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Kozo Tomita

Publications and source records attributed to Kozo Tomita.

9 recordsLinked to original sources
PubMed2006

Crystal structures of leucyl/phenylalanyl-tRNA-protein transferase and its complex with an aminoacyl-tRNA analog.

Eubacterial leucyl/phenylalanyl-tRNA protein transferase (L/F-transferase), encoded by the aat gene, conjugates leucine or phenylalanine to the N-terminal Arg or Lys residue of proteins, using Leu-tRNA(Leu) or Phe-tRNA(Phe) as a substrate. The resulting N-terminal Leu or Phe acts as a degradation signal for the ClpS-ClpAP-mediated N-end rule protein degradation pathway. Here, we present the crystal structures of Escherichia coli L/F-transferase and its complex with an aminoacyl-tRNA analog, puromycin. The C-terminal domain of L/F-transferase consists of the GCN5-related N-acetyltransferase fold, commonly observed in the acetyltransferase superfamily. The p-methoxybenzyl group of puromycin, corresponding to the side chain of Leu or Phe of Leu-tRNA(Leu) or Phe-tRNA(Phe), is accommodated in a highly hydrophobic pocket, with a shape and size suitable for hydrophobic amino-acid residues lacking a branched beta-carbon, such as leucine and phenylalanine. Structure-based mutagenesis of L/F-transferase revealed its substrate specificity. Furthermore, we present a model of the L/F-transferase complex with tRNA and substrate proteins bearing an N-terminal Arg or Lys.

Amino Acid Sequence↗
PubMed2006

Complete crystallographic analysis of the dynamics of CCA sequence addition.

CCA-adding polymerase matures the essential 3'-CCA terminus of transfer RNA without any nucleic-acid template. However, it remains unclear how the correct nucleotide triphosphate is selected in each reaction step and how the polymerization is driven by the protein and RNA dynamics. Here we present complete sequential snapshots of six complex structures of CCA-adding enzyme and four distinct RNA substrates with and without CTP (cytosine triphosphate) or ATP (adenosine triphosphate). The CCA-lacking RNA stem extends by one base pair to force the discriminator nucleoside into the active-site pocket, and then tracks back after incorporation of the first cytosine monophosphate (CMP). Accommodation of the second CTP clamps the catalytic cleft, inducing a reorientation of the turn, which flips C74 to allow CMP to be accepted. In contrast, after the second CMP is added, the polymerase and RNA primer are locked in the closed state, which directs the subsequent A addition. Between the CTP- and ATP-binding stages, the side-chain conformation of Arg 224 changes markedly; this is controlled by the global motion of the enzyme and position of the primer terminus, and is likely to achieve the CTP/ATP discrimination, depending on the polymerization stage. Throughout the CCA-adding reaction, the enzyme tail domain firmly anchors the TPsiC-loop of the tRNA, which ensures accurate polymerization and termination.

Adenosine Monophosphate↗
PubMed2004

Structural basis for template-independent RNA polymerization.

The 3'-terminal CCA nucleotide sequence (positions 74-76) of transfer RNA is essential for amino acid attachment and interaction with the ribosome during protein synthesis. The CCA sequence is synthesized de novo and/or repaired by a template-independent RNA polymerase, 'CCA-adding enzyme', using CTP and ATP as substrates. Despite structural and biochemical studies, the mechanism by which the CCA-adding enzyme synthesizes the defined sequence without a nucleic acid template remains elusive. Here we present the crystal structure of Aquifex aeolicus CCA-adding enzyme, bound to a primer tRNA lacking the terminal adenosine and an incoming ATP analogue, at 2.8 A resolution. The enzyme enfolds the acceptor T helix of the tRNA molecule. In the catalytic pocket, C75 is adjacent to ATP, and their base moieties are stacked. The complementary pocket for recognizing C74-C75 of tRNA forms a 'protein template' for the penultimate two nucleotides, mimicking the nucleotide template used by template-dependent polymerases. These results are supported by systematic analyses of mutants. Our structure represents the 'pre-insertion' stage of selecting the incoming nucleotide and provides the structural basis for the mechanism underlying template-independent RNA polymerization.

