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Effect of Changes in the Flexible Arm on tRNase Z Processing Kinetics*

  • Louis Levinger
    Correspondence
    To whom correspondence should be addressed: 94-20 Guy R. Brewer Blvd., Jamaica, NY 11451. Tel.:718-262-2704; Fax:718-262-2369;
    Affiliations
    Department of Biology, York College of the City University of New York, Jamaica, New York 11451
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  • Author Footnotes
    2 Present address: Dept. of Microbiology and Immunology, University of Michigan Medical School.
    Angela Hopkinson
    Footnotes
    2 Present address: Dept. of Microbiology and Immunology, University of Michigan Medical School.
    Affiliations
    Department of Biology, York College of the City University of New York, Jamaica, New York 11451
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  • Rohini Desetty
    Affiliations
    Department of Biology, York College of the City University of New York, Jamaica, New York 11451
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  • Christopher Wilson
    Affiliations
    Department of Biology, York College of the City University of New York, Jamaica, New York 11451
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants S06GM08153, R15CA120072, and SC3GM084764. This work was also supported by grants from the Professional Staff Congress-City University of New York.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Figs. 1–3.
    2 Present address: Dept. of Microbiology and Immunology, University of Michigan Medical School.
Open AccessPublished:April 07, 2009DOI:https://doi.org/10.1074/jbc.M900745200
      tRNAs are transcribed as precursors and processed in a series of reactions culminating in aminoacylation and translation. Central to tRNA maturation, the 3′ end trailer can be endonucleolytically removed by tRNase Z. A flexible arm (FA) extruded from the body of tRNase Z consists of a structured ααββ hand that binds the elbow of pre-tRNA. Deleting the FA hand causes an almost 100-fold increase in Km with little change in kcat, establishing its contribution to substrate recognition/binding. Remarkably, a 40-residue Ala scan through the FA hand reveals a conserved leucine at the ascending stalk/hand boundary that causes practically the same increase in Km as the hand deletion, thus nearly eliminating its ability to bind substrate. Km also increases with substitutions in the GP (α4–α5) loop and at other conserved residues in the FA hand predicted to contact substrate based on the co-crystal structure. Substitutions that reduce kcat are clustered in the β10–β11 loop.
      tRNAs are transcribed as precursors with a 5′ end leader and 3′ end trailer. The 5′ end leader is removed by RNase P. The 3′ end trailer can be endonucleolytically removed by tRNase Z, which cleaves following the unpaired nucleotide just beyond the 3′ side of the acceptor stem (the discriminator) leaving a 3′-OH ready for CCA addition. In some bacteria and in all archaea and eukaryotes (including their organelles), CCA at the 3′ end of mature tRNAs is not transcriptionally encoded, and a CCA-adding enzyme is required (
      • Aebi M.
      • Kirchner G.
      • Chen J.Y.
      • Vijayraghavan U.
      • Jacobson A.
      • Martin N.C.
      • Abelson J.
      ); endonucleolytic processing by tRNase Z is thus a precise and probably essential reaction in the pathway to a mature 3′ end (
      • Tavtigian S.V.
      • Simard J.
      • Teng D.H.
      • Abtin V.
      • Baumgard M.
      • Beck A.
      • Camp N.J.
      • Carillo A.R.
      • Chen Y.
      • Dayananth P.
      • Desrochers M.
      • Dumont M.
      • Farnham J.M.
      • Frank D.
      • Frye C.
      • Ghaffari S.
      • Gupte J.S.
      • Hu R.
      • Iliev D.
      • Janecki T.
      • Kort E.N.
      • Laity K.E.
      • Leavitt A.
      • Leblanc G.
      • McArthur-Morrison J.
      • Pederson A.
      • Penn B.
      • Peterson K.T.
      • Reid J.E.
      • Richards S.
      • Schroeder M.
      • Smith R.
      • Snyder S.C.
      • Swedlund B.
      • Swensen J.
      • Thomas A.
      • Tranchant M.
