Terminus Of tRNA Research Articles

Methionyl-tRNA synthetase gene from an extreme thermophile, Thermus thermophilus HB8. Molecular cloning, primary-structure analysis, expression in Escherichia coli, and site-directed mutagenesis.

The gene for the methionyl-tRNA synthetase (MetRS) from an extreme thermophile, Thermus thermophilus HB8, was cloned and sequenced. By expression of the T. thermophilus MetRS gene in Escherichia coli cells, thermostable MetRS was overproduced and purified to homogeneity by heat treatment and one-step column chromatography. The amino acid sequence of T. thermophilus MetRS showed low identities (approximately 25%) with those of MetRSs from E. coli, and cytoplasm and mitochondria of Saccharomyces cerevisiae. However, the amino acid residues in the binding sites for ATP and the anticodon and the 3' terminus of tRNA(Met) are highly conserved among the four MetRSs. T. thermophilus MetRS has a zinc finger-like sequence with all the three cysteine residues and a histidine residue. By site-directed mutagenesis of one of the cysteine residues (Cys127) of T. thermophilus MetRS, the SH group was found to be important for methionyl-tRNA synthesis. Just upstream of the structural gene for T. thermophilus MetRS there is a short open reading frame which codes for a methionine-rich peptide and is partly overlapped with an alternative terminator/antiterminator structure, suggesting that transcription of this gene is regulated by attenuation. Further upstream a region contains a nucleotide sequence homologous to that of the 5' half of T. thermophilus initiator tRNA(Met).

Purification and characterization of RNase P from Clostridium sporogenes

RNase P is a multi-subunit enzyme responsible for the accurate processing of the 5' terminus of all tRNAs. The RNA subunit from Clostridium sporogenes has been partially purified and characterized. The RNA is approximately 400 nucleotides long and makes a precise endonucleolytic cleavage at the mature 5' terminus of tRNA. The RNA requires moderate concentrations of Mg2+ (20 mM) and relatively high concentrations of NH4Cl (800 mM) for optimal activity. Mn2+ effectively substitutes for Mg2+ at 2 mM. Zn2+, Ni2+, Ca2+, and Co2+ are ineffective at stimulating activity. Monovalent ions are, in general, more effective the greater the ionic radius (NH+4 greater than Cs greater than Rb greater than K greater than Na). In contrast to the activity of Bacillus subtilis, C. sporogenes RNase P RNA is significant more active in (NH4)2SO4 than in NH4Cl.

Binding of the 3' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF-G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3' end of tRNAPhe, which considerably decrease the affinity of E-site binding, lower the translocation rate up to 40-fold. Furthermore, 3'-end modifications lower or abolish the stimulation by P site-bound tRNA of the GTPase activity of EF-G on the ribosome. The results suggest that a hydrogen-bonding interaction of the 3'-terminal adenine of the leaving tRNA in the E site, most likely base-pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF-G on the ribosome. We propose the following molecular model of translocation: after the binding of EF-G.GTP, the P site-bound tRNA, by a movement of the 3'-terminal single-stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co-operative interaction between the E site and the EF-G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA.

Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites

Three sets of conserved nucleotides in 23 rRNA are protected from chemical probes by binding of tRNA to the ribosomal A, P, and E sites, respectively. They are located almost exclusively in domain V, primarily in or adjacent to the loop identified with the peptidyl transferase function. Some of these sites are also protected by antibiotics such as chloramphenicol, which could explain how these drugs interfere with protein synthesis. Certain tRNA-dependent protections are abolished when the 3′-terminal A or CA or 2′,3′-linked acyl group is removed, providing direct evidence for the interaction of the conserved CCA terminus of tRNA with 23S rRNA. When the EF-Tu·GTP·aminoacyl-tRNA ternary complex is bound to the ribosome, no tRNA-dependent A site protections are detected in 23S rRNA until EF-Tu is released. Thus, EF-Tu prevents interaction of the 3′ terminus of the incoming aminoacyl-tRNA with the peptidyl transferase region of the ribosome during anticodon selection, thereby permitting translational proofreading.

