Mackie, George A

Research Interest

It is increasingly recognized that “ribo-regulation” is a key determinant of gene expression and often relies on selective turnover of RNAs to modulate their activity. My trainees and I employ model systems for investigating this process using appropriate biochemical and molecular biological tools. Our goal is to explain how the properties of the relevant enzymes and RNA chaperones, the functional state of the mRNA (e.g., its efficiency of translation) and the secondary or tertiary structure of the RNA substrate determine its fate. Turnover of mRNA in Escherichia coli is believed to be initiated by specific endonucleolytic cleavages followed by exonucleolytic “scavenging” of the newly created 3′ ends. We have cloned, over-expressed, and purified the key ribonucleases as well as several accessory proteins including an RNA helicase and RNA chaperones. A major success has been the reconstitution of mRNA degradation in vitro from purified enzymes and substrates. Our work has shown that RNase E, the principle endonuclease, is 5′-end-dependent, the first such example. This property can explain several aspects of mRNA decay, including the reason why intermediates in the decay process are so transient. Our current work is aimed at identifying the key residues in the RNA binding and catalytic domains of RNase E, its close relative RNase G, and the exonuclease, polynucleotide phosphorylase, a model for the eukaryotic exosome. We use a variety of techniques including mutagenesis, deletion mapping, partial proteolysis and structural determination. We have also created substrates with defined secondary structures or novel conformations (e.g., circular RNAs) to unravel the pathways of mRNA turnover. Our research is funded by CIHR.

Publications

Comprehensive List

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Selected Publications

1. G.A. Mackie. Determinants in the rpsT mRNAs recognized by the 5’-sensor domain of RNase E.  Mol. Microbiol. 89, 388-401 (2013).
2. A.G. Wong, K.L McBurney, K.J. Thompson, L.M Stickney and G.A. Mackie. S1 and KH domains of polynucleotide phosphorylase determine the efficiency of RNA binding and autoregulation.  J. Bacteriol. 195,2021-2031 (2013).
3. G.A. Mackie. RNase E: at the interface of bacterial RNA processing and decay.  Nature Rev. Microbiol. 11, 45-57 (2013).
4. S.M. Garrey and G.A. Mackie. The physiological role of the 5’-sensor domain in RNase E.  Mol. Microbiol. 80, 1613-1624 (2011).
5. J.S. Hankins, H. Denroche, and G.A. Mackie. Interactions of the RNA binding protein, Hfq, with cspA mRNA encoding the major cold-shock protein.  J. Bacteriol. 192, 2482-2490 (2010).
6. S.M. Garrey, M. Blech, J. Riffel, J.S. Hankins, L.M. Stickney, M. Diver, Y.-H.R. Hsu, V. Kunanithy and G.A. Mackie. Substrate binding and active site residues in RNases E and G: the role of the 5′-sensor. J. Biol. Chem. 284, 31843-31850 (2009).
7. S.A. Chang, M. Cozad, G.A. Mackie and G.H. Jones. Kinetics of polynucleotide phosphorylase: comparison of enzymes from Streptomyces and Escherichia coli and effects of nucleoside diphosphates. J. Bacteriol. 190, 98-106 (2008)
8. J.S. Hankins, C. Zappavigna, A.Prud’homme-Généreux, and G.A. Mackie. The role of RNA structure and susceptibility to RNase E in the regulation of a cold-shock mRNA, cspA mRNA. J. Bacteriol. 189, 4353-4358 (2007)
9. L.M. Stickney, J.S. Hankins, X. Miao and G.A. Mackie. Function of the conserved S1 and KH domains in polynucleotide phosphorylase. J. Bacteriol. 187, 7214-7221 (2005)
10. A. Prud’homme-Généreux, R.K. Beran, I. Iost, C.S. Ramey, G.A. Mackie and R.W. Simons. Physical and functional interactions among RNase E, polynucleotide phosphorylase and the cold-shock protein, CsdA: evidence for a “cold-shock degradosome”. Mol. Microbiol. 54, 1409-1421 (2004)
11. M. Schubert, R.E. Edge, P. Lario, M.A. Cook, N.C.J. Strynadka, G.A. Mackie, and L.P. McIntosh. Structural Characterization of the RNase E S1 Domain and Identification of its Oligonucleotide-Binding and Dimerization Interfaces. J.Mol. Biol. 341, 37-54 (2004)
12. G.H. Jones, M.F. Symmons, J.S. Hankins and G.A. Mackie. Overexpression and purification of untagged polynucleotide phosphorylases. Protein Expression and Purification 32, 202-209 (2003)
13. D.J. Briant, J.S. Hankins, M.A. Cook and G.A. Mackie. The quaternary structure of RNase G from Escherichia coli. Mol. Microbiol. 50, 1381-1390 (2003)
14. K.E. Baker and G.A. Mackie. Ectopic RNase E sites promote bypass of 5′-end-dependent mRNA decay in Escherichia coli. Mol. Microbiol. 47, 75-88 (2003)
15. R.K. Beran, A. Prud’homme-Généreux, K.E. Baker, X. Miao, R.W. Simons and G.A. Mackie. mRNA decay in Escherichia coli: enzymes, mechanisms and adaptation. Chapter 9, in Translation Mechanisms (J. Lapointe and L. Brakier-Gingras, eds), Landes Bioscience (2003)
16. G.A. Mackie, G.A. Coburn, X. Miao, D.J. Briant, and A. Prud’homme-Généreux. Preparation of the Escherichia coli Rne protein and reconstitution of the RNA degradosome. Methods in Enzymology 342, 346-356 (2001)
17. C. Spickler, V. Stronge and G.A. Mackie. Preferential cleavage of degradative intermediates of rpsT mRNA by the Escherichia coli RNA degradosome.J. Bacteriol. 183, 1106-1109 (2001)
18. G.A. Mackie. Stabilization of circular rpsT mRNA demonstrates the 5′-end dependence of RNase E action in vivo. J. Biol. Chem. 275, 25069-25072 (2000)
19. C. Spickler and G.A. Mackie. The action of ribonuclease II and polynucleotide phosphorylase against RNAs containing stem-loops of defined structure. J. Bacteriol.182, 2422-2427 (2000)
20. G.A. Coburn, X. Miao, D.J. Briant and G.A. Mackie. Reconstitution of a minimal RNA degradosome demonstrates functional coordination between a 3′-exonuclease and a DEAD-box RNA helicase. Genes & Development 13, 2594-2603 (1999)
21. G.A. Coburn and G.A. Mackie. Degradation of mRNA in E. coli: an old problem with some new twists. In Progress in Nucleic Acids Research and Molecular Biology V. 62, pp 55-108, W.E. Cohn and K. Moldave, editors, Academic Press, San Diego, (1999)
22. G.A. Mackie. Ribonuclease E is a 5′-end dependent endonuclease. Nature 395, 720-723 (1998)
23. G.A. Coburn and G.A. Mackie. Reconstitution of the degradation of the mRNA for ribosomal protein S20 with purified enzymes. J. Mol. Biol. 279, 1061-1074 (1998)
24. G.A. Mackie, J.L. Genereaux, and S. Masterman. Modulation of the activity of RNase E in vitro by RNA sequences and secondary structures 5′ to cleavage sites. J. Biol. Chem. 272, 609-616 (1997)