Purification and properties of yeast ATP (CTP):tRNA nucleotidyltransferase from wild type and overproducing cells

ATP (CTP):tRNA nucleotidyltransferase (EC 2.7.7.25) has been purified from wild type cells of the yeast Saccharomyces cerevisiae, as well as from a strain that overproduces the activity. Purification from the wild type strain was accomplished with a multistep protocol including ammonium sulfate fractionation, anion exchange chromatography, gel filtration, and affinity chromatography. The purified enzyme is near homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at 59,000 Da is smaller than reported previously. A similar molecular mass is obtained by gel filtration demonstrating that the enzyme is active as a monomer. The pH optimum for the enzyme is around 9.5. The apparent KM values for ATP and CTP were determined to be 5.6 x 10(-4) M and 1.8 x 10(-4) M, respectively. Purification of the enzyme from the overproducing cells was accomplished by a three step protocol with high yield. The nucleotidyltransferase activity from the overproducing cells had a KM for CTP indistinguishable from that of the wild type enzyme, and the mobility of the protein on sodium dodecyl sulfate gels was the same regardless of the source. Thus, the overproducing strain appears to be a good source for large amounts of yeast nucleotidyltransferase for further biochemical and structural studies.     

Purines in tRNAs required for recognition by ATP/CTP:tRNA nucleotidyltransferase from rabbit liver

Recognition of tRNA by the enzyme ATP/CTP:tRNA nucleotidyltransferase from rabbit liver was studied using 12 tRNAs, previously treated with the chemical modifier diethylpyrocarbonate (DEP). Such chemically modified tRNAs were labeled with 32P by nucleotidyltransferase, using alpha-[32P]ATP as a cosubstrate. A carbethoxylated purine at position 57 in the psi-loop interfered with recognition of the tRNA in all instances. DEP-modified purines at other positions (58 in the psi-loop, 52 or 53 in the psi-stem, and 71-73 in the acceptor stem), also interfered with the interaction, but in only a few tRNAs. The mammalian enzyme was more similar to the homologous enzyme from yeast than that from bacteria, in its requirements for chemically unmodified purines. The extent of exclusion of modified bases from 32P-labeled material diminished as the concentration of enzyme increased, demonstrating that interference was not due to the inability of the chemically altered tRNA to refold into a recognizable conformation. The degree of purification of the enzyme did not affect the identity of bases that inhibited the reaction when modified.     

Preparation of oligonucleotides corresponding to the acceptor stem of yeast tRNAPhe and their interaction with yeast ATP(CTP):tRNA nucleotidyltransferase

Seven oligonucleotides corresponding to the 3' and 5' sequences of the acceptor stem of yeast tRNAPhe have been prepared by chemical synthesis, chemical-enzymatic synthesis or by isolation from tRNA hydrolysates. The oligonucleotides have been examined as substrates for phosphodiester bond synthesis in the presence of ATP as catalysed by yeast ATP (CTP): tRNA nucleotidyltransferase. Oligonucleotides which correspond to the sequence of the 3'-strand of the tRNA acceptor stem and possess no secondary structure exhibit little or no activity with the enzyme. The ability of the enzyme to catalyse the synthesis of a phosphodiester linkage using ATP and an oligonucleotide corresponding to the 3'-strand of the acceptor stem is in general dramatically increased when an oligonucleotide corresponding to the sequence of the 5'-strand of tRNA acceptor stem is present. In cases where significant activity was observed kinetic parameters have been determined.     

Transfer RNA pyrophosphorolysis with CTP(ATP):tRNA nucleotidyltransferase. A direct route to tRNAs modified at the 3' terminus

The pyrophosphorolysis of tRNA by yeast CTP-(ATP):tRNA nucleotidyltransferase has been studied in an effort to define the behavior of the enzyme and the experimental parameters that lead to net loss of the 3'-terminal nucleotide or to nucleotide exchange. It was found that removal of AMP from the terminus of tRNA proceeded optimally at 1.0 mM PPi; incorporation of 2'- or 3'-dAMP was also studied and shown to proceed optimally at a 6.0 mM concentration of deoxynucleoside triphosphate. CTP was shown to inhibit the pyrophosphorolysis and nucleotide exchange observed when starting from intact tRNA, but apparently not by inhibiting removal of CMP from tRNA missing the 3'-terminal adenosine moiety. The optimized conditions for nucleotide exchange were used for the preparative conversion of tRNAs to species terminating in 2'- and 3'-deoxyadenosine.     

