tRNA Biogenesis, Function and Quality Control

One major lab project focuses on understanding the biology of tRNA. tRNA plays a crucial role in translation, and numerous factors participate in its biogenesis, function, intracellular trafficking, and quality control. In the yeast Saccharomyces cerevisiae, cytoplasmic tRNAs have an average of 13 modifications, and there are a total of 25 chemically distinct modifications, many of which are highly conserved in different organisms, and of unknown function. We previously used a biochemical genomics approach to identify the genes encoding a number of different modification enzymes. We are currently focusing on the assignment of other activities to genes, and on two aspects of the biology of modifications: First, we are investigating the roles of modifications. We recently found evidence that lack of certain modifications can lead to rapid degradation of specific mature tRNAs, suggesting the existence of a quality control pathway that monitors the integrity of tRNA. We are currently working to define the components of this rapid tRNA decay pathway, the mechanism by which tRNAs are targeted for turnover, and the conditions under which this pathway is used. Second, we are defining the mechanisms by which modification enzymes recognize and act on their specific tRNA substrates. Since modifications have crucial roles in the cell, incorrect modification of a tRNA is likely to have catastrophic effects. We recently found that recognition of the tRNAHis anticodon is necessary and sufficient for Thg1 to add a guanine nucleotide to the 5' end of the tRNA, a reaction that is essential in wild type cells for proper translation. We are currently examining other modification enzymes to uncover the different mechanisms by which specificity is attained.

Functional Genomics

A second major lab project focuses on the design, construction and implementation of genomic methods to analyze protein structure and function (in collaboration with Elizabeth Grayhack). To facilitate parallel biochemical analysis of the proteome, we previously constructed two genomic libraries of strains in which nearly every yeast gene is expressed as a tagged fusion protein under regulated control. We are currently using these libraries to define new biochemical activities, and to help define cell circuitry by systematic analysis of genetic suppression caused by overexpression of genes. In addition, in collaboration with investigators at the Hauptman-Woodward Institute, we are developing methodology for high throughput expression and purification of protein complexes from yeast, for structural analysis by x-ray crystallography.

• Preston MA, D'Silva S, Kon Y, Phizicky EM. tRNAHis 5-methylcytidine levels increase in response to several growth arrest conditions in Saccharomyces cerevisiae. RNA. 2013 Feb; 19(2):243-56.
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• Guy MP, Podyma BM, Preston MA, Shaheen HH, Krivos KL, Limbach PA, Hopper AK, Phizicky EM. Yeast Trm7 interacts with distinct proteins for critical modifications of the tRNAPhe anticodon loop. RNA. 2012 Oct; 18(10):1921-33.
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• Dewe JM, Whipple JM, Chernyakov I, Jaramillo LN, Phizicky EM. The yeast rapid tRNA decay pathway competes with elongation factor 1A for substrate tRNAs and acts on tRNAs lacking one or more of several modifications. RNA. 2012 Oct; 18(10):1886-96.
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• Grant TD, Snell EH, Luft JR, Quartley E, Corretore S, Wolfley JR, Elizabeth Snell M, Hadd A, Perona JJ, Phizicky EM, Grayhack EJ. Structural conservation of an ancient tRNA sensor in eukaryotic glutaminyl-tRNA synthetase. Nucleic Acids Res. 2012 Apr 1; 40(8):3723-31.
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• Wilusz JE, Whipple JM, Phizicky EM, Sharp PA. tRNAs marked with CCACCA are targeted for degradation. Science. 2011 Nov 11; 334(6057):817-21.
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• Whipple JM, Lane EA, Chernyakov I, D'Silva S, Phizicky EM. The yeast rapid tRNA decay pathway primarily monitors the structural integrity of the acceptor and T-stems of mature tRNA. Genes Dev. 2011 Jun 1; 25(11):1173-84.
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• D'Silva S, Haider SJ, Phizicky EM. A domain of the actin binding protein Abp140 is the yeast methyltransferase responsible for 3-methylcytidine modification in the tRNA anti-codon loop. RNA. 2011 Jun; 17(6):1100-10.
