Dataset Information

Effect of changes in the flexible arm on tRNase Z processing kinetics.

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.

SUBMITTER: Levinger L

PROVIDER: S-EPMC2708865 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Effect of changes in the flexible arm on tRNase Z processing kinetics.

Levinger Louis L Hopkinson Angela A Desetty Rohini R Wilson Christopher C

The Journal of biological chemistry 20090407 23

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/ ...[more]

PMID: 19351879

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Project description:BACKGROUND: tRNase Z removes the 3'-trailer sequences from precursor tRNAs, which is an essential step preceding the addition of the CCA sequence. tRNase Z exists in the short (tRNase ZS) and long (tRNase ZL) forms. Based on the sequence characteristics, they can be divided into two major types: bacterial-type tRNase ZS and eukaryotic-type tRNase ZL, and one minor type, Thermotoga maritima (TM)-type tRNase ZS. The number of tRNase Zs is highly variable, with the largest number being identified experimentally in the flowering plant Arabidopsis thaliana. It is unknown whether multiple tRNase Zs found in A. thaliana is common to the plant kingdom. Also unknown is the extent of sequence and structural conservation among tRNase Zs from the plant kingdom. RESULTS: We report the identification and analysis of candidate tRNase Zs in 27 fully sequenced genomes of green plants, the great majority of which are flowering plants. It appears that green plants contain multiple distinct tRNase Zs predicted to reside in different subcellular compartments. Furthermore, while the bacterial-type tRNase ZSs are present only in basal land plants and green algae, the TM-type tRNase ZSs are widespread in green plants. The protein sequences of the TM-type tRNase ZSs identified in green plants are similar to those of the bacterial-type tRNase ZSs but have distinct features, including the TM-type flexible arm, the variant catalytic HEAT and HST motifs, and a lack of the PxKxRN motif involved in CCA anti-determination (inhibition of tRNase Z activity by CCA), which prevents tRNase Z cleavage of mature tRNAs. Examination of flowering plant chloroplast tRNA genes reveals that many of these genes encode partial CCA sequences. Based on our results and previous studies, we predict that the plant TM-type tRNase ZSs may not recognize the CCA sequence as an anti-determinant. CONCLUSIONS: Our findings substantially expand the current repertoire of the TM-type tRNase ZSs and hint at the possibility that these proteins may have been selected for their ability to process chloroplast pre-tRNAs with whole or partial CCA sequences. Our results also support the coevolution of tRNase Zs and tRNA 3'-trailer sequences in plants.

Dataset Information

Effect of changes in the flexible arm on tRNase Z processing kinetics.

Publications

Effect of changes in the flexible arm on tRNase Z processing kinetics.

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