Transfer RNA is a substrate for RNase D in vivo.

RNase D is a 3'-exonuclease whose in vitro specificity has suggested a role in tRNA processing. However, since mutant Escherichia coli strains devoid of RNase D display a normal phenotype, it has not been possible to ascertain the enzyme's function or even to determine which RNA is its substrate in vivo. Here we show that transformation of strains devoid of tRNA nucleotidyltransferase with a multicopy plasmid carrying the rnd+ gene leads to extremely slow growth due to elevated levels of RNase D activity. Analysis of such a slow-growing strain revealed that less tRNA is present in the cell and that the tRNA that could be recovered is substantially damaged. These studies demonstrate that RNase D can act at the 3' terminus of tRNA in vivo, and they support the conclusion that RNase D participates in tRNA metabolism.

Estradiol regulation of reactions involved in turnover of the amino acid acceptor terminus of tRNA in the rat uterus

Estradiol (E2) increases the specific amino acid acceptor activity of raterine tRNAs by increasing the proportion of certain tRNAs with intact and functional 3′-CCA acceptor termini. Activities of tRNA nucleotidyltransferase and 3′-exoribonuclease which synthesize and degrade this terminus, respectively, were measured and neither enzyme was modified by hormone treatment. Since cytidine triphosphate (CTP) levels are below reported Km values for nucleotidyltransferase, changes in CTP concentrations may regulate nucleotidyltransferase activity. An E2-induced 3-fold increase was seen in CTP synthetase activity (conversion of uridine triphosphate, UTP, into CTP). Uterine CTP levels in controls are minute (9 nmol/uterus, approx. 90 μM), and are increase 2.5-fold in E2(12h)-treated rats. The rate of incorporation of [3H]UTP into the 3′-CCA terminus of tRNA was measured as coupled CTP synthetase-nucleotidyltransferase reactions and a 2.5-fold increase in incorporation occurred 8–12 h after E2 treatment. Injection of azaserine, (inhibitor of CTP synthetase) reduced E2-induced increases in CTP levels, CTP synthetase activity, and leucine acceptor activity of tRNAs. These results indicate that E2 regulates CTP levels by modulation of CTP synthetase activity, and that regulation of synthesis and/or repair of the 3′-CCA terminus of tRNA is proportional to E2-induced uterine cytosolic CTP levels.

Estradiol regulation of reactions involved in turnover of the amino acid acceptor terminus of tRNA in the rat uterus

Estradiol (E2) increases the specific amino acid acceptor activity of rat uterine tRNAs by increasing the proportion of certain tRNAs with intact and functional 3'-CCA acceptor termini. Activities of tRNA nucleotidyltransferase and 3'-exoribonuclease which synthesize and degrade this terminus, respectively, were measured and neither enzyme was modified by hormone treatment. Since cytidine triphosphate (CTP) levels are below reported Km values for nucleotidyltransferase, changes in CTP concentrations may regulate nucleotidyltransferase activity. An E2-induced 3-fold increase was seen in CTP synthetase activity (conversion of uridine triphosphate, UTP, into CTP). Uterine CTP levels in controls are minute (9 nmol/uterus, approx. 90 microM), and are increased 2.5-fold in E2(12 h)-treated rats. The rate of incorporation of [3H]UTP into the 3'-CCA terminus of tRNA was measured as coupled CTP synthetase-nucleotidyltransferase reactions and a 2.5-fold increase in incorporation occurred 8-12 h after E2 treatment. Injection of azaserine, (inhibitor of CTP synthetase) reduced E2-induced increases in CTP levels, CTP synthetase activity, and leucine acceptor activity of tRNAs. These results indicate that E2 regulates CTP levels by modulation of CTP synthetase activity, and that regulation of synthesis and/or repair of the 3'-CCA terminus of tRNA is proportional to E2-induced uterine cytosolic CTP levels.