Adenosine Monophosphate↗
PubMed2003

Divergent evolutions of trinucleotide polymerization revealed by an archaeal CCA-adding enzyme structure.

CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase], a template-independent RNA polymerase, adds the defined 'cytidine-cytidine-adenosine' sequence onto the 3' end of tRNA. The archaeal CCA-adding enzyme (class I) and eubacterial/eukaryotic CCA-adding enzyme (class II) show little amino acid sequence homology, but catalyze the same reaction in a defined fashion. Here, we present the crystal structures of the class I archaeal CCA-adding enzyme from Archaeoglobus fulgidus, and its complexes with CTP and ATP at 2.0, 2.0 and 2.7 A resolutions, respectively. The geometry of the catalytic carboxylates and the relative positions of CTP and ATP to a single catalytic site are well conserved in both classes of CCA-adding enzymes, whereas the overall architectures, except for the catalytic core, of the class I and class II CCA-adding enzymes are fundamentally different. Furthermore, the recognition mechanisms of substrate nucleotides and tRNA molecules are distinct between these two classes, suggesting that the catalytic domains of class I and class II enzymes share a common origin, and distinct substrate recognition domains have been appended to form the two presently divergent classes.

Adenosine Triphosphate↗
PubMed2002

Crystal structures of the Bacillus stearothermophilus CCA-adding enzyme and its complexes with ATP or CTP.

CCA-adding enzymes polymerize CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template. The 3.0 A resolution crystal structures of the CCA-adding enzyme from Bacillus stearothermophilus and its complexes with ATP or CTP reveal a seahorse-shaped subunit consisting of four domains: head, neck, body, and tail. The head is structurally homologous to the palm domain of DNA polymerase beta but has additional structural features and functions. The neck, body, and tail represent new protein folding motifs. The neck provides a specific template for the incoming ATP or CTP, whereas the body and tail may bind tRNA. Each subunit has one active site capable of switching its base specificity between ATP and CTP, an important component of the CCA-adding mechanism.

Adenosine Triphosphate↗
PubMed2002

Closely related CC- and A-adding enzymes collaborate to construct and repair the 3'-terminal CCA of tRNA in Synechocystis sp. and Deinococcus radiodurans.

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 CC-adding 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.

Amino Acid Sequence↗
PubMed2002

The cephalopod Loligo bleekeri mitochondrial genome: multiplied noncoding regions and transposition of tRNA genes.

We previously reported the sequence of a 9260-bp fragment of mitochondrial (mt) DNA of the cephalopod Loligo bleekeri [J. Sasuga et al. (1999) J. Mol. Evol. 48:692-702]. To clarify further the characteristics of Loligo mtDNA, we have sequenced an 8148-bp fragment to reveal the complete mt genome sequence. Loligo mtDNA is 17,211 bp long and possesses a standard set of metazoan mt genes. Its gene arrangement is not identical to any other metazoan mt gene arrangement reported so far. Three of the 19 noncoding regions longer than 10 bp are 515, 507, and 509 bp long, and their sequences are nearly identical, suggesting that multiplication of these noncoding regions occurred in an ancestral Loligo mt genome. Comparison of the gene arrangements of Loligo, Katharina tunicata, and Littorina saxatilis mt genomes revealed that 17 tRNA genes of the Loligo mt genome are adjacent to noncoding regions. A majority (15 tRNA genes) of their counterparts is found in two tRNA gene clusters of the Katharina mt genome. Therefore, the Loligo mt genome (17 tRNA genes) may have spread over the genome, and this may have been coupled with the multiplication of the noncoding regions. Maximum likelihood analysis of mt protein genes supports the clade Mollusca + Annelida + Brachiopoda but fails to infer the relationships among Katharina, Loligo, and three gastropod species.

Animals↗
PubMed2001

U2 small nuclear RNA is a substrate for the CCA-adding enzyme (tRNA nucleotidyltransferase).

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.

Adenosine Triphosphate↗