Preparation of synthetic tRNA precursors with tRNA nucleotidyltransferase.

Rabbit liver tRNA nucleotidyltransferase can be used to substitute nucleotides within the -C-C-A sequence of tRNA or to add nucleotides following this sequence. These anomolous reactions of the enzyme have been used to prepare radioactively-labeled synthetic tRNA precursors which mimic the structure of the natural precursors. Under appropriate conditions synthetic precursors of defined structure can be made. In this paper we describe the synthesis of tRNA-C-[14C]U and tRNA-C-C-A-[14C]C-C, which are representative of tRNA precursors containing altered residues within the -C-C-A sequence or with extra residues following the normal 3'terminus. A variety of other possible precursors can also be prepared. These synthetic tRNA precursors have already proved useful for isolation of possible tRNA processing nucleases.     

In vitro processing of E. coli tRNA precursors

Using E. coli tRNA precursors isolated from an RNAase P mutant strain, we have studied the steps required for the formation of tRNAs having a mature primary sequence in vitro. Our results suggest that at least three different enzymatic activities can participate in the processing of tRNA precursors.     

Stable tRNA precursors in HeLa cells.

Two tRNA precursors were isolated from 32P-labeled or unlabeled HeLa cells by two dimensional polyacrylamide gel electrophoresis, and were sequenced. These were the precursors of tRNAMet and tRNALeu, and both contained four extra nucleotides including 5'-triphosphates at their 5'-end and nine extra nucleotides including oligo U at their 3'-end. These RNAs are the first naturally occurring tRNA precursors from higher eukaryotes whose sequences have been determined. In these molecules, several modified nucleosides such as m2G, t6A and ac4C in mature tRNAs were undermodified. Two additional hydrogen bonds were formed in the clover leaf structures of these tRNA precursors. These extra hydrogen bonds may be responsible for the stabilities of these tRNA precursors.     

Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis

In contrast to Escherichia coli, where the 3' ends of tRNAs are primarily generated by exoribonucleases, maturation of the 3' end of tRNAs is catalysed by an endoribonuclease, known as RNase Z (or 3' tRNase), in many eukaryotic and archaeal systems. RNase Z cleaves tRNA precursors 3' to the discriminator base. Here we show that this activity, previously unsuspected in bacteria, is encoded by the yqjK gene of Bacillus subtilis. Decreased yqjK expression leads to an accumulation of a population of B.subtilis tRNAs in vivo, none of which have a CCA motif encoded in their genes, and YqjK cleaves tRNA precursors with the same specificity as plant RNase Z in vitro. We have thus renamed the gene rnz. A CCA motif downstream of the discriminator base inhibits RNase Z activity in vitro, with most of the inhibition due to the first C residue. Lastly, tRNAs with long 5' extensions are poor substrates for cleavage, suggesting that for some tRNAs, processing of the 5' end by RNase P may have to precede RNase Z cleavage.     

Identification by affinity labeling of potential sites in 23-S rRNA interacting with the 3'' end of tRNA

Tyrosine 3-monooxygenase was purified to homogeneity, as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, from rat adrenal. The specific activity of the final preparation was approximately 1,600 nmol min-1 mg protein-1, which was much higher than the highest yet reported. The enzyme was markedly stabilized in the presence of glycerol, Tween 80 and EDTA. As judged by gel filtration on Ultrogel AcA 34, sodium dodecyl sulfate/polyacrylamide gel electrophoresis and cross-linking studies, the enzyme appeared to be composed of four identical subunits, each possessing a molecular weight of 59,000. The isoelectric point of the enzyme was estimated to be 6.7 in the presence of 8 M urea and 6.6 in its absence. Amino acid analysis of the enzyme revealed a fairly high content of serine residues in this protein. Purification of the enzyme caused changes in the kinetic properties of the enzyme. The Km for 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine decreased from 220 microM to 58 microM. The pH profile for the enzyme activity became more broad and the pH optimum was changed from an acid pH to a neutral pH. Although polyanions, such as heparin and dextran sulfate, markedly stimulated the activity of crude enzyme by increasing the V, they were much less effective in the activation of purified enzyme. A marked stimulation of the enzyme activity by phospholipids, such as phosphatidylserine, phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine, were not observed in both pure and crude preparations even at low concentrations of the pterin cofactor.     

Specific ribonucleases involved in processing of tRNA precursors of Escherichia coli. Partial purification and some properties.

