Effects of conserved D/T loop substitutions in the pre-tRNA substrate on tRNase Z catalysis

Hopkinson, Angela; Levinger, Louis

doi:10.4161/rna.5.2.6086

Cited by 7 publications

(8 citation statements)

References 32 publications

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“…These conserved features and the characteristic length of the coaxially stacked acceptor and T stems present potential characteristics for recognition by tRNase Z and other enzymes in the tRNA maturation pathway that distinguish tRNAs from other RNA molecules without discriminating between individual tRNAs (36 -38). Moderate effects on K m of human tRNase Z L were observed with substitutions in the D and T loops of the substrate (28), consistent with a function in tRNase Z recognition for these structural elements. Importance of the elbow and T loop for tRNase Z reaction has been questioned (39), but this and similar studies were performed with a much higher enzyme concentration than in the present study.…”

Section: Discussionmentioning

confidence: 68%

“…These results demonstrate that the FA of tRNase Z is involved with substrate recognition and binding remote from its active site and from the scissile bond of the substrate. Some of the substrate D/T loop substitutions in human tRNA Arg also cause the K m for tRNase Z L to increase, suggesting effects on binding (28). The FA of Thermotoga maritima tRNase Z (a short form with an atypical cleavage site and FA) (29,30) is dispensable, but some of the mutations affected its cleavage site (31).…”

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confidence: 99%

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Effect of Changes in the Flexible Arm on tRNase Z Processing Kinetics

Levinger

Hopkinson

Desetty

et al. 2009

Journal of Biological Chemistry

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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 ␣␣␤␤ hand that binds the elbow of pre-tRNA. Deleting the FA hand causes an almost 100-fold increase in K m with little change in k cat , 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 K m as the hand deletion, thus nearly eliminating its ability to bind substrate. K m also increases with substitutions in the GP (␣4 -␣5) loop and at other conserved residues in the FA hand predicted to contact substrate based on the co-crystal structure. Substitutions that reduce k cat are clustered in the ␤10 -␤11 loop.tRNAs are transcribed as precursors with a 5Ј end leader and 3Ј end trailer. The 5Ј end leader is removed by RNase P. The 3Ј end trailer can be endonucleolytically removed by tRNase Z, which cleaves following the unpaired nucleotide just beyond the 3Ј side of the acceptor stem (the discriminator) leaving a 3Ј-OH ready for CCA addition. In some bacteria and in all archaea and eukaryotes (including their organelles), CCA at the 3Ј end of mature tRNAs is not transcriptionally encoded, and a CCA-adding enzyme is required (1); endonucleolytic processing by tRNase Z is thus a precise and probably essential reaction in the pathway to a mature 3Ј end (2, 3).Interestingly, the 3Ј end CCA is an anti-determinant for tRNase Z that discourages the recycling of mature tRNAs (4 -7), although not in every case (8). Additional functions have been suggested for tRNase Z, including a possible role in human prostate cancer susceptibility (2, 9 -12). In some instances, tRNase Z can recognize and cleave RNAs that are structurally related to pre-tRNAs with 3Ј end extensions (10, 12).tRNase Z is an ancient member of the ␤-lactamase superfamily of metal-dependent hydrolases (2, 13). The signature sequence of this family, the His domain (HXHXDH, Motif II), in conjunction with histidines in Motifs III and V and aspartate in Motif IV, contributes side chains that coordinate two divalent metal ions (14, 15). Additionally, the Glu side chain in HEAT and His in the HST loop (located between Motifs IV and V) apparently function as a pair to facilitate proton transfer at the final stage of reaction (16,17). A Glu-His pair in CPSF-73, the long sought endonuclease responsible for pre-mRNA cleavage and a member of the tRNase Z class of RNA endonucleases (13,16,18), displays the same structure relative to the active site and presumably functions identically in catalysis.Substitutions in Motifs II-V, HEAT, and HST loop residues did not show increases in K m (17,19); thus, these residues apparently contribute to metal ion binding and catalysis without being involved with substrate reco...

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Section: Discussionmentioning

confidence: 68%

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confidence: 99%

Effect of Changes in the Flexible Arm on tRNase Z Processing Kinetics

Levinger

Hopkinson

Desetty

et al. 2009

Journal of Biological Chemistry

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“…First, the flexible arm has been suggested to contribute to cleavage site selection and the inhibitory effect of the CCA sequence [62]. This tRNase Z-specific element is located on the opposite side of the active site and binds primarily the D and T loops of the pre-tRNA [28,39,63]. Notably, the flexible arms of the T. maritima and plant tRNase Z S s lack the GP motif but contain the KL motif.…”

Section: Discussionmentioning

confidence: 99%

A survey of green plant tRNA 3'-end processing enzyme tRNase Zs, homologs of the candidate prostate cancer susceptibility protein ELAC2

et al. 2011

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BackgroundtRNase 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.ResultsWe 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.ConclusionsOur 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.

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“…Position A 58 was the most sensitive in a survey of nuclear encoded tRNA Arg with D and T loop substitutions. 42 These changes may impair the tRNA-tRNase Z interaction, as suggested by the increases in K M ( Table 2).…”

Section: Next Is Trnamentioning

confidence: 99%

“…Previous work 31,42 suggests that structural determinants for tRNase Z activity are present in the T-loop, effects of substitutions in the D-loop being modulatory and less definitive. This investigation of mutant mitochondrial tRNAs was therefore limited to the T-loop.…”

Section: Introductionmentioning

confidence: 99%

Pathogenesis-related mutations in the T-loops of human mitochondrial tRNAs affect 3′ end processing and tRNA structure

Levinger

Serjanov²

2012

RNA Biology

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Numerous mutations in the mitochondrial genome are associated with maternally transmitted diseases and syndromes that affect muscle and other high energy-demand tissues. The mitochondrial genome encodes 13 polypeptides, 2 rRNAs and 22 interspersed tRNAs via long bidirectional polycistronic primary transcripts, requiring precise excision of the tRNAs. Despite making up only~10% of the mitochondrial genome, tRNA genes harbor most of the pathogenesis-related mutations. tRNase Z endonucleolytically removes the pre-tRNA 3' trailer. The flexible arm of tRNase Z recognizes and binds the elbow (including the T-loop) of pre-tRNA. Pathogenesis-related T-loop mutations in mitochondrial tRNAs could thus affect tRNA structure, reduce tRNase Z binding and 3' processing, and consequently slow mitochondrial protein synthesis. Here we inspect the effects of pathogenesis-related mutations in the T-loops of mitochondrial tRNAs on pretRNA structure and tRNase Z processing. Increases in K M arising from 59A . G substitutions in mitochondrial tRNA Gly and tRNA Ile accompany changes in T-loop structure, suggesting impaired substrate binding to enzyme.

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Effects of conserved D/T loop substitutions in the pre-tRNA substrate on tRNase Z catalysis

Cited by 7 publications

References 32 publications

Effect of Changes in the Flexible Arm on tRNase Z Processing Kinetics

Effect of Changes in the Flexible Arm on tRNase Z Processing Kinetics

A survey of green plant tRNA 3'-end processing enzyme tRNase Zs, homologs of the candidate prostate cancer susceptibility protein ELAC2

Pathogenesis-related mutations in the T-loops of human mitochondrial tRNAs affect 3′ end processing and tRNA structure

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