26] Probing ribosome structure and function using short oligodeoxyribonucleotides

To probe the function of these regions, small complementary oligo deoxyribonucleotides (cDNA probes) have been used to hybridize specific regions of the ribosomal RNA in the ribosomal subunits after which various functions of the ribosome are checked with the probe in place. The methods used to perform these studies will be discussed in this chapter. This chapter presents various specific applications such as subunit association sites, tRNA binding sites, and other sites. That certain rRNA regions may be involved in subunit association has been suggested by several different studies. Recent evidence suggested that the 807–809 region of the 23S rRNA might base-pair with the CCA 3´-terminus of tRNA. There are many other assays that can be used to determine the function of various regions of the rRNA. Clearly, binding of mRNA can be assayed as it comes in juxtaposition with the ribosome. All in all it appears that this technique offers a way of providing substantive insight into the structure and function of regions of rRNA in the ribosome.

TRNA-like structures tag the 3' ends of genomic RNA molecules for replication: implications for the origin of protein synthesis.

Single-stranded RNA viruses often have 3'-terminal tRNA-like structures that serve as substrates for the enzymes of tRNA metabolism, including the tRNA synthases and the CCA-adding enzyme. We propose that such 3'-terminal tRNA-like structures are in fact molecular fossils of the original RNA world, where they tagged genomic RNA molecules for replication and also functioned as primitive telomeres to ensure that 3'-terminal nucleotides were not lost during replication. This picture suggests that the CCA-adding activity was originally an RNA enzyme, that modern DNA telomeres with the repetitive structure CmAn are the direct descendants of the CCA terminus of tRNA, and that the precursor of the modern enzyme RNase P evolved to convert genomic into functional RNA molecules by removing this 3'-terminal tRNA-like tag. Because early RNA replicases would have been catalytic RNA molecules that used the 3'-terminal tRNA-like tag as a template for the initiation of RNA synthesis, these tRNA-like structures could have been specifically aminoacylated with an amino acid by an aberrant activity of the replicase. We show that it is mechanistically reasonable to suppose that this aminoacylation occurred by the same sequence of reactions found in protein synthesis today. The advent of such tRNA synthases would thus have provided a pathway for the evolution of modern protein synthesis.

Possible Regulation by Nucleosidediphosphate Kinase Involvement of the Synthesis of tRNA 3′-Terminal –pCpCpA in Mammalian Cells

When the cytosol of Ehrlich ascites tumor cells was fractionated by chromatofocusing in the pH range of 9 to 6, two active peaks (I and II) of tRNA nucleotidyltransferase were obtained. Fraction I was a multiple complex with a high molecular weight (M.W. greater than 300K) and fraction II comprised components derived from fraction I. Fraction II was separated into tRNA nucleotidyltransferase (M.W., ca. 46,000) and nucleosidediphosphate kinase (M.W., ca. 74,000) by subsequent Sephacryl S-200 chromatography. The two enzymes appeared to be associated loosely with each other. Using the above fraction II or a mixture of the purified tRNA nucleotidyltransferase and nucleosidediphosphate kinase, it was possible to effectively synthesize the 3'-terminal -pCpCpA of tRNA in a reaction mixture containing [3H]-CDP plus XTP or [3H]ADP plus XTP as substrate. Among the XTPs investigated, dTTP was most effective. In addition, it was found that [3H]AMP + XTP also serves as a substrate. [14C]CMP plus XTP, however, was not utilized. From the antagonism of cold CDP against [3H]CTP, and that of cold ADP and AMP against [3H]ATP with the purified tRNA nucleotidyltransferase, the affinity of CDP to the enzyme was estimated to be 1/100 of that of CTP, while the affinities of ADP and AMP to the enzyme were 3 and 30 times higher, respectively, than that of ATP, suggesting that the subsite which binds ATP also binds ADP or AMP. The tRNA nucleotidyltransferase, which had bound ADP or AMP, could not completely synthesize the 3'-terminus of tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

High-level overexpression, rapid purification, and properties of Escherichia coli tRNA nucleotidyltransferase.