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• Phizicky EM, Hopper AK. tRNA biology charges to the front. Genes Dev. 2010 Sep 1; 24(17):1832-60.
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• Preston MA, Phizicky EM. The requirement for the highly conserved G-1 residue of Saccharomyces cerevisiae tRNAHis can be circumvented by overexpression of tRNAHis and its synthetase. RNA. 2010 May; 16(5):1068-77.
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• Phizicky EM, Alfonzo JD. Do all modifications benefit all tRNAs? FEBS Lett. 2010 Jan 21; 584(2):265-71.
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• Murthi A, Shaheen HH, Huang HY, Preston MA, Lai TP, Phizicky EM, Hopper AK. Regulation of tRNA bidirectional nuclear-cytoplasmic trafficking in Saccharomyces cerevisiae. Mol Biol Cell. 2010 Feb 15; 21(4):639-49.
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• Quartley E, Alexandrov A, Mikucki M, Buckner FS, Hol WG, DeTitta GT, Phizicky EM, Grayhack EJ. Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding. J Struct Funct Genomics. 2009 Sep; 10(3):233-47.
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• Phizicky EM. Waste not, want not: a case for tRNA repair. Nat Struct Mol Biol. 2008 Aug; 15(8):783-4.
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• Merritt EA, Holmes M, Buckner FS, Van Voorhis WC, Quartly E, Phizicky EM, Lauricella A, Luft J, DeTitta G, Neely H, Zucker F, Hol WG. Structure of a Trypanosoma brucei alpha/beta-hydrolase fold protein with unknown function. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Jun 1; 64(Pt 6):474-8.
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• Chernyakov I, Whipple JM, Kotelawala L, Grayhack EJ, Phizicky EM. Degradation of several hypomodified mature tRNA species in Saccharomyces cerevisiae is mediated by Met22 and the 5'-3' exonucleases Rat1 and Xrn1. Genes Dev. 2008 May 15; 22(10):1369-80.
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• Jackman JE, Phizicky EM. Identification of critical residues for G-1 addition and substrate recognition by tRNA(His) guanylyltransferase. Biochemistry. 2008 Apr 22; 47(16):4817-25.
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• Chernyakov I, Baker MA, Grayhack EJ, Phizicky EM. Chapter 11. Identification and analysis of tRNAs that are degraded in Saccharomyces cerevisiae due to lack of modifications. Methods Enzymol. 2008; 449:221-37.
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• Jackman JE, Grayhack EJ, Phizicky EM. The use of Saccharomyces cerevisiae proteomic libraries to identify RNA-modifying proteins. Methods Mol Biol. 2008; 488:383-93.
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• Fan E, Baker D, Fields S, Gelb MH, Buckner FS, Van Voorhis WC, Phizicky E, Dumont M, Mehlin C, Grayhack E, Sullivan M, Verlinde C, Detitta G, Meldrum DR, Merritt EA, Earnest T, Soltis M, Zucker F, Myler PJ, Schoenfeld L, Kim D, Worthey L, Lacount D, Vignali M, Li J, Mondal S, Massey A, Carroll B, Gulde S, Luft J, Desoto L, Holl M, Caruthers J, Bosch J, Robien M, Arakaki T, Holmes M, Le Trong I, Hol WG. Structural genomics of pathogenic protozoa: an overview. Methods Mol Biol. 2008; 426:497-513.
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• Kotelawala L, Grayhack EJ, Phizicky EM. Identification of yeast tRNA Um(44) 2'-O-methyltransferase (Trm44) and demonstration of a Trm44 role in sustaining levels of specific tRNA(Ser) species. RNA. 2008 Jan; 14(1):158-69.
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• Malkowski MG, Quartley E, Friedman AE, Babulski J, Kon Y, Wolfley J, Said M, Luft JR, Phizicky EM, DeTitta GT, Grayhack EJ. Blocking S-adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing. Proc Natl Acad Sci U S A. 2007 Apr 17; 104(16):6678-83.
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• Wilkinson ML, Crary SM, Jackman JE, Grayhack EJ, Phizicky EM. The 2'-O-methyltransferase responsible for modification of yeast tRNA at position 4. RNA. 2007 Mar; 13(3):404-13.
