Tethered Domains and Flexible Regions in tRNase ZL, the Long Form of tRNase Z

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 ZS) is the only one found in bacteria and archaebacteria and is also present in some eukaryotes. The homologous long form (tRNase ZL), exclusively found in eukaryotes, consists of related amino- and carboxy-domains, suggesting that tRNase ZL arose from a tandem duplication of tRNase ZS followed by interdependent divergence of the domains. X-ray crystallographic structures of tRNase ZS 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 ZL structures have been solved; alternative biophysical studies are therefore needed to illuminate its functional characteristics. Structural analyses of tRNase ZL 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 ZL function

Sequence and predicted secondary structure of <i>H. sapiens</i> tRNase Z^L (Psipred).

<p>Enclosed regions N_dom, Tether and C_dom and annotation of functional motifs are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066942#pone-0066942-g004" target="_blank">Figure 4</a>. Polymorphisms and isoforms (identified by enclosure in blue circles and orange ellipses, respectively) are described in detail in Supplement.</p

Two dimensional gel electrophoresis of <i>D. melanogaster</i> tRNase Z-trypsin polypeptides.

<p>A three min. digest like those illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066942#pone-0066942-g003" target="_blank">Figure 3</a> was electrophoresed on a first dimension isoelectric focusing tube gel. The second dimension SDS-PAGE was the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066942#pone-0066942-g003" target="_blank">Figure 3</a>.</p

Maldi-ion trap mass spectra of exhaustive in-gel trypsin digest products.

<p>Spots were excised from the two dimensional gel (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066942#pone-0066942-g005" target="_blank">Figure 5</a>). (A) Full length <i>D. melanogaster</i> tRNase Z. (B) A representative C_dom2 partial proteolysis polypeptide. Start and end residue numbers are indicated for all peaks that can be positively assigned to tRNase Z^L tryptic peptides. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066942#pone-0066942-t001" target="_blank">table 1</a> for details. Peptides present in both samples are labeled in black; those present in the full length protein but not C_dom2 are labeled in red.</p

Sequence and predicted secondary structure of <i>D. melanogaster</i> tRNase Z (Psipred).

<p>Large rectangular enclosures indicate amino domain, tether and carboxy domain. Dashed rectangular enclosures indicate functionally characterized motifs (discussed in detail and further annotated with a multiple sequence alignment in Supplemental Figure SF2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066942#pone.0066942.s003" target="_blank">File S1</a>).</p

tRNase Z purifies as a monomer.

<p>(A) Affinity purified <i>D. melanogaster</i> tRNase Z was separated by SEC using a Superose 6 10/300 GL column on the Akta Purifier 10 platform (GE Life Sciences). Symbols above the absorbance profile indicate elution peaks for Ovalbumin (45 kD), BSA (66 kD) and Alcohol Dehydrogenase (141 kD). tRNase Z (green triangles) chromatograms as a monomer true to its ∼84 kD molecular weight. * identifies a shoulder in the chromatogram coinciding with expected position of the tRNase Z^L dimer which is apparently low in quantity and enzyme activity. Lines beneath axis indicate peak fractions. B) Protein gel. UF: Unfractionated tRNase Z. Pool – peak fractions were pooled and concentrated. C) Processing assay of three peak fractions compared with UF indicates that gel filtration improves enzyme specific activity. Each fraction was incubated with end-labeled substrate for 15 min. and electrophoresed on a denaturing polyacrylamide gel. S, P at right designate the 90 nt <i>D. melanogaster</i> pre-tRNA^Arg substrate and 73 nt product, respectively. Lane 0: no processing control.</p

Tethered Domains and Flexible Regions in tRNase Z^L, the Long Form of tRNase Z

<div><p>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.</p></div

Tether alignments.

<p>The long form of tRNase Z from <i>H. sapiens</i> (Hsa; Accession # NP_060597), <i>D. melanogaster</i> (Dme; Q8MKW7), <i>A. thaliana</i> (Ath; AAM51378), <i>C. elegans</i> (Cel; O4476) and <i>S. cereviseae</i> (Sce; NP013005.1) were aligned using Clustalw. The full length alignments are presented in Supplemental Figure SF2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066942#pone.0066942.s003" target="_blank">File S1</a>. Numbers at top are for <i>H. sapiens</i> tRNase ZL. Regions presented begin with the last identified homology block (N_dom-T_prox) in the amino domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066942#pone.0066942-Wang1" target="_blank">[41]</a> proximal to tether and include two smaller homology blocks in tether (rectangular enclosures).//indicates omitted sequence. Small bold rectangles enclose hydrophilic patches that mark the N_dom-tether boundary.</p

<i>D. melanogaster</i> tRNase Z Peptide Analysis.

<p>Tryptic Peptides from 2D gel spots were analyzed by MALDI-Ion trap MS. All peptides listed are found in full length tRNase Z.</p><p>Bold: N_dom and tether peptides are found in full length tRNase Z but not in C_dom2 region.</p><p>Italic: Absence of peptide 365–384 from C_dom2 defines the amino boundary of the C_dom2 polypeptide.</p>1<p>First seven residues are leader following TEV site.</p

Two dimensional gel electrophoresis of <i>H. sapiens</i> tRNase Z^L-trypsin polypeptides.

<p>A ten min. digest like those illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066942#pone-0066942-g005" target="_blank">Figure 5</a> was electrophoresed on a first dimension isoelectric focusing tube gel. The second dimension SDS-PAGE was as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066942#pone-0066942-g005" target="_blank">Figure 5</a>.</p