Cytidines in tRNAs that are required intact by ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and Saccharomyces cerevisiae.

Individual species of tRNA from Escherichia coli were treated with hydrazine/3 M NaCl to modify cytidine residues. The chemically modified tRNAs were used as substrate for ATP/CTP: tRNA nucleotidyltransferases from E. coli and yeast, with [alpha-32P]ATP as cosubstrate. tRNAs that were labeled were analyzed for their content of modified cytidines. Cytidines at positions 74 and 75 were found to be required chemically intact for interaction with both enzymes. C56 was also required intact by the E. coli enzyme in all tRNAs, and by the yeast enzyme in several instances. C61 was found to be important in seven of 14 tRNAs with the E. coli enzyme but only in four of 13 tRNAs with that from yeast. Our results support a model in which nucleotidyltransferase extends from the 3' end of its tRNA substrate across the top of the stacked array of bases in the accepter- and psi-stems to the corner of the molecule where the D- and psi-loops are juxtaposed.     

Recognition of the tRNA-like structure in tobacco mosaic viral RNA by ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and Saccharomyces cerevisiae

The 3'-terminal tRNA-like structure of the tobacco mosaic virus RNA interacts with ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli or yeast in much the same manner as do tRNAs. Primary sites of interaction cluster near the 3' end and in the loop proposed to be analogous to the psi-loop of a tRNA. Some modified bases in the tRNA-like structure inhibit interaction with nucleotidyltransferase, yet the analogous bases in a tRNA do not. The location of some of these nucleotides within the analog to the psi-loop suggests that this structure differs slightly from its counterpart in a tRNA. The location of other such bases in the helical stem near the 3' end can be explained if the pseudoknot is disrupted by these modified bases or if the tertiary structure of the RNA is altered in the enzyme-RNA complex. A partially denatured secondary structure that persists on denaturing gels is proposed.     

19F nuclear magnetic resonance of 5-fluorouridine-substituted tRNA1Val from Escherichia coli.

The 19F NMR spectrum of Escherichia coli tRNA1Val in which [5-19F]uridine replaces 93% of all uridine and uridine-derived residues has been examined at 93.6 and 235 MHz. The resolution of 11 peaks and visibility of two additional shoulders at either frequency for the 14 FUra residues in the molecule attests to the excellence of 19F as a probe for the structure of tRNA1Val in solution. No significant gain in resolution was attained at the higher frequency. A comparison of the relative areas in the different regions of the 19F spectrum of mixed [FUra]tRNAs with that of [FUra]tRNA1Val suggests that the three single resonances at lowest field in the region 86.5 to 88.5 ppm upfield from trifluoroacetate correspond to the three invariant bases which form tertiary hydrogen bonds in all tRNAs, namely, 8 (U or s4U), 54 (T), and 55 (phi) in unsubstituted tRNAs.     

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.     

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.     

The first human genes for tRNA(ArgICG), tRNA(GlyUCC), and tRNA(ThrIGU) and more tRNA(Val) pseudogenes: expression and pre-tRNA maturation in HeLa cell-free extracts.

A functional tRNA(Val) gene, which codes for the major tRNA(ValIAC) isoacceptor species, and three new tRNA(Val) pseudogenes have been isolated from human genomic DNA. Two tRNA(Val) pseudogenes and a tRNA(Val) variant gene were found to be associated with tRNA genes encoding tRNA(ArgICG), tRNA(GlyUCC), and tRNA(ThrIGU), respectively, on distinct DNA fragments. All tRNA genes, including the pseudogenes, are actively transcribed in HeLa nuclear extract. Pre-tRNAs of tRNA(Val), tRNA(Arg), tRNA(Thr), and tRNA(Gly) genes are correctly processed to mature-sized tRNAs, whereas the three tRNA(Val) pseudogenes yield stable pre-tRNAs in vitro. These findings reveal that, together with the three known pseudogenes, half of the members of the human tRNA(Val) gene family are pseudogenes, all of which are active in homologous nuclear extracts in vitro and presumably also in vivo.     

Unusual anticodon loop structure found in E.coli lysine tRNA.

Although both tRNA(Lys) and tRNA(Glu) of E. coli possess similar anticodon loop sequences, with the same hypermodified nucleoside 5-methylaminomethyl-2-thiouridine (mnm5s2U) at the first position of their anticodons, the anticodon loop structures of these two tRNAs containing the modified nucleoside appear to be quite different as judged from the following observations. (1) The CD band derived from the mnm5s2U residue is negative for tRNA(Glu), but positive for tRNA(Lys). (2) The mnm5s2U monomer itself and the mnm5s2U-containing anticodon loop fragment of tRNA(Lys) show the same negative CD bands as that of tRNA(Glu). (3) The positive CD band of tRNA(Lys) changes to negative when the temperature is raised. (4) The reactivity of the mnm5s2U residue toward H2O2 is much lower for tRNA(Lys) than for tRNA(Glu). These features suggest that tRNA(Lys) has an unusual anticodon loop structure, in which the mnm5s2U residue takes a different conformation from that of tRNA(Glu); whereas the mnm5s2U base of tRNA(Glu) has no direct bonding with other bases and is accessible to a solvent, that of tRNA(Lys) exists as if in some way buried in its anticodon loop. The limited hydrolysis of both tRNAs by various RNases suggests that some differences exist in the higher order structures of tRNA(Lys) and tRNA(Glu). The influence of the unusual anticodon loop structure observed for tRNA(Lys) on its function in the translational process is also discussed.     