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• Jackman JE, Kotelawala L, Grayhack EJ, Phizicky EM. Identification and characterization of modification enzymes by biochemical analysis of the proteome. Methods Enzymol. 2007; 425:139-52.
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• Phizicky EM, Grayhack EJ. Proteome-scale analysis of biochemical activity. Crit Rev Biochem Mol Biol. 2006 Sep-Oct; 41(5):315-27.
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• Jackman JE, Phizicky EM. tRNAHis guanylyltransferase catalyzes a 3'-5' polymerization reaction that is distinct from G-1 addition. Proc Natl Acad Sci U S A. 2006 Jun 6; 103(23):8640-5.
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• Jackman JE, Phizicky EM. tRNAHis guanylyltransferase adds G-1 to the 5' end of tRNAHis by recognition of the anticodon, one of several features unexpectedly shared with tRNA synthetases. RNA. 2006 Jun; 12(6):1007-14.
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• Arakaki T, Le Trong I, Phizicky E, Quartley E, DeTitta G, Luft J, Lauricella A, Anderson L, Kalyuzhniy O, Worthey E, Myler PJ, Kim D, Baker D, Hol WG, Merritt EA. Structure of Lmaj006129AAA, a hypothetical protein from Leishmania major. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Mar 1; 62(Pt 3):175-9.
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• Alexandrov A, Chernyakov I, Gu W, Hiley SL, Hughes TR, Grayhack EJ, Phizicky EM. Rapid tRNA decay can result from lack of nonessential modifications. Mol Cell. 2006 Jan 6; 21(1):87-96.
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• Gelperin DM, White MA, Wilkinson ML, Kon Y, Kung LA, Wise KJ, Lopez-Hoyo N, Jiang L, Piccirillo S, Yu H, Gerstein M, Dumont ME, Phizicky EM, Snyder M, Grayhack EJ. Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes Dev. 2005 Dec 1; 19(23):2816-26.
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• Shull NP, Spinelli SL, Phizicky EM. We are writing to express our concerns regarding the recently published article by Kumaran et al. Protein Sci. 2005 Dec; 14(12):3145; author reply 3145.
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• Gu W, Hurto RL, Hopper AK, Grayhack EJ, Phizicky EM. Depletion of Saccharomyces cerevisiae tRNA(His) guanylyltransferase Thg1p leads to uncharged tRNAHis with additional m(5)C. Mol Cell Biol. 2005 Sep; 25(18):8191-201.
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• Phizicky EM. Have tRNA, will travel. Proc Natl Acad Sci U S A. 2005 Aug 9; 102(32):11127-8.
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• Cartlidge RA, Knebel A, Peggie M, Alexandrov A, Phizicky EM, Cohen P. The tRNA methylase METTL1 is phosphorylated and inactivated by PKB and RSK in vitro and in cells. EMBO J. 2005 May 4; 24(9):1696-705.
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• Alexandrov A, Grayhack EJ, Phizicky EM. tRNA m7G methyltransferase Trm8p/Trm82p: evidence linking activity to a growth phenotype and implicating Trm82p in maintaining levels of active Trm8p. RNA. 2005 May; 11(5):821-30.
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• Shull NP, Spinelli SL, Phizicky EM. A highly specific phosphatase that acts on ADP-ribose 1''-phosphate, a metabolite of tRNA splicing in Saccharomyces cerevisiae. Nucleic Acids Res. 2005; 33(2):650-60.
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• Hiley SL, Jackman J, Babak T, Trochesset M, Morris QD, Phizicky E, Hughes TR. Detection and discovery of RNA modifications using microarrays. Nucleic Acids Res. 2005; 33(1):e2.
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• Steiger MA, Jackman JE, Phizicky EM. Analysis of 2'-phosphotransferase (Tpt1p) from Saccharomyces cerevisiae: evidence for a conserved two-step reaction mechanism. RNA. 2005 Jan; 11(1):99-106.