      • Woodland A.M.
      • Labrie F.
      • Skolnick M.H.
      • Neuhausen S.
      • Rommens J.
      • Cannon-Albright L.A.
      ,
      • Chen Y.
      • Beck A.
      • Davenport C.
      • Chen Y.
      • Shattuck D.
      • Tavtigian S.V.
      ).
      Interestingly, the 3′ end CCA is an anti-determinant for tRNase Z that discourages the recycling of mature tRNAs (
      • Nashimoto M.
      ,
      • Mohan A.
      • Whyte S.
      • Wang X.
      • Nashimoto M.
      • Levinger L.
      ,
      • Pellegrini O.
      • Nezzar J.
      • Marchfelder A.
      • Putzer H.
      • Condon C.
      ,
      • Zareen N.
      • Hopkinson A.
      • Levinger L.
      ), although not in every case (
      • Schiffer S.
      • Rösch S.
      • Marchfelder A.
      ). Additional functions have been suggested for tRNase Z, including a possible role in human prostate cancer susceptibility (
      • Tavtigian S.V.
      • Simard J.
      • Teng D.H.
      • Abtin V.
      • Baumgard M.
      • Beck A.
      • Camp N.J.
      • Carillo A.R.
      • Chen Y.
      • Dayananth P.
      • Desrochers M.
      • Dumont M.
      • Farnham J.M.
      • Frank D.
      • Frye C.
      • Ghaffari S.
      • Gupte J.S.
      • Hu R.
      • Iliev D.
      • Janecki T.
      • Kort E.N.
      • Laity K.E.
      • Leavitt A.
      • Leblanc G.
      • McArthur-Morrison J.
      • Pederson A.
      • Penn B.
      • Peterson K.T.
      • Reid J.E.
      • Richards S.
      • Schroeder M.
      • Smith R.
      • Snyder S.C.
      • Swedlund B.
      • Swensen J.
      • Thomas A.
      • Tranchant M.
      • Woodland A.M.
      • Labrie F.
      • Skolnick M.H.
      • Neuhausen S.
      • Rommens J.
      • Cannon-Albright L.A.
      ,
      • Korver W.
      • Guevara C.
      • Chen Y.
      • Neuteboom S.
      • Bookstein R.
      • Tavtigian S.
      • Lees E.
      ,
      • Takaku H.
      • Minagawa A.
      • Takagi M.
      • Nashimoto M.
      ,
      • Smith M.M.
      • Levitan D.J.
      ,
      • Hölzle A.
      • Fischer S.
      • Heyer R.
      • Schütz S.
      • Zacharias M.
      • Walther P.
      • Allers T.
      • Marchfelder A.
      ). In some instances, tRNase Z can recognize and cleave RNAs that are structurally related to pre-tRNAs with 3′ end extensions (
      • Takaku H.
      • Minagawa A.
      • Takagi M.
      • Nashimoto M.
      ,
      • Hölzle A.
      • Fischer S.
      • Heyer R.
      • Schütz S.
      • Zacharias M.
      • Walther P.
      • Allers T.
      • Marchfelder A.
      ).
      tRNase Z is an ancient member of the β-lactamase superfamily of metal-dependent hydrolases (
      • Tavtigian S.V.
      • Simard J.
      • Teng D.H.
      • Abtin V.
      • Baumgard M.
      • Beck A.
      • Camp N.J.
      • Carillo A.R.
      • Chen Y.
      • Dayananth P.
      • Desrochers M.
      • Dumont M.
      • Farnham J.M.
      • Frank D.
      • Frye C.
      • Ghaffari S.
      • Gupte J.S.
      • Hu R.
      • Iliev D.
      • Janecki T.
      • Kort E.N.
      • Laity K.E.
      • Leavitt A.
      • Leblanc G.
      • McArthur-Morrison J.
      • Pederson A.
      • Penn B.
      • Peterson K.T.
      • Reid J.E.
      • Richards S.
      • Schroeder M.
