ABSTRACT
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 alphaalphabetabeta 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 (alpha4-alpha5) 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 beta10-beta11 loop.
Subject(s)
Drosophila Proteins/genetics , Endoribonucleases/genetics , Amino Acid Sequence , Animals , Bacteria/enzymology , Bacteria/genetics , Conserved Sequence , DNA Primers , Drosophila/enzymology , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Endoribonucleases/chemistry , Endoribonucleases/metabolism , Genetic Variation , Glycine , Humans , Kinetics , Methionine , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence AlignmentABSTRACT
tRNAs are transcribed as precursors with a 5' end leader and a 3' end trailer. In the course of tRNA maturation, RNase P removes the 5' end leader and tRNase Z can endonucleolytically remove the 3' end trailer. A domain remote from the active site of tRNase Z recognizes and binds substrate, principally through contacts with the elbow (D/T loops) of the tRNA. To evaluate possible contacts, processing kinetics was performed using human nuclear encoded pre-tRNA(Arg) with substitutions in conserved D and T loop nucleotides. Changes in K(M) observed with some of the substitutions suggest contacts between tRNase Z and substrate tRNA in this region, and changes in tRNA structure provide an additional basis for interpretation of the kinetic effects.
Subject(s)
Conserved Sequence , Endoribonucleases/metabolism , Mutation/genetics , Nucleic Acid Conformation , RNA Precursors/chemistry , RNA Precursors/genetics , Bacillus subtilis/enzymology , Base Sequence , Catalysis , Humans , Kinetics , Molecular Sequence Data , RNA Processing, Post-Transcriptional , RNA, Transfer, Arg/chemistry , RNA, Transfer, Arg/genetics , RNA, Transfer, Val/chemistry , RNA, Transfer, Val/genetics , Substrate SpecificityABSTRACT
tRNAs are transcribed as precursors and processed in a series of required reactions leading to aminoacylation and translation. The 3'-end trailer can be removed by the pre-tRNA processing endonuclease tRNase Z, an ancient, conserved member of the beta-lactamase superfamily of metal-dependent hydrolases. The signature sequence of this family, the His domain (HxHxDH, Motif II), and histidines in Motifs III and V and aspartate in Motif IV contribute seven side chains for the coordination of two divalent metal ions. We previously investigated the effects on catalysis of substitutions in Motif II and in the PxKxRN loop and Motif I on the amino side of Motif II. Herein, we present the effects of substitutions on the carboxy side of Motif II within Motifs III, IV, the HEAT and HST loops, and Motif V. Substitution of the Motif IV aspartate reduces catalytic efficiency more than 10,000-fold. Histidines in Motif III, V, and the HST loop are also functionally important. Strikingly, replacement of Glu in the HEAT loop with Ala reduces efficiency by approximately 1000-fold. Proximity and orientation of this Glu side chain relative to His in the HST loop and the importance of both residues for catalysis suggest that they function as a duo in proton transfer at the final stage of reaction, characteristic of the tRNase Z class of RNA endonucleases.
Subject(s)
Endoribonucleases/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , Base Sequence , Catalysis , Conserved Sequence , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Endoribonucleases/genetics , Histidine/chemistry , Histidine/genetics , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Sequence AlignmentABSTRACT
tRNase Z, which can endonucleolytically remove pre-tRNA 3'-end trailers, possesses the signature His domain (HxHxDH; Motif II) of the beta-lactamase family of metal-dependent hydrolases. Motif II combines with Motifs III-V on its carboxy side to coordinate two divalent metal ions, constituting the catalytic core. The PxKxRN loop and Motif I on the amino side of Motif II have been suggested to modulate tRNase Z activity, including the anti-determinant effect of CCA in mature tRNA. Ala walks through these two homology blocks reveal residues in which the substitutions unexpectedly reduce catalytic efficiency. While substitutions in Motif II can drastically affect k(cat) without affecting k(M), five- to 15-fold increases in k(M) are observed with substitutions in several conserved residues in the PxKxRN loop and Motif I. These increases in k(M) suggest a model for substrate binding. Expressed tRNase Z processes mature tRNA with CCA at the 3' end approximately 80 times less efficiently than a pre-tRNA possessing natural sequence of the 3'-end trailer, due to reduced k(cat) with no effect on k(M), showing the CCA anti-determinant to be a characteristic of this enzyme.
Subject(s)
Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Endoribonucleases/chemistry , Endoribonucleases/metabolism , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , RNA, Transfer/metabolism , Alanine/genetics , Alanine/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Arginine/genetics , Arginine/metabolism , Bacillus subtilis/enzymology , Drosophila Proteins/genetics , Endoribonucleases/genetics , Glycine/genetics , Glycine/metabolism , Histidine/chemistry , Histidine/genetics , Molecular Sequence Data , Point Mutation , Protein Structure, Secondary , Protein Structure, Tertiary , RNA Precursors/genetics , Sequence Alignment , Sequence Homology, Amino Acid , beta-Lactamases/metabolismABSTRACT
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.
