BSc (Physiology and Biochemistry), University of Toronto, 1967
PhD (Biochemistry and Molecular Biology), Cornell University, 1971
Tel: 604-822-5943
Email: gamackie@interchange.ubc.ca
Research Interests
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.
Selected Publications
Modulation of the activity of RNase E in vitro by RNA sequences and secondary structures 5' to cleavage sites.
G.A. Mackie, J.L. Genereaux, and S. Masterman
J. Biol. Chem. 272, 609-616 (1997)
Reconstitution of the degradation of the mRNA for ribosomal protein S20 with purified enzymes.
G.A. Coburn and G.A. Mackie
J. Mol. Biol. 279, 1061-1074 (1998)
Ribonuclease E is a 5'-end dependent endonuclease.
G.A. Mackie
Nature 395, 720-723 (1998)
Degradation of mRNA in E. coli: an old problem with some new twists.
G.A. Coburn and G.A. Mackie
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)
Reconstitution of a minimal RNA degradosome demonstrates functional coordination between a 3'-exonuclease and a DEAD-box RNA helicase.
G.A. Coburn, X. Miao, D.J. Briant and G.A. Mackie
Genes & Development 13, 2594-2603 (1999)
The action of ribonuclease II and polynucleotide phosphorylase against RNAs containing stem-loops of defined structure.
C. Spickler and G.A. Mackie
J. Bacteriol. 182, 2422-2427 (2000)
Stabilization of circular rpsT mRNA demonstrates the 5'-end dependence of RNase E action in vivo.
G.A. Mackie
J. Biol. Chem. 275, 25069-25072 (2000)
Preferential cleavage of degradative intermediates of rpsT mRNA by the Escherichia coli RNA degradosome.
C. Spickler, V. Stronge and G.A. Mackie
J. Bacteriol. 183, 1106-1109 (2001)
Preparation of the Escherichia coli Rne protein and reconstitution of the RNA degradosome.
G.A. Mackie, G.A. Coburn, X. Miao, D.J. Briant, and A. Prud'homme-Généreux
Methods in Enzymology 342, 346-356 (2001)
mRNA decay in Escherichia coli: enzymes, mechanisms and adaptation
R.K. Beran, A. Prud'homme-Généreux, K.E. Baker, X. Miao, R.W. Simons and G.A. Mackie
Chapter 9, in Translation Mechanisms (J. Lapointe and L. Brakier-Gingras, eds), Landes Bioscience (2003)
Ectopic RNase E sites promote bypass of 5'-end-dependent mRNA decay in Escherichia coli
K.E. Baker and G.A. Mackie
Mol. Microbiol. 47, 75-88 (2003)
The quaternary structure of RNase G from Escherichia coli
D.J. Briant, J. S. Hankins, M.A. Cook and G.A. Mackie
Mol. Microbiol. 50, 1381-1390 (2003)
Overexpression and purification of untagged polynucleotide phosphorylases
G.H. Jones, M.F. Symmons, J.S. Hankins and G.A. Mackie
Protein Expression and Purification 32, 202-209 (2003)
Structural Characterization of the RNase E S1 Domain and Identification of its Oligonucleotide-Binding and Dimerization Interfaces
M. Schubert, R. E. Edge, P. Lario, M. A. Cook, N.C.J. Strynadka, G. A. Mackie, and L. P. McIntosh
J.Mol. Biol. 341 , 37-54 (2004) .
Physical and functional interactions among RNase E, polynucleotide phosphorylase and the cold-shock protein, CsdA: evidence for a “cold-shock degradosome”
A. Prud'homme-G J n J reux, R. Beran, I. Iost, C. S. Ramey, G.A. Mackie and R.W. Simons
Mol. Microbiol . 54 , 1409-1421 (2004)
Function of the conserved S1 and KH domains in polynucleotide phosphorylase.
L. M. Stickney, J.S. Hankins, X. Miao and G.A. Mackie
J. Bacteriol. 187 , 7214-7221 (2005)
The role of RNA structure and susceptibility to RNase E in the regulation of a cold-shock mRNA, cspA mRNA
J.S. Hankins, C. Zappavigna, A. Prud'homme-G J n J reux and G.A. Mackie.
J. Bacteriol. 189, 4353-4358 (2007)
Kinetics of polynucleotide phosphorylase: comparison of enzymes from Streptomyces and Escherichia coli and effects of nucleoside diphosphates.
Samantha A. Chang, Madeline Cozad, George A. Mackie and George H. Jones.
J. Bacteriol. 190, 98-106 (2008)
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