Unusual synthesis by the Escherichia coli CCA-adding enzyme

Hou, Ya‐Ming

doi:10.1017/s1355838200000686

Cited by 35 publications

(57 citation statements)

References 40 publications

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“…These products are, respectively, tRNA-C74C75 and tRNA-C74C75C76 (where the incorporated residues are underlined). The synthesis of the latter product is consistent with the polyC activity of CCA enzymes, when CTP is used as the sole nucleotide substrate (Hou 2000;Seth et al 2002;Hou et al 2005).…”

Section: Resultssupporting

confidence: 73%

Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3′ end

Zhang¹,

Liu²,

Christian³

et al. 2008

RNA

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All mature tRNA molecules have the conserved CCA sequence at the 39 end with a range of dynamic conformations that are important for tRNA functions. We present here the details of a general approach to fluorescent labeling of the CCA sequence with the fluorescent base analog pyrrolo-C (PyC) at position 75 as a molecular probe for monitoring the dynamics of the tRNA 39 end. Using Escherichia coli tRNACys as an example, we achieve such labeling by first synthesizing the tRNA as a transcript up to C74 and then employing the tRNA CCA-adding enzyme to incorporate PyC75 and A76, using pyrrolo-CTP (PyCTP) and ATP as the respective substrates. PyC-labeled full-length tRNA Cys , separated from the unlabeled precursor tRNA by reverse phase highpressure liquid chromatography, is an efficient substrate for aminoacylation by E. coli cysteinyl-tRNA synthetase (CysRS). Fluorescence binding measurement of the PyC-labeled tRNACys with E. coli CysRS reveals an equilibrium K d closely similar to the value determined from the fluorescence of intrinsic enzyme tryptophans. Kinetic measurements of translocation of the PyClabeled tRNA from the ribosomal A to P sites identify a kinetic intermediate with a rate of formation and decay similar to the values reported for tRNAs labeled with the fluorescent proflavin at the tertiary core. These results highlight the potential of PyC to probe the dynamics of the tRNA CCA end in reactions ranging from aminoacylation to those on the ribosome.

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Section: Resultssupporting

confidence: 73%

Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3′ end

Zhang¹,

Liu²,

Christian³

et al. 2008

RNA

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“…Because it is not yet possible to distinguish Class II CCA-adding enzymes from poly(A) polymerases based solely on sequence analysis (38), we included only those enzymes whose enzymatic activities had been experimentally demonstrated or could be confidently assumed. These are the H. influenzae CCAadding enzyme and poly(A) polymerase, both of which are almost identical to their experimentally characterized E. coli counterparts (21,23,39), the B. subtilis and M. leprae CCA-adding enzymes (38), and the CCA-adding enzyme from M. tuberculosis, which is almost identical to M. leprae enzyme (31). A multiple alignment of the 25-kDa core regions of these Class II enzymes, including the active site signature, is shown in Fig.…”

Section: A Phylogeny Of Eubacterial Cca- Cc- and A-adding Enzymes Amentioning

confidence: 73%

Closely Related CC- and A-adding Enzymes Collaborate to Construct and Repair the 3′-Terminal CCA of tRNA in Synechocystis sp. and Deinococcus radiodurans

Tomita¹,

Weiner

2002

Journal of Biological Chemistry

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The 3-terminal CCA sequence of tRNA is faithfully constructed and repaired by the CCA-adding enzyme (ATP(CTP):tRNA nucleotidyltransferase) using CTP and ATP as substrates but no nucleic acid template. Until recently, all CCA-adding enzymes from all three kingdoms appeared to be composed of a single kind of polypeptide with dual specificity for adding both CTP and ATP; however, we recently found that in Aquifex aeolicus, which lies near the deepest root of the eubacterial 16 S rRNA-based phylogenetic tree, CCA addition represents a collaboration between closely related CCadding and A-adding enzymes (Tomita, K. and Weiner, A. M. (2001) Science 294, 1334 -1336). Here we show that in Synechocystis sp. and Deinococcus radiodurans, as in A. aeolicus, CCA is added by homologous CC-and A-adding enzymes. We also find that the eubacterial CCA-, CC-, and A-adding enzymes, as well as the related eubacterial poly(A) polymerases, each fall into phylogenetically distinct groups derived from a common ancestor. Intriguingly, the Thermatoga maritima CCA-adding enzyme groups with the A-adding enzymes, suggesting that these distinct tRNA nucleotidyltransferase activities can intraconvert over evolutionary time.The 3Ј-terminal CCA sequence (positions 74 -76 in the standard cloverleaf representation) is universally present in the tRNAs of all organisms (1) and is important for many aspects of gene expression. The CCA terminus is required for the aminoacylation of tRNA (2, 3) and for translation on the ribosome where the CCA sequences of the aminoacyl-and peptidyl-tRNA pair with the large ribosomal RNA near the peptidyltransferase center (4 -6). In eubacteria, the CCA sequence is required for the efficient maturation of the 5Ј-end of tRNA by RNase P (7,8). In eukaryotes, the CCA sequence serves as an antideterminant to block 3Ј-exonuclease activity (9), and it is essential for the export of mature tRNA from the nucleus to the cytoplasm (10, 11).The CCA-adding enzyme (ATP(CTP):tRNA nucleotidyltransferase) builds and repairs the 3Ј-terminal CCA sequence of all tRNAs (12). CCA-adding activity has been identified in all three kingdoms, suggesting conservation of function and perhaps structure throughout evolution (13). CCA-adding activity is essential in some eubacteria as well as in all archaea and eukaryotes where some or all tRNA genes do not encode CCA (14). Yet, even in organisms such as Escherichia coli where all tRNA genes do encode CCA, CCA-adding activity confers a substantial selective advantage, probably by repairing tRNAs that have been subject to errant nucleolytic attack (15).The CCA-adding enzyme belongs to the nucleotidyltransferase (NTR) 1 family, a large protein superfamily that encompasses template-dependent DNA polymerases (DNA polymerase ␤) and template-independent RNA and DNA polymerases (poly(A) polymerase, terminal deoxynucleotidyltransferase, and CCA-adding enzymes) as well as metabolic regulators (GlnB uridylyltransferase, glutamine synthase adenylyltransferase) and antibiotic resistance factors (kanamycin nucleo...