The cloned Escherichia coli cca gene, described in the accompanying paper (Cudny, H., Lupski, J. R., Godson, G. N., and Deutscher, M. P. (1986) J. Biol. Chem. 261, 6444-6449), has been used to construct strains that overproduce tRNA nucleotidyltransferase, the enzyme that synthesizes the CCA terminus of tRNA. Strain UT481 (pEC4), which contains a 1.9-kilobase cca gene insert in plasmid pUC8, overproduces the enzyme by about 100-150-fold, probably under the control of the cca gene promoter. A second strain, containing a plasmid with a 1.5-kilobase insert, overproduces tRNA nucleotidyltransferase by about 650-fold, to a level of about 3-4% of the soluble cell protein. In this case, overexpression was dependent on the lac promoter of the plasmid. A rapid, two-step procedure was developed to purify large amounts of the enzyme from strain UT481 (pEC4) that was about 40% pure, free of ribonucleases, and suitable for use as a reagent for modification of tRNA molecules. Preparation of milligram quantities of homogeneous tRNA nucleotidyltransferase was accomplished by two further chromatographic steps. The structural and catalytic properties of this purified enzyme were similar to those from partially purified preparations previously described. The availability of large amounts of pure tRNA nucleotidyltransferase will not permit a variety of structural and functional studies of the enzyme that previously were not possible.

Accurate transcription and processing of 19 Euglena chloroplast tRNAs in a Euglena soluble extract

The transcription and accurate processing of 19 different Euglena gracilis chloroplast tRNAs in a homologous chloroplast soluble extract is described. The chloroplast DNA dependent-RNA polymerase present in the extract selectively transcribes the tRNA genes (Greenberg et al., 1984, J. Biol. Chem., 259: 14880-14887). Two dimensional polyacrylamide gel electrophoresis and RNA fingerprint analysis were used to show that the tRNAs are correctly processed at the 5'- and 3'-ends. The Euglena chloroplast soluble extract contains a 5'-processing or 'RNase P-like' activity and RNases responsible for processing tRNA termini. However, it was not determined if the 3'-CCA was added. Therefore, the soluble extract contains activities that are quite similar to an extract of spinach chloroplasts (Greenberg et al. (1984), Plant Mol. Biol., 3: 97-109). After transcription of total chloroplast DNA in the Euglena soluble extract, thirty-three tRNA sized products were resolved by two dimensional polyacrylamide gel electrophoresis. Nineteen tRNAs could be identified in this mixture.

Purification and characterization of Escherichia coli RNase T.

RNase T, a nuclease thought to be involved in end-turnover of tRNA, has been purified about 4,000-fold from extracts of Escherichia coli. At this stage of purification, the enzyme was judged to be at least 95% pure based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight of RNase T determined from gel filtration and sedimentation analyses is about 50,000, whereas the monomer molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 25,000, suggesting that the protein is an alpha 2 dimer. Purified RNase T is extremely sensitive to inactivation by oxidation, sulfhydryl group reagents, and temperature. The ribonuclease activity against tRNA-C-C-[14C]A is optimal at pH 8-9 in the presence of 2-5 mM MgCl2 and ionic strengths of less than 50mM. Although RNase T is highly specific for intact tRNA-C-C-A as a substrate and can hydrolyze all species in a mixed population of tRNA, it is inhibited by other RNAs, such as poly(A), rRNA, 5 S RNA, and tRNA-C-C. RNase T is an exoribonuclease which initiates attack at a free 3' terminus of tRNA and releases AMP; aminoacyl-tRNA is not a substrate. The role of RNase T in the end-turnover of tRNA and its possible involvement in other aspects of RNA metabolism are discussed.

Ribonuclease T: new exoribonuclease possibly involved in end-turnover of tRNA.