Recognition of tRNA by the enzyme ATP/CTP:tRNA nucleotidyltransferase. Interference by nucleotides modified with diethyl pyrocarbonate or hydrazine

Treatment of tRNA with diethyl pyrocarbonate or hydrazine prior to incubation with the enzyme ATP/CTP:tRNA nucleotidyltransferase and [alpha-32P]ATP results in exclusion of modified bases from labeled molecules. Purines modified with diethyl pyrocarbonate, which interfere with enzyme recognition, cluster at the corner of the tRNA molecule, where the D- and psi-loops are juxtaposed in all 15 tRNAs used in this study. When the enzyme is isolated from Escherichia coli, few other sites of interference are evident near the 3'-end; when the homologous enzyme from yeast is used, more exclusions are apparent near the 3'-end. Modification of uridines with hydrazine has no effect on interaction with the enzyme, except for one uridine near the 3'-end of tRNA(Gly). Interference of enzyme activity by modified bases can be overcome by longer incubation times or increased concentrations of enzyme.     

tRNA processing in human mitochondrial disorders

Many human mitochondrial disorders are associated with mutations in tRNA genes or with deletions of regions containing tRNA genes, all of which may be suspected to play a role in recognition by RNase P. Here we describe the analysis of five such mutations. The results presented here demonstrate that none of thse mutations result in errors in RNase P function. Further studies of mutations in tRNAs need to be pursued to elucidate the identity elements for RNase P function in mammalian mitochondria.     

tRNA-nucleotidyltransferases: Highly unusual RNA polymerases with vital functions

tRNA-nucleotidyltransferases are fascinating and unusual RNA polymerases responsible for the synthesis of the nucleotide triplet CCA at the 3′-terminus of tRNAs. As this CCA end represents an essential functional element for aminoacylation and translation, these polymerases (CCA-adding enzymes) are of vital importance in all organisms. With a possible origin of ancient telomerase-like activity, the CCA-adding enzymes obviously emerged twice during evolution, leading to structurally different, but functionally identical enzymes. The evolution as well as the unique polymerization features of these interesting proteins will be discussed in this review.     

The pre-tRNA nucleotide base and 2'-hydroxyl at N(-1) contribute to fidelity in tRNA processing by RNase P

Fidelity in tRNA processing by the RNase P RNA from Escherichia coli depends, in part, on interactions with the nucleobase and 2' hydroxyl group of N(-1), the nucleotide immediately upstream of the site of RNA strand cleavage. Here, we report a series of biochemical and structure-function studies designed to address how these interactions contribute to cleavage site selection. We find that simultaneous disruption of cleavage site nucleobase and 2' hydroxyl interactions results in parallel reactions leading to correct cleavage and mis-cleavage one nucleotide upstream (5') of the correct site. Changes in Mg(2+) concentration and pH can influence the fraction of product that is incorrectly processed, with pH effects attributable to differences in the rate-limiting steps for the correct and mis-cleavage reaction pathways. Additionally, we provide evidence that interactions with the 2' hydroxyl group adjacent to the reactive phosphate group also contribute to catalysis at the mis-cleavage site. Finally, disruption of the adjacent 2'-hydroxyl contact has a greater effect on catalysis when pairing between the ribozyme and N(-1) is also disrupted, and the effects of simultaneously disrupting these contacts on binding are also non-additive. One implication of these results is that mis-cleavage will result from any combination of active site modifications that decrease the rate of correct cleavage beyond a certain threshold. Indeed, we find that inhibition of correct cleavage and corresponding mis-cleavage also results from disruption of any combination of active site contacts including metal ion interactions and conserved pairing interactions with the 3' RCCA sequence. Such redundancy in interactions needed for maintaining fidelity may reflect the necessity for multiple substrate recognition in vivo. These studies provide a framework for interpreting effects of substrate modifications on RNase P cleavage fidelity and provide evidence for interactions with the nucleobase and 2' hydroxyl group adjacent to the reactive phosphate group in the transition state.     

A tRNA's fate is decided at its 3′ end: Collaborative actions of CCA-adding enzyme and RNases involved in tRNA processing and degradation

tRNAs are key players in translation and are additionally involved in a wide range of distinct cellular processes. The vital importance of tRNAs becomes evident in numerous diseases that are linked to defective tRNA molecules. It is therefore not surprising that the structural intactness of tRNAs is continuously scrutinized and defective tRNAs are eliminated. In this process, erroneous tRNAs are tagged with single-stranded RNA sequences that are recognized by degrading exonucleases. Recent discoveries have revealed that the CCA-adding enzyme – actually responsible for the de novo synthesis of the 3′-CCA end – plays an indispensable role in tRNA quality control by incorporating a second CCA triplet that is recognized as a degradation tag. In this review, we give an update on the latest findings regarding tRNA quality control that turns out to represent an interplay of the CCA-adding enzyme and RNases involved in tRNA degradation and maturation. In particular, the RNase-induced turnover of the CCA end is now recognized as a trigger for the CCA-adding enzyme to repeatedly scrutinize the structural intactness of a tRNA. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.     