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• Alexandrov A, Vignali M, LaCount DJ, Quartley E, de Vries C, De Rosa D, Babulski J, Mitchell SF, Schoenfeld LW, Fields S, Hol WG, Dumont ME, Phizicky EM, Grayhack EJ. A facile method for high-throughput co-expression of protein pairs. Mol Cell Proteomics. 2004 Sep; 3(9):934-8.
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• Xing F, Hiley SL, Hughes TR, Phizicky EM. The specificities of four yeast dihydrouridine synthases for cytoplasmic tRNAs. J Biol Chem. 2004 Apr 23; 279(17):17850-60.
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• Gu W, Jackman JE, Lohan AJ, Gray MW, Phizicky EM. tRNAHis maturation: an essential yeast protein catalyzes addition of a guanine nucleotide to the 5' end of tRNAHis. Genes Dev. 2003 Dec 1; 17(23):2889-901.
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• Jackman JE, Montange RK, Malik HS, Phizicky EM. Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9. RNA. 2003 May; 9(5):574-85.
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• Phizicky E, Bastiaens PI, Zhu H, Snyder M, Fields S. Protein analysis on a proteomic scale. Nature. 2003 Mar 13; 422(6928):208-15.
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• Hopper AK, Phizicky EM. tRNA transfers to the limelight. Genes Dev. 2003 Jan 15; 17(2):162-80.
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• Alexandrov A, Martzen MR, Phizicky EM. Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA. RNA. 2002 Oct; 8(10):1253-66.
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• Xing F, Martzen MR, Phizicky EM. A conserved family of Saccharomyces cerevisiae synthases effects dihydrouridine modification of tRNA. RNA. 2002 Mar; 8(3):370-81.
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• Phizicky EM, Martzen MR, McCraith SM, Spinelli SL, Xing F, Shull NP, Van Slyke C, Montagne RK, Torres FM, Fields S, Grayhack EJ. Biochemical genomics approach to map activities to genes. Methods Enzymol. 2002; 350:546-59.
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• Steiger MA, Kierzek R, Turner DH, Phizicky EM. Substrate recognition by a yeast 2'-phosphotransferase involved in tRNA splicing and by its Escherichia coli homolog. Biochemistry. 2001 Nov 20; 40(46):14098-105.
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• Grayhack EJ, Phizicky EM. Genomic analysis of biochemical function. Curr Opin Chem Biol. 2001 Feb; 5(1):34-9.
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• Polevoda B, Martzen MR, Das B, Phizicky EM, Sherman F. Cytochrome c methyltransferase, Ctm1p, of yeast. J Biol Chem. 2000 Jul 7; 275(27):20508-13.
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• Kierzek R, Steiger MA, Spinelli SL, Turner DH, Phizicky EM. The chemical synthesis of oligoribonucleotides with selectively placed 2'-O-phosphates. Nucleosides Nucleotides Nucleic Acids. 2000 May-Jun; 19(5-6):917-33.
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• Martzen MR, McCraith SM, Spinelli SL, Torres FM, Fields S, Grayhack EJ, Phizicky EM. A biochemical genomics approach for identifying genes by the activity of their products. Science. 1999 Nov 5; 286(5442):1153-5.
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• Spinelli SL, Kierzek R, Turner DH, Phizicky EM. Transient ADP-ribosylation of a 2'-phosphate implicated in its removal from ligated tRNA during splicing in yeast. J Biol Chem. 1999 Jan 29; 274(5):2637-44.
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• Spinelli SL, Malik HS, Consaul SA, Phizicky EM. A functional homolog of a yeast tRNA splicing enzyme is conserved in higher eukaryotes and in Escherichia coli. Proc Natl Acad Sci U S A. 1998 Nov 24; 95(24):14136-41.
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• Robinson MK, Phizicky EM. Purification and assay of the Ptc1/Tpd1 protein phosphatase 2C from the yeast Saccharomyces cerevisiae. Methods Mol Biol. 1998; 93:235-42.
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• Spinelli SL, Consaul SA, Phizicky EM. A conditional lethal yeast phosphotransferase (tpt1) mutant accumulates tRNAs with a 2'-phosphate and an undermodified base at the splice junction. RNA. 1997 Dec; 3(12):1388-400.
