;48(1):119–131. doi: 10.1016/0378-1119(86)90358-6. [DOI] [PubMed] [Google Scholar]
- Jaeger J. A., Turner D. H., Zuker M. Improved predictions of secondary structures for RNA. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7706–7710. doi: 10.1073/pnas.86.20.7706. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jaeger J. A., Turner D. H., Zuker M. Predicting optimal and suboptimal secondary structure for RNA. Methods Enzymol. 1990;183:281–306. doi: 10.1016/0076-6879(90)83019-6. [DOI] [PubMed] [Google Scholar]
- KELLY M., CLARKE P. H. An inducible amidase produced by a strain of Pseudomonas aeruginosa. J Gen Microbiol. 1962 Feb;27:305–316. doi: 10.1099/00221287-27-2-305. [DOI] [PubMed] [Google Scholar]
- Lowe N., Rice P. M., Drew R. E. Nucleotide sequence of the aliphatic amidase regulator gene (amiR) of Pseudomonas aeruginosa. FEBS Lett. 1989 Mar 27;246(1-2):39–43. doi: 10.1016/0014-5793(89)80249-2. [DOI] [PubMed] [Google Scholar]
- Mahadevan S., Wright A. A bacterial gene involved in transcription antitermination: regulation at a rho-independent terminator in the bgl operon of E. coli. Cell. 1987 Jul 31;50(3):485–494. doi: 10.1016/0092-8674(87)90502-2. [DOI] [PubMed] [Google Scholar]
- Pearl L., O'Hara B., Drew R., Wilson S. Crystal structure of AmiC: the controller of transcription antitermination in the amidase operon of Pseudomonas aeruginosa. EMBO J. 1994 Dec 15;13(24):5810–5817. doi: 10.1002/j.1460-2075.1994.tb06924.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roberts J. W. Phage lambda and the regulation of transcription termination. Cell. 1988 Jan 15;52(1):5–6. doi: 10.1016/0092-8674(88)90523-5. [DOI] [PubMed] [Google Scholar]
- Squires C. L., Greenblatt J., Li J., Condon C., Squires C. L. Ribosomal RNA antitermination in vitro: requirement for Nus factors and one or more unidentified cellular components. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):970–974. doi: 10.1073/pnas.90.3.970. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilson S. A., Chayen N. E., Hemmings A. M., Drew R. E., Pearl L. H. Crystallization of and preliminary X-ray data for the negative regulator (AmiC) of the amidase operon of Pseudomonas aeruginosa. J Mol Biol. 1991 Dec 20;222(4):869–871. doi: 10.1016/0022-2836(91)90579-u. [DOI] [PubMed] [Google Scholar]
- Wilson S. A., Drew R. E. Transcriptional analysis of the amidase operon from Pseudomonas aeruginosa. J Bacteriol. 1995 Jun;177(11):3052–3057. doi: 10.1128/jb.177.11.3052-3057.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilson S. A., Wachira S. J., Drew R. E., Jones D., Pearl L. H. Antitermination of amidase expression in Pseudomonas aeruginosa is controlled by a novel cytoplasmic amide-binding protein. EMBO J. 1993 Sep;12(9):3637–3642. doi: 10.1002/j.1460-2075.1993.tb06037.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilson S., Drew R. Cloning and DNA sequence of amiC, a new gene regulating expression of the Pseudomonas aeruginosa aliphatic amidase, and purification of the amiC product. J Bacteriol. 1991 Aug;173(16):4914–4921. doi: 10.1128/jb.173.16.4914-4921.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zuker M. On finding all suboptimal foldings of an RNA molecule. Science. 1989 Apr 7;244(4900):48–52. doi: 10.1126/science.2468181. [DOI] [PubMed] [Google Scholar]