Analysis of the Functional Modules of the tRNA 3′ Endonuclease (tRNase Z)

Bettina Späth,Silvia Kirchner,Andreas Vogel,Sylvia Schubert,Petra Meinlschmidt,Simone Aymanns,Jamel Nezzar,Anita Marchfelder

doi:10.1074/jbc.m506418200

Abstract

tRNA 3' processing is one of the essential steps during tRNA maturation. The tRNA 3'-processing endonuclease tRNase Z was only recently isolated, and its functional domains have not been identified so far. We performed an extensive mutational study to identify amino acids and regions involved in dimerization, tRNA binding, and catalytic activity. 29 deletion and point variants of the tRNase Z enzyme were generated. According to the results obtained, variants can be sorted into five different classes. The first class still had wild type activity in all three respects. Members of the second and third class still formed dimers and bound tRNAs but had reduced catalytic activity (class two) or no catalytic activity (class three). The fourth class still formed dimers but did not bind the tRNA and did not process precursors. Since this class still formed dimers, it seems that the amino acids mutated in these variants are important for RNA binding. The fifth class did not have any activity anymore. Several conserved amino acids could be mutated without or with little loss of activity.

Highlights

It has been shown that in Escherichia coli, tRNA 3Ј maturation is a multistep process involving endo- as well as exonucleases, the final steps being performed by an exonuclease [3]
The tRNase ZS proteins are present in all kingdoms, the tRNase ZL enzymes can only be found in eukarya
Characteristics of the tRNase Z Enzyme From A. thaliana—The TRZ1 protein is a homodimer, as are the homologous proteins from E. coli [18], T. maritima [21], and B. subtilis [20], and it tightly binds to tRNAs and is catalyzing tRNA 3Ј end processing

Summary

Introduction

It has been shown that in Escherichia coli, tRNA 3Ј maturation is a multistep process involving endo- as well as exonucleases, the final steps being performed by an exonuclease [3]. TRZ1 belongs to the family of metal-dependent ␤-lactamases [9], a group of metalloproteins that perform a variety of diverse functions (10 –12). This metalloprotein family was classified into 16 subgroups [12], and the tRNase Z enzymes are part of the Elac1/Elac subgroup. Other subgroups include the 3Ј mRNA cleavage and adenylation specificity factors [13], SNM1 ( named PSO2), and Artemis [14, 15], proteins that are involved in DNA repair [16] Another subgroup consists of cAMP phosphodiesterase enzymes [17], which catalyze the hydrolysis of cAMP to the corresponding nucleoside 5Ј monophosphate. The exosite (an element outside the active site that participates in substrate binding) protrudes from the main protein body pointing toward the solvent

Results

Discussion

Conclusion