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• Culver GM, McCraith SM, Consaul SA, Stanford DR, Phizicky EM. A 2'-phosphotransferase implicated in tRNA splicing is essential in Saccharomyces cerevisiae. J Biol Chem. 1997 May 16; 272(20):13203-10.
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• Phizicky EM, Fields S. Protein-protein interactions: methods for detection and analysis. Microbiol Rev. 1995 Mar; 59(1):94-123.
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• Culver GM, Consaul SA, Tycowski KT, Filipowicz W, Phizicky EM. tRNA splicing in yeast and wheat germ. A cyclic phosphodiesterase implicated in the metabolism of ADP-ribose 1",2"-cyclic phosphate. J Biol Chem. 1994 Oct 7; 269(40):24928-34.
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• Robinson MK, van Zyl WH, Phizicky EM, Broach JR. TPD1 of Saccharomyces cerevisiae encodes a protein phosphatase 2C-like activity implicated in tRNA splicing and cell separation. Mol Cell Biol. 1994 Jun; 14(6):3634-45.
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• Culver GM, McCraith SM, Zillmann M, Kierzek R, Michaud N, LaReau RD, Turner DH, Phizicky EM. An NAD derivative produced during transfer RNA splicing: ADP-ribose 1"-2" cyclic phosphate. Science. 1993 Jul 9; 261(5118):206-8.
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• Phizicky EM, Greer CL. Pre-tRNA splicing: variation on a theme or exception to the rule? Trends Biochem Sci. 1993 Jan; 18(1):31-4.
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• Zillmann M, Gorovsky MA, Phizicky EM. HeLa cells contain a 2'-phosphate-specific phosphotransferase similar to a yeast enzyme implicated in tRNA splicing. J Biol Chem. 1992 May 25; 267(15):10289-94.
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• Phizicky EM, Consaul SA, Nehrke KW, Abelson J. Yeast tRNA ligase mutants are nonviable and accumulate tRNA splicing intermediates. J Biol Chem. 1992 Mar 5; 267(7):4577-82.
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• Zillmann M, Gorovsky MA, Phizicky EM. Conserved mechanism of tRNA splicing in eukaryotes. Mol Cell Biol. 1991 Nov; 11(11):5410-6.
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• McCraith SM, Phizicky EM. An enzyme from Saccharomyces cerevisiae uses NAD+ to transfer the splice junction 2'-phosphate from ligated tRNA to an acceptor molecule. J Biol Chem. 1991 Jun 25; 266(18):11986-92.
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• McCraith SM, Phizicky EM. A highly specific phosphatase from Saccharomyces cerevisiae implicated in tRNA splicing. Mol Cell Biol. 1990 Mar; 10(3):1049-55.
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• Xu Q, Phizicky EM, Greer CL, Abelson JN. Purification of yeast transfer RNA ligase. Methods Enzymol. 1990; 181:463-71.
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• Westaway SK, Phizicky EM, Abelson J. Structure and function of the yeast tRNA ligase gene. J Biol Chem. 1988 Mar 5; 263(7):3171-6.
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• Phizicky EM, Schwartz RC, Abelson J. Saccharomyces cerevisiae tRNA ligase. Purification of the protein and isolation of the structural gene. J Biol Chem. 1986 Feb 25; 261(6):2978-86.
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• Roberts JW, Phizicky EM, Burbee DG, Roberts CW, Moreau PL. A brief consideration of the SOS inducing signal. Biochimie. 1982 Aug-Sep; 64(8-9):805-7.
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• Phizicky EM, Roberts JW. Induction of SOS functions: regulation of proteolytic activity of E. coli RecA protein by interaction with DNA and nucleoside triphosphate. Cell. 1981 Jul; 25(1):259-67.
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• Phizicky EM, Roberts JW. Kinetics of RecA protein-directed inactivation of repressors of phage lambda and phage P22. J Mol Biol. 1980 May 25; 139(3):319-28.
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• Roberts JW, Roberts CW, Craig NL, Phizicky EM. Activity of the Escherichia coli recA-gene product. Cold Spring Harb Symp Quant Biol. 1979; 43 Pt 2:917-20.
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