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“…The 3Ј-terminal stem/loop IV of U2 snRNA alone was also a good substrate (Fig. 2, C and D), suggesting that the 3Ј end of U2 snRNA resembles a tRNA minihelix (57,58) and that the TC loop contributes marginally, if at all, to substrate recognition (12).…”

Section: Resultsmentioning

confidence: 99%

“…Thus, the SRP RNA adenylating enzyme behaves like a poly(A) polymerase and is unlikely to play a role in U2 snRNA biosynthesis. These results suggest that the 3Ј stem/loop of U2 snRNA does in fact resemble a tRNA minihelix, the smallest efficient substrate for class I and II CCA-adding enzymes (12,57), and that CCA addition to U2 snRNA could take place in vivo.…”

mentioning

confidence: 94%

“…tRNA consists of two structurally and functionally independent domains: a "top half" or minihelix consisting of the acceptor stem stacked on the TC stem/loop, and a "bottom half" consisting of the DHU stem/loop stacked on the anticodon stem/loop (77,78). Not only does a tRNA minihelix serve as substrate for CCA-adding enzymes (12,57), but single-stranded RNA viruses often have 3Ј-terminal tRNA-like structures that serve as substrates for enzymes of tRNA metabolism including tRNA synthetases, the CCA-adding enzyme, and RNase P (11, 79 -81). Maize mitochondrial mRNAs also contain posttranscriptionally added 3Ј-terminal nucleotides that could be the product of a mitochondrial form of the CCA-adding enzyme (82).…”

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confidence: 99%

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U2 Small Nuclear RNA Is a Substrate for the CCA-adding Enzyme (tRNA Nucleotidyltransferase)

Cho

Tomita

Suzuki

et al. 2002

Journal of Biological Chemistry

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The CCA-adding enzyme builds and repairs the 3 terminus of tRNA. Approximately 65% of mature human U2 small nuclear RNA (snRNA) ends in 3-terminal CCA, as do all mature tRNAs; the other 35% ends in 3 CC or possibly 3 C. The 3-terminal A of U2 snRNA cannot be encoded because the 3 end of the U2 snRNA coding region is CC/CC, where the slash indicates the last encoded nucleotide. The first detectable U2 snRNA precursor contains 10 -16 extra 3 nucleotides that are removed by one or more 3 exonucleases. Thus, if 3 exonuclease activity removes the encoded 3 CC during U2 snRNA maturation, as appears to be the case in vitro, the cell may need to build or rebuild the 3-terminal A, CA, or CCA of U2 snRNA. We asked whether homologous and heterologous class I and class II CCA-adding enzymes could add 3-terminal A, CA, or CCA to human U2 snRNA lacking 3-terminal A, CA, or CCA. The naked U2 snRNAs were good substrates for the human CCA-adding enzyme but were inactive with the Escherichia coli enzyme; activity was also observed on native U2 snRNPs. We suggest that the 3 stem/loop of U2 snRNA resembles a tRNA minihelix, the smallest efficient substrate for class I and II CCA-adding enzymes, and that CCA addition to U2 snRNA may take place in vivo after snRNP assembly has begun.

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Unusual synthesis by the Escherichia coli CCA-adding enzyme

Cited by 35 publications

References 40 publications

Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3′ end

Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3′ end

Closely Related CC- and A-adding Enzymes Collaborate to Construct and Repair the 3′-Terminal CCA of tRNA in Synechocystis sp. and Deinococcus radiodurans

U2 Small Nuclear RNA Is a Substrate for the CCA-adding Enzyme (tRNA Nucleotidyltransferase)

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