      • Smith R.
      • Snyder S.C.
      • Swedlund B.
      • Swensen J.
      • Thomas A.
      • Tranchant M.
      • Woodland A.M.
      • Labrie F.
      • Skolnick M.H.
      • Neuhausen S.
      • Rommens J.
      • Cannon-Albright L.A.
      ,
      • Dominski Z.
      ). The signature sequence of this family, the His domain (HXHXDH, Motif II), in conjunction with histidines in Motifs III and V and aspartate in Motif IV, contributes side chains that coordinate two divalent metal ions (
      • Vogel A.
      • Schilling O.
      • Meyer-Klaucke W.
      ,
      • de la Sierra-Gallay I.L.
      • Pellegrini O.
      • Condon C.
      ). Additionally, the Glu side chain in HEAT and His in the HST loop (located between Motifs IV and V) apparently function as a pair to facilitate proton transfer at the final stage of reaction (
      • Mandel C.R.
      • Kaneko S.
      • Zhang H.
      • Gebauer D.
      • Vethantham V.
      • Manley J.L.
      • Tong L.
      ,
      • Karkashon S.
      • Hopkinson A.
      • Levinger L.
      ). A Glu-His pair in CPSF-73, the long sought endonuclease responsible for pre-mRNA cleavage and a member of the tRNase Z class of RNA endonucleases (
      • Dominski Z.
      ,
      • Mandel C.R.
      • Kaneko S.
      • Zhang H.
      • Gebauer D.
      • Vethantham V.
      • Manley J.L.
      • Tong L.
      ,
      • Dominski Z.
      • Yang X.C.
      • Marzluff W.F.
      ), displays the same structure relative to the active site and presumably functions identically in catalysis.
      Substitutions in Motifs II–V, HEAT, and HST loop residues did not show increases in Km (
      • Karkashon S.
      • Hopkinson A.
      • Levinger L.
      ,
      • Zareen N.
      • Yan H.
      • Hopkinson A.
      • Levinger L.
      ); thus, these residues apparently contribute to metal ion binding and catalysis without being involved with substrate recognition/binding. This interpretation is supported by the co-crystal structure (
      • de la Sierra-Gallay I.L.
      • Mathy N.
      • Pellegrini O.
      • Condon C.
      ), in which tRNase Z with a Motif II His→Ala substitution (introduced to reduce catalytic activity) bound only one metal ion/subunit but displayed structurally reasonable tRNA binding. Increases in Km observed with several of the substitutions in the PXKXRN loop and Motif I region (
      • Zareen N.
      • Hopkinson A.
      • Levinger L.
      ) were suggested to be involved with CCA anti-determination and with substrate recognition and binding around the acceptor stem (
      • Zareen N.
      • Hopkinson A.
      • Levinger L.
      ,
      • Zareen N.
      • Yan H.
      • Hopkinson A.
      • Levinger L.
      ). Results of previous studies of processing kinetics (
      • Zareen N.
      • Hopkinson A.
      • Levinger L.
      ,
      • Karkashon S.
      • Hopkinson A.
      • Levinger L.
      ,
      • Zareen N.
      • Yan H.
      • Hopkinson A.
      • Levinger L.
      ) left open for further detailed analysis the initiating substrate recognition/binding events leading to catalysis by tRNase Z, centered on the flexible arm (
      • de la Sierra-Gallay I.L.
      • Pellegrini O.
      • Condon C.
      ,
      • de la Sierra-Gallay I.L.
      • Mathy N.
      • Pellegrini O.
      • Condon C.
      ,
      • Schilling O.
      • Späth B.
      • Kostelecky B.
      • Marchfelder A.
      • Meyer-Klacke W.
      • Vogel A.
      ).

      Acknowledgments

      We acknowledge the technical assistance of Drupattie Dial, Victoria Edwards, Shay Karkashon, and Asif Rizwan. We also acknowledge Kevin Ryan (City College) and Liang Tong (Columbia University) for helpful discussions.

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