Residues in the conserved His domain of fruit fly tRNase Z that function in catalysis are not involved in substrate recognition or binding.
ABSTRACT Transfer RNAs are transcribed as precursors with extensions at both the 5' and 3' ends. RNase P removes endonucleolytically the 5' end leader. tRNase Z can remove endonucleolytically the 3' end trailer as a necessary step in tRNA maturation. CCA is not transcriptionally encoded in the tRNAs of eukaryotes, archaebacteria and some bacteria and must be added by a CCA-adding enzyme after removal of the 3' end trailer. tRNase Z is a member of the beta-lactamase family of metal-dependent hydrolases, the signature sequence of which, the conserved histidine cluster (HxHxDH), is essential for activity. Starting with baculovirus-expressed fruit fly tRNase Z, we completed an 18 residue Ala scan of the His cluster to analyze the functional landscape of this critical region. Residues in and around the His cluster fall into three categories based on effects of the substitutions on processing efficiency: substitutions in eight residues have little effect, five substitutions reduce efficiency moderately (approximately 5-50-fold), while substitutions in five conserved residues, one serine, three histidine and one aspartate, severely reduce efficiency (approximately 500-5000-fold). Wild-type and mutant dissociation constants (Kd values), determined using gel shifts, displayed no substantial differences, and were of the same order as kM (2-20 nM). Lower processing efficiencies arising from substitutions in the His domain are almost entirely due to reduced kcat values; conserved, functionally important residues within the His cluster of tRNase Z are thus involved in catalysis, and substrate recognition and binding functions must reside elsewhere in the protein.
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ABSTRACT: tRNase Z, a member of the metallo-β-lactamase family, endonucleolytically removes the pre-tRNA 3' trailer in a step central to tRNA maturation. The short form (tRNase Z(S)) is the only one found in bacteria and archaebacteria and is also present in some eukaryotes. The homologous long form (tRNase Z(L)), exclusively found in eukaryotes, consists of related amino- and carboxy-domains, suggesting that tRNase Z(L) arose from a tandem duplication of tRNase Z(S) followed by interdependent divergence of the domains. X-ray crystallographic structures of tRNase Z(S) reveal a flexible arm (FA) extruded from the body of tRNase Z remote from the active site that binds tRNA far from the scissile bond. No tRNase Z(L) structures have been solved; alternative biophysical studies are therefore needed to illuminate its functional characteristics. Structural analyses of tRNase Z(L) performed by limited proteolysis, two dimensional gel electrophoresis and mass spectrometry establish stability of the amino and carboxy domains and flexibility of the FA and inter-domain tether, with implications for tRNase Z(L) function.PLoS ONE 07/2013; 8(7):e66942. · 3.53 Impact Factor
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ABSTRACT: We study the Kerr nonlinearity associated with cross-phase modulation based on electromagnetically induced transparency in asymmetric double quantum wells. It is shown that, different from atomic system, not only the nonlinear dispersion and absorption but also the linear absorption depends on the relative phase of the laser fields because of the Fano interference. By choosing the parameters appropriately, large cross-phase modulation with nearly vanishing two-photon absorption, even π phase shift with single photon level, could be achieved in the asymmetric quantum wells.Optics Communications 01/2011; 284(1):276-281. · 1.54 Impact Factor
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ABSTRACT: RNase Z is a dinuclear zinc enzyme that catalyzes the removal of the tRNA 3′-end trailer. Density functional theory is used to investigate the phosphodiester hydrolysis mechanism of this enzyme with a model of the active site constructed on the basis of the crystal structure. The calculations imply that the reaction proceeds through two steps. The first step is a nucleophilic attack by a bridging hydroxide coupled with protonation of the leaving group by a Glu-His diad. Subsequently, a water molecule activated by the same Glu-His diad makes a reverse attack, regenerating the bridging hydroxide. The second step is calculated to be the rate-limiting step with a barrier of 18 kcal/mol, in good agreement with experimental kinetic studies. Both zinc ions participate in substrate binding and orientation, facilitating nucleophilic attack. In addition, they act as electrophilic catalysts to stabilize the pentacoordinate trigonal-bipyramidal transition states.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)Berichte der deutschen chemischen Gesellschaft 06/2009; 2009(20):2967 - 2972. · 2.97 Impact Factor