Correlations between fluorine-19 nuclear magnetic resonance chemical shift and the secondary and tertiary structure of 5-fluorouracil-substituted tRNA.

To complete assignment of the 19F nuclear magnetic resonance (NMR) spectrum of 5-fluorouracil-substituted Escherichia coli tRNA(Val), resonances from 5-fluorouracil residues involved in tertiary interactions have been identified. Because these assignments could not be made directly by the base-replacement method used to assign 5-fluorouracil residues in loop and stem regions of the tRNA, alternative assignment strategies were employed. FU54 and FU55 were identified by 19F homonuclear Overhauser experiments and were then assigned by comparison of their 19F NMR spectra with those of 5-fluorouracil-labeled yeast tRNA(Phe) mutants having FU54 replaced by adenine and FU55 replaced by cytosine. FU8 and FU12, were assigned from the 19F NMR spectrum of the tRNA(Val) mutant in which the base triple G9-C23-G12 substituted for the wild-type A9-A23-FU12. Although replacement of the conserved U8 (FU8) with A or C disrupts the tertiary structure of tRNA(Val), it has only a small effect on the catalytic turnover number of valyl-tRNA synthetase, while reducing the affinity of the tRNA for enzyme. Analysis of the 19F chemical shift assignments of all 14 resonances in the spectrum of 5-fluorouracil-substituted tRNAVal indicated a strong correlation to tRNA secondary and tertiary structure. 5-Fluorouracil residues in loop regions gave rise to peaks in the central region of the spectrum, 4.4 to 4.9 parts per million (p.p.m.) downfield from free 5-fluorouracil. However, the signal from FU59, in the T-loop of tRNA(Val), was shifted more than 1 p.p.m. downfield, to 5.9 p.p.m., presumably because of the involvement of this fluorouracil in the tertiary interactions between the T and D-loops. The 19F chemical shift moved upfield, to the 2.0 to 2.8 p.p.m. range, when fluorouracil was base-paired with adenine in helical stems. This upfield shift was less pronounced for the fluorine of the FU7.A66 base-pair, located at the base of the acceptor stem, an indication that FU7 is only partially stacked on the adjacent G49 in the continuous acceptor stem/T-stem helix. An unanticipated finding was that the 19F resonances of 5-fluorouracil residues wobble base-paired with guanine were shifted 4 to 5 p.p.m. downfield of those from fluorouracil residues paired with A. In the 19F NMR spectra of all fluorinated tRNAs studied, the farthest downfield peak corresponded to FU55, which replaced the conserved pseudouridine normally found at this position.     

19F NMR of 5-fluorouracil-substituted transfer RNA transcribed in vitro: resonance assignment of fluorouracil-guanine base pairs.

5-Fluorouracil is readily incorporated into active tRNA(Val) transcribed in vitro from a recombinant phagemid containing a synthetic E. coli tRNA(Val) gene. This tRNA has the expected sequence and a secondary and tertiary structure resembling that of native 5-fluorouracil-substituted tRNA(Val), as judged by 19F NMR spectroscopy. To assign resonances in the 19F spectrum, mutant phagemids were constructed having base changes in the tRNA gene. Replacement of fluorouracil in the T-stem with cytosine, converting a FU-G to a C-G base pair, results in the loss of one downfield peak in the 19F NMR spectrum of the mutant tRNA(Val). The spectra of other mutant tRNAs having guanine for adenine substitutions that convert FU-A to FU-G base pairs all have one resonance shifted 4.5 to 5 ppm downfield. These results allow assignment of several 19F resonances and demonstrate that the chemical shift of the 19F signal from base-paired 5-fluorouracil differs considerably between Watson-Crick and wobble geometry.     

Nucleotide sequence of a spinach chloroplast valine tRNA.

The nucleotide sequence of a spinach chloroplast valine tRNA (sp. chl. tRNA Val) has been determined. This tRNA shows essentially equal homology to prokaryotic valine tRNAs (58-65% homology) and to the mitochondrial valine tRNAs of lower eukaryotes (yeast and N. crassa, 61-62% homology). Sp. chl. tRNA Val shows distinctly lower homology to mouse mitochondrial valine tRNA (53% homology) and to eukaryotic cytoplasmic valine tRNAs (47-53% homology). Sp. chl. tRNA Val, like all other chloroplast tRNAs sequenced, contains a methylated GG sequence in the dihydrouridine loop and lacks unusual structural features which have been found in several mitochondrial tRNAs.     