Examination of double mutants lacking one of the exoribonucleases, RNase II, RNase D, RNase BN, or RNase R, and also devoid of tRNA nucleotidyltransferase has suggested that none of these RNases participates in the end-turnover of tRNA. This prompted a search for and identification of a new exoribonuclease, termed RNase T. RNase T could be detected in mutant Escherichia coli strains lacking as many as three of the known exoribonucleases, and it could be separated from each of the four previously described RNases. RNase T is optimally active at pH 8-9 and requires a divalent cation for activity. The enzyme is sensitive to ionic strengths greater than 50 mM and is rapidly inactivated by heating at 45 degrees C. Its preferred substrate is tRNA-C-C-[14C]A, with much less activity shown against tRNA-C-C. RNase T is an exoribonuclease that initiates attack at the 3' hydroxyl terminus of tRNA and releases AMP in a random mode of hydrolysis. The possible involvement of RNase T in end-turnover of tRNA and in RNA metabolism in general are discussed.

Incorporation of 1,N6-ethenoadenosine into the 3' terminus of tRNA using T4 RNA ligase. 2. Preparation and ribosome interaction of fluorescent Escherichia coli tRNAMetf.

A fluorescent derivative of tRNAMetf from Escherichia coli has been prepared which contains 1,N6-etheno-adenosine (epsilon A) in the place of adenosine 73, the fourth residue from the 3' end. The labeled tRNA, tRNAMetf epsilon A73, is fully active with respect to aminoacylation, formylation and formylmethionyl transfer to puromycin. The preparation procedure entails the chemical removal of four nucleotides from the 3' end of tRNAMetf, ligation of the truncated molecule with epsilon A 3',5'-bisphosphate by use of T4 RNA ligase and repair of the C-C-A end with nucleotidyl transferase. The fluorescence of fMet-tRNAMetf epsilon A73 has been exploited for studying tRNA-ribosome complexes. Upon binding the tRNA into the ribosomal P site, the fluorophor experiences a change of its molecular environment as indicated by an increased fluorescence intensity. On the other hand, iodide quenching experiments indicate that, in the complex, the fluorophor is not shielded against solvent access. The results suggest that (a) adenosine 73 is not involved in direct contacts with the ribosome and (b) the stacking of the 3'-terminal A-C-C-A sequence is changed upon binding to the ribosome.

Incorporation of 1,N6-ethenoadenosine into the 3' terminus of tRNA using T4 RNA ligase. 1. Preparation of yeast tRNAPhe derivatives.

The 3'-terminal A-C-C-A sequence of yeast tRNAPhe has been modified by replacing either adenosine 76 or 73 with the fluorescent analogues 1,N6-ethenoadenosine (epsilon A) or 2-aza-1,N6-ethenoadenosine (aza-epsilon A). T4 RNA ligase was used to join the nucleoside 3',5'-bisphosphates to the 3' end of the tRNA which was shortened by one [tRNAPhe(-A)] or four [tRNAPhe(-ACCA)] nucleotides. It was found that the base-paired 3'-terminal cytidine 72 in tRNAPhe(-ACCA) is a more efficient acceptor in the ligation reaction than the unpaired cytidine 75 at the A-C-C terminus of tRNAPhe(-A). This finding indicates that the mobility of the accepting nucleoside substantially influences the ligation reaction, the efficiency being higher the lower the mobility. This conclusion is corroborated by the observation that the ligation reaction with the double-stranded substrate exhibits a positive temperature dependence rather than a negative one as found for single-stranded acceptors. The replacement of the 3'-terminal adenosine 76 with epsilon A and aza-epsilon A leads to moderately fluorescent tRNAPhe derivatives, which are inactive in the aminoacylation reaction. A number of other tRNAs (Met, Ser, Glu, Lys and Leu-specific tRNAs both from yeast and Escherichia coli) are also inactivated by epsilon A incorporation. Replacement of adenosine 73 followed by repair of the C-C-A end using nucleotidyl transferase leads to tRNAPhe derivatives which are fully active in the aminoacylation reaction and in polyphenylalanine synthesis. The fluorescence of epsilon A and aza-epsilon A at position 73 is virtually completely quenched, suggesting a stacked arrangement of bases around this position. There is no fluorescence increase when the epsilon A-labeled tRNAPhe is complexed with phenylalanyl-tRNA synthetase, elongation factor Tu, or ribosomes. These observations indicate that the stacked conformation of the 3' terminus is not changed appreciably in these complexes.