Ribonucleases O and Q, the two putative nucleolytic activities which we detected previously in the crude extract from a thermosensitive ribonuclease P mutant (TS241) of Escherichia coli and which were shown to function in the processing of tRNA precursors in vitro, were partially purified from the 1000000 x g supernatant fraction of E. coli Q13. In the course of purification of these enzymes, the total RNAs synthesized in the thermosensitive mutant at the restrictive temperature were used as the substrates and the activities were identified from disappearance or alteration of specific tRNA precursor molecules in polyacrylamide gel electrophoresis. The purified ribonuclease O preparation cleaved specifically the multimeric tRNA precursors at the spacer regions. The purified ribonuclease Q preparation removed, in accordance with the definition of this enzyme, extra nucleotides from the 3'-terminal ends of monomeric tRNA precursors. Some properties of these two nucleases were investigated. In addition to these nucleases, another exonuclease (tentatively designated ribonuclease Y) and ribonuclease P, a well-characterized endonuclease, were also purified. The sequential mode of the processing of tRNA precursors, originally observed in the cleavage reactions with the crude extracts in vitro, was supported by studies with the purified enzyme preparations.     

Purification of Rat 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP, EC 3.1.4.37) has been isolated from rat brain myelin by chromatography on successive columns of phenyl-Sepharose CL-4B, CM-Sepharose CL-6B, and 8-(6-aminohexyl) amino-2'AMP-Sepharose 4B. From 15 g of rat brain, approximately 400 micrograms of pure CNP was obtained, with a specific activity of 1,200 (2',3'-cyclic AMP) units/mg protein. The Km of the rat enzyme was 3.7 mM, using 2',3'-cAMP as the substrate. Isoelectric focusing of the enzyme indicated a broad isoelectric range of 8.5-9.0. On SDS polyacrylamide gels, rat CNP appears as two protein bands of approximately 48,000 and 50,000 M.W., with an upper band intensity of about 1/10 that of the lower band. The relative intensities of the bands for CNP and the molecular weights correspond to the Wolfgram proteins W1 and W2 described by other investigators. The amino acid analysis of the purified rat enzyme compared favorably with reported determinations for the bovine enzyme and also with reported values for the rat Wolfgram proteins W1 and W2.     

Chloroplast mRNA 3'' end processing requires a nuclear-encoded RNA-binding protein.

The protein coding regions of plastid mRNAs in higher plants are generally flanked by 3' inverted repeat sequences. In spinach chloroplast mRNAs, these inverted repeat sequences can fold into stem-loop structures and serve as signals for the correct processing of the mature mRNA 3' ends. The inverted repeat sequences are also required to stabilize 5' upstream mRNA segments, and interact with chloroplast protein in vitro. To dissect the molecular components involved in chloroplast mRNA 3' end processing and stability, a spinach chloroplast protein extract containing mRNA 3' end processing activity was fractionated by FPLC and RNA affinity chromatography. The purified fraction consisted of several proteins and was capable of processing the 3' ends of the psbA, rbcL, petD and rps14 mRNAs. This protein fraction was enriched for a 28 kd RNA-binding protein (28RNP) which interacts with both the precursor and mature 3' ends of the four mRNAs. Using specific antibodies to this protein, the poly(A) RNA-derived cDNA for the 28RNP was cloned and sequenced. The predicted amino acid sequence for the 28RNP reveals two conserved RNA-binding domains, including the consensus sequences RNP-CS1 and CS2, and a novel acidic and glycine-rich N-terminal domain. The accumulation of the nuclear-encoded 28RNP mRNA and protein are developmentally regulated in spinach cotyledons, leaves, root and stem, and are enhanced during light-dependent chloroplast development. The general correlation between accumulation of the 28RNP and plastid mRNA during development, together with the result that depletion of the 28RNP from the chloroplast protein extract interferes with the correct 3' end processing of several chloroplast mRNAs, suggests that the 28RNP is required for plastid mRNA 3' end processing and/or stability.     

3'-terminal processing of ribosomal RNA precursors in mammalian cells.