A chemical interference study on the interaction of ribosomal protein L11 from Escherichia coli with RNA molecules containing its binding site from 23S rRNA.

The interaction between ribosomal protein L11 from Escherichia coli and in vitro synthesized RNA containing its binding site from 23S rRNA was characterized by identifying nucleotides that interfered with complex formation when chemically modified by diethylpyrocarbonate or hydrazine. Chemically modified RNA was incubated with L11 under conditions appropriate for specific binding of L11 and the resulting protein-RNA complex was separated from unbound RNA on Mg(2+)-containing polyacrylamide gels. The ability to isolate L11 complexes on such gels was affected by the extent of modification by either reagent. Protein-bound and free RNAs were recovered and treated with aniline to identify their content of modified bases. Exclusion of RNA containing chemically altered bases from L11-associated material occurred for 29 modified nucleotides, located throughout the region corresponding to residues 1055-1105 in 23S rRNA. Ten bases within this region did not reproducibly inhibit binding when modified. Multiple bands of RNA were consistently observed on the nondenaturing gels, suggesting that significant intermolecular RNA-RNA interactions had occurred.     

tRNA leucine identity and recognition sets

Transfer RNAs (tRNAs) are grouped into two classes based on the structure of their variable loop. In Escherichia coli, tRNAs from three isoaccepting groups are classified as type II. Leucine tRNAs comprise one such group. We used both in vivo and in vitro approaches to determine the nucleotides that are required for tRNA(Leu) function. In addition, to investigate the role of the tRNA fold, we compared the in vivo and in vitro characteristics of type I tRNA(Leu) variants with their type II counterparts.A minimum of six conserved tRNA(Leu) nucleotides were required to change the amino acid identity and recognition of a type II tRNA(Ser) amber suppressor from a serine to a leucine residue. Five of these nucleotides affect tRNA tertiary structure; the G15-C48 tertiary "Levitt base-pair" in tRNA(Ser) was changed to A15-U48; the number of nucleotides in the alpha and beta regions of the D-loop was changed to achieve the positioning of G18 and G19 that is found in all tRNA(Leu); a base was inserted at position 47n between the base-paired extra stem and the T-stem; in addition the G73 "discriminator" base of tRNA(Ser) was changed to A73. This minimally altered tRNA(Ser) exclusively inserted leucine residues and was an excellent in vitro substrate for LeuRS. In a parallel experiment, nucleotide substitutions were made in a glutamine-inserting type I tRNA (RNA(SerDelta); an amber suppressor in which the tRNA(Ser) type II extra-stem-loop is replaced by a consensus type I loop). This "type I" swap experiment was successful both in vivo and in vitro but required more nucleotide substitutions than did the type II swap. The type I and II swaps revealed differences in the contributions of the tRNA(Leu) acceptor stem base-pairs to tRNA(Leu) function: in the type I, but not the type II fold, leucine specificity was contingent on the presence of the tRNA(Leu) acceptor stem sequence. The type I and II tRNAs used in this study differed only in the sequence and structure of the variable loop. By altering this loop, and thereby possibly introducing subtle changes into the overall tRNA fold, it became possible to detect otherwise cryptic contributions of the acceptor stem sequence to recognition by LeuRS. Possible reasons for this effect are discussed.     

ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences

A computer program, ARAGORN, identifies tRNA and tmRNA genes. The program employs heuristic algorithms to predict tRNA secondary structure, based on homology with recognized tRNA consensus sequences and ability to form a base-paired cloverleaf. tmRNA genes are identified using a modified version of the BRUCE program. ARAGORN achieves a detection sensitivity of 99% from a set of 1290 eubacterial, eukaryotic and archaeal tRNA genes and detects all complete tmRNA sequences in the tmRNA database, improving on the performance of the BRUCE program. Recently discovered tmRNA genes in the chloroplasts of two species from the ‘green’ algae lineage are detected. The output of the program reports the proposed tRNA secondary structure and, for tmRNA genes, the secondary structure of the tRNA domain, the tmRNA gene sequence, the tag peptide and a list of organisms with matching tmRNA peptide tags.     