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.

Dissection of the active site of rabbit liver tRNA nucleotidyltransferase. Specificity and properties of the tRNA and acceptor subsites determined with model acceptor substrates.

The specificity of rabbit liver tRNA nucleotidyltransferase with respect to its interaction with acceptor residues at the 3' end of tRNA was analyzed using a model acceptor system consisting of dinucleoside monophosphates or nucleosides. Of all the dinucleoside monophosphates tested, only CpC was an active AMP acceptor, indicating that the specificity of the enzyme conforms exactly to the structure present at the 3' terminus of the natural acceptor, tRNA-C-C. Similarly, CMP incorporation into model acceptors closely paralleled the specificity seen with tRNA-C and tRNA-X. Competition studies between the model acceptors and tRNAs with modified 3' termini suggested that the model compounds bind to the enzyme at the site normally recognizing the 3' terminus of tRNA. Comparison of nucleotide incorporation into tRNAs and into the model acceptors revealed a number of differences which allowed us to separate effects on tRNA structure from direct effects on the reaction. These studies enabled us to distinguish several subsites on the enzyme: an ATP-donor site, two sites specifically recognizing the 2 terminal C residues on tRNA, and a site recognizing the nonreacting part of the tRNA. Thus, these results support several features of the multisite model previously proposed (Deutscher, M. P. (1972) J. Biol. Chem. 247, 459-468) to explain tRNA nucleotidyltransferase action.

Conformational flexibility of the 3' acceptor end of transfer ribonucleic acid.

The intimate details of the conformational features and dynamics of the trinucleoside diphosphates CpCpA and ApCpC in aqueous solution have been arrived at by the complete analysis of their proton magnetic resonance spectra. In addition to the right-handed stacked species in which the phosphodiester torsions conform to the gauche-gauche domains, sugar puckers 3E, C4'-C5 approximately equal to 60 degrees, C5'-O5' approximately 180 degrees, C3'-O3' approximately equal to 205 degrees, and chi CN approximately equal to 40 degrees, the trimers display a variety of spatial configurations, an important one being a bulged configuration in which the central nucleotide unit is bulged out, enabling stacking interactions between the end units. It is further shown that the 3' acceptor end of tRNA, CpCpA, displays considerable flexibility for the terminal adenine nucleotide unit. Theoretical NMR calculations demonstrate that the predominant solution conformation does not conform to the CCA terminus of tRNA as reported by four independent crystallographic studies of tRNAPhe. It is shown that the preferred intramolecular order of CCA in solution is such that chi 1 = chi 2 = chi 3 = 40 degrees, all the three sugars are in 3E, psi 1 = psi 2 = psi 3 = 60 degrees, phi 2 and phi 3 = 170 and 180 degrees, respectively, phi 1 = phi 2' = 205 degrees, and omega 1/omega 1 and omega 2/omega 2' = 240/205 degrees and 295/265 degrees, respectively.

An interferon-induced phosphodiesterase degrading (2'-5') oligoisoadenylate and the C-C-A terminus of tRNA.

A phosphodiesterase characterized by a generally higher activity on 2'-5' than on 3'-5' phosphodiester bonds was isolated from mouse L cells treated with interferon. A similar enzyme was purified from mouse reticulocytes. The phosphodiesterase 2'-PDi splits the 2'-phosphate bond of pppA2'p5'A2'p5'A, the oligonucleotide activator of ribonuclease F. The level of phosphodiesterase 2'-PDi is increased by interferon treatment of L cells. The phosphodiesterase was also shown to degrade the C-C-A terminus of tRNA and to reduce the amino acid acceptance of tRNA in cell-free extracts, thereby causing a tRNA-reversible inhibition of mRNA translation.