Sequence motifs that distinguish ATP(CTP):tRNA nucleotidyl transferases from eubacterial poly(A) polymerases

ATP(CTP):tRNA nucleotidyl transferases, tRNA maturing enzymes found in all organisms, and eubacterial poly(A) polymerases, enzymes involved in mRNA degradation, are so similar that until now their biochemical functions could not be distinguished by their amino acid sequence. BLAST searches and analysis with the program "Sequence Space" for the prediction of functional residues revealed sequence motifs which define these two protein families. One of the poly(A) polymerase defining motifs specifies a structure that we propose to function in binding the 3' terminus of the RNA substrate. Similar motifs are found in other homopolyribonucleotidyl transferases. Phylogenetic classification of nucleotidyl tranferases from sequenced genomes reveals that eubacterial poly(A) polymerases have evolved relatively recently and are found only in a small group of bacteria and surprisingly also in plants, where they may function in organelles.     

tRNA核酸内切酶的研究进展

tRNA在蛋白质合成过程中起着极其重要的作用。在所有的生物体内,tRNA首先以前体形式转录,然后必需经过一系列的加工后才能成为有功能的tRNA分子。tRNaseZ、RNaseP和tRNA剪接内切酶是参与tRNA前体加工的三种主要的核酸内切酶,分别参与tRNA前体3′末端、tRNA前体5′末端和内含子剪接的加工。这三种酶具有不同的结构特征,并且利用完全不同的催化机制水解磷酸二酯键。tRNaseZ和RNaseP都是金属酶,活性中心分别需要Zn^2＋和Mg^2＋的参与;而tRNA剪接内切酶活性中心不需要金属离子,是一个由不同催化亚基上的关键氨基酸残基构成的组合式活性中心。     

The length of the 5' leader of Escherichia coli tRNA precursors influences bacterial growth

Based on a computational analysis of the 5' regions of tRNA-encoding genes, the average length of the 5' leaders in tRNA precursors in Escherichia coli appears to be 17-18 residues long. An in vivo assay based on tRNA nonsense suppression was developed and used to investigate the function of the 5' leader of the tRNA precursors on tRNA processing and bacterial growth. Our data indicate that the 5' leader influences bacterial growth but is surprisingly not absolutely necessary for growth. These findings are consistent with previous in vitro data where it was demonstrated that the 5' leader plays a role in the interaction with RNase P, the endoribonuclease responsible for removing the 5' leader in the cell. We discuss the plausible role of the 5' leader in processing and tRNA gene expression.     

Differential coupling efficiency of chemically activated amino acid to tRNA

Summary Interaction based on possible chemical affinity of an amino acid for tRNA was examined as a model for the aminoacylation of primitive tRNA without aid of an enzyme system. Two types of reaction were carried out and compared. One was the acyl linkage of amino acid to the 5-terminal phosphate of a tRNA activated as an imidazolide. The other was the incorporation of an amino acid activated as an imidazolide into 2(3)-hydroxyl groups of intact tRNA. Both types of reaction indicated that none of the amino acids tested had any selectivity for the tRNAs examined. However, the rates of reaction with a given tRNA were different among amino acids. In the second type of reaction, amino acids were found mainly at loop-out regions of tRNA, but not at either its 5- or 3-terminal sitesOneA ₂₆₀ unit is defined as an amount of material which gives an absorption of 1.0 at 260 nm when dissolved in 1 ml water and measured with a 1-cm light path     

RNase E cleavage in the 5' leader of a tRNA precursor

In this study, we have used various tRNA(Tyr)Su3 precursor (pSu3) derivatives that are processed less efficiently by RNase P to investigate if the 5' leader is a target for RNase E. We present data that suggest that RNase E cleaves the 5' leader of pSu3 both in vivo and in vitro. The site of cleavage in the 5' leader corresponds to the cleavage site for a previously identified endonuclease activity referred to as RNase P2/O. Thus, our findings suggest that RNase P2/O and RNase E activities are of the same origin. These data are in keeping with the suggestion that the structure of the 5' leader influences tRNA expression by affecting tRNA processing and indicate the involvement of RNase E in the regulation of cellular tRNA levels.