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: essential elements for recognition of tRNA substrates within the anticodon stem-loop

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the alkylation of the exocyclic amine of A37 by a dimethylallyl unit in tRNAs with an adenosine in the third anticodon position (position 36). By use of purified recombinant enzyme, steady- state kinetic studies were conducted with chemically synthesized RNA oligoribonucleotides to determine the essential elements within the tRNA anticodon stem-loop structure required for recognition by the enzyme. A 17-base oligoribonucleotide corresponding to the anticodon stem-loop of E. coli tRNA(Phe) formed a stem-loop minihelix (minihelix(Phe)) when annealed rapidly on ice, while the same molecule formed a duplex structure with a central loop when annealed slowly at higher concentrations. Both the minihelix and duplex structures gave k(cat)s similar to that for the normal substrate (full-length tRNA(Phe) unmodified at A37), although the K(m) for minihelix(Phe) was approximately 180-fold higher than full-length tRNA. The A36-A37-A38 motif, which is completely conserved in tRNAs modified by the enzyme, was found to be important for modification. Changing A36 to G in the minihelix resulted in a 260-fold reduction in k(cat) compared to minihelix(Phe) and a 13-fold increase in K(m). An A38G variant was modified with a 9-fold reduction in k(cat) and a 5-fold increase in K(m). A random coil 17-base oligoribonucleotide in which the loop sequence of E. coli tRNA(Phe) was preserved, but the 5 base pair helix stem was completely disrupted and showed no measurable activity, indicating that a helix-loop structure is essential for recognition. Finally, altering the identity of several base pairs in the helical stem did not have a major effect on catalytic efficiency, suggesting that the enzyme does not make base-specific contacts important for binding or catalysis in this region.     

Affinity electrophoretic detection of primary amino groups in nucleic acids: application to modified bases of tRNA and to aminoacylation.

Thiolation of primary amino groups in tRNA with the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate gives rise to species which are retarded during electrophoresis in organomercury-containing polyacrylamide gels. Since such amino groups occur, as far as is known, only as part of the modified bases 3-(3-amino-3-carboxypropyl)uridine and N-2-(5-amino-5-carboxypentyl)cytidine or as the alpha-amino group of aminoacylated tRNAs, this extension of the principle of affinity electrophoresis can be used for the detection and analysis of a specific functional group in both single tRNA species and in a mixed population. The strength of the interaction may be quantified and provides information on the chemical environment/conformation of the derivatized bases.     

Effect of zinc ions on tRNA structure: imino proton NMR spectroscopy

The structure of tRNA in solution was explored by NMR spectroscopy to evaluate the effect of divalent cations, especially zinc, which has a profound effect on the chromatographic behaviour of tRNAs in certain systems. The divalent ions Mg2+ and Zn2+ have specific effects on the imino proton region of the 1H NMR spectrum of valine transfer RNA (tRNA(Val] of Escherichia coli and of phenylalanine transfer RNA (tRNA(Phe] of yeast. The dependence of the imino proton spectra of the two tRNAs was examined as a function of Zn2+ concentration. In both tRNAs the tertiary base pair (G-15).(C-48) was markedly affected by Zn2+ (shifted downfield possibly by as much as 0.4 ppm); this is the terminal base pair in the augmented dihydrouridine helix (D-helix). Base pair (U-8).(A-14) in yeast tRNA(Phe) or (s4U-8).(A-14) in tRNA1(Val), which are stacked on (G-15).(C-48), was not affected by Zn2+, except when 1-2 Mg2+ ions per tRNA were also present. Another imino proton that may be affected by Zn2+ in both tRNAs is that of the tertiary base pair (G-19).(C-46). The assignment of this resonance in yeast tRNA(Phe) is tentative since it is located in the region of highly overlapping resonances between 12.6 and 12.3 ppm. This base pair helps to anchor the D-loop to the T psi C loop.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phylogenetic comparative chemical footprint analysis of the interaction between ribonuclease P RNA and tRNA.

Ribonuclease P RNA is the catalytic moiety of the ribonucleoprotein enzyme that endonucleolytically cleaves precursor sequences from the 5' ends of pre-tRNAs. The bacterial RNase P RNA-tRNA complex was examined with a footprinting approach, utilizing chemical modification to determine RNase P RNA nucleotides that potentially contact tRNA. RNase P RNA was modified with dimethylsulfate or kethoxal in the presence or absence of tRNA, and sites of modification were detected by primer extension. Comparison of the results reveals RNase P bases that are protected from modification upon binding tRNA. Analyses were carried out with RNase P RNAs from three different bacteria: Escherichia coli, Chromatium vinosum and Bacillus subtilis. Discrete bases of these RNAs that lie within conserved, homologous portions of the secondary structures are similarly protected. One protection among all three RNAs was attributed to the precursor segment of pre-tRNA. Experiments using pre-tRNAs containing precursor segments of variable length demonstrate that a precursor segment of only 2-4 nucleotides is sufficient to confer this protection. Deletion of the 3'-terminal CCA sequence of tRNA correlates with loss of protection of a particular loop in the RNase P RNA secondary structure. Analysis of mutant tRNAs containing sequential 3'-terminal deletions suggests a relative orientation of the bound tRNA CCA to that loop.