The 3'-terminal structures of ribosomal 28S RNA and its precursors from rat and mouse were analyzed by means of periodate oxidation followed by reduction with 3H-borohydride. 3'-terminal labeled nucleoside derivatives produced by RNase T2 digestion were determined by thin-layer chromatography and oligonucleotides generated by RNase T1 digestion were analyzed by DEAE-Sephadex chromatography. In the rat, the major 3'-terminal sequences of ribosomal 28S RNA, nucleolar 28S, 32S, 41S, and 45S RNAs were YGUoh, GZ2Uoh, GZ12Uoh, GZ2Uoh, and GZ7Goh, respectively, whereas in the mouse corresponding sequences were YGUoh, GZ1,2, or 3Uoh, Goh, Uoh and GZ 13Uoh, respectively. (Y: pyrimidine nucleoside, Z: any nucleoside other than guanosine) These results suggest that a "transcribed spacer" sequence is present at the 3'-terminus of the 45S pre-ribosomal RNA, which is gradually removed during the steps of processing.     

Minimum requirements for substrates of mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes a 3' trailer after the discriminator nucleotide from precursor tRNA (pre-tRNA). To elucidate the minimum requirements for 3' tRNase substrates, we tested small pre-tRNA(Arg) substrates lacking the D and anticodon stem-loop domain for cleavage by purified pig 3' tRNase. A small pre-tRNA (R-ATW) composed of an acceptor stem, an extra loop, a T stem-loop domain, a discriminator nucleotide, and a 3' trailer was cleaved more efficiently than the full-length wild type. The catalytic efficiencies of three R-ATW derivatives, which were constructed to destroy the original T stem base pairs, were also higher than that of the full-length wild type. Pig 3' tRNase efficiently processed a "minihelix" (R-ATM5) that consists of a T stem-loop domain, an acceptor stem, a discriminator nucleotide, and a 3' trailer, while the enzyme never cleaved a "microhelix" that is composed of a T loop, an acceptor stem, a discriminator nucleotide, and a 3' trailer. Five R-ATM5 derivatives that have one to seven base substitutions in the T loop were all cleaved slightly more efficiently than the full-length wild type and slightly less efficiently than R-ATM5. A helix ("minihelixDelta1") one base pair smaller than minihelices was a good substrate, while small helices containing a continuous 10-base pair stem were poor substrates. The cleavage of these three small substrates occurred after the discriminator and one to three nucleotides downstream of the discriminator. From these results, we conclude that minimum substrates for efficient cleavage by mammalian 3' tRNase are minihelices or minihelicesDelta1, in which there seem to be no essential bases.     

A synthetic alanyl-initiator tRNA with initiator tRNA properties as determined by fluorescence measurements: comparison to a synthetic alanyl-elongator tRNA.

Two synthetic tRNAs have been generated that can be enzymatically aminoacylated with alanine and have AAA anticodons to recognize a poly(U) template. One of the tRNAs (tRNA(eAla/AAA)) is nearly identical to Escherichia coli elongator tRNA(Ala). The other has a sequence similar to Escherichia coli initiator tRNA(Met) (tRNA(iAla/AAA)). Although both tRNAs can be used in poly(U)-directed nonenzymatic initiation at 15 mM Mg2+, only the elongator tRNA can serve for peptide elongation and polyalanine synthesis. Only the initiator tRNA can be bound to 30S ribosomal subunits or 70S ribosomes in the presence of initiation factor 2 (IF-2) and low Mg2+ suggesting that it can function in enzymatic peptide initiation. A derivative of coumarin was covalently attached to the alpha amino group of alanine of these two Ala-tRNA species. The fluorescence spectra, quantum yield and anisotropy for the two Ala-tRNA derivatives are different when they are bound to 70S ribosomes (nonenzymatically in the presence of 15 mM Mg2+) indicating that the local environment of the probe is different. Also, the effect of erythromycin on their fluorescence is quite different, suggesting that the probes and presumably the alanine moiety to which they are covalently linked are in different positions on the ribosomes.     

Splicing of yeast tRNA precursors: a two-stage reaction.

Soluble extracts of S. cerevisiae splice tRNA precursors which contain intervening sequences. The reaction goes to completion and requires ATP for the production of mature sequence tRNA. In the absence of ATP, half-tRNA molecules accumulate. Similar half-tRNA molecules appear as kinetic intermediates and accumulate if splicing is inhibited with pure, mature tRNA. Half-tRNA molecules have been purified. These half-tRNAs are efficiently ligated in an ATP-dependent reaction that is inhibited by added mature tRNA. The product of ligation is the expected mature sequence tRNA. The excised intervening sequence has also been identified. These results suggest an enzymatic mechanism for splicing which involves two independent steps.