Binding and cleavage of pre-tRNA by the Xenopus splicing endonuclease: two separable steps of the intron excision reaction

We have constructed three base-substitution mutants of the yeast tRNALeu3 gene. In two of them the ability to form an extended anticodon stem is lost. In the first mutant the bases encoding the anticodon change from TTG to GAC (positions 37, 36, 35); in the second, the nucleotides encoding the region of the intron that base-pair with the anticodon change from CAA to GTC (positions 48, 47, 46). The third is a double mutant characterized by both substitutions described above so that its ability to form an extended anticodon stem is restored. The precursors derived from the two single mutants are accurately spliced in the X. laevis germinal vesicles (GV) extract: pairing of the anticodon with the intron, therefore, is not required for the splicing reaction. The precursor derived from the double mutant is not spliced, indicating that the new extended anticodon stem exerts an inhibitory action. Since the double mutant precursor binds to the purified splicing endonuclease, binding and cleavage occur as two separable steps in the intron excision reaction.     

A G43 to U43 mutation in E. coli tRNAtyrsu3+ which affects processing by RNase P.

A novel mutation in the anticodon stem of E. coli tRNA1Tyrsu3+ (G43 to U43) has been characterized. The gene coding for the mutant tRNA, carried by phage phi 80DHA61.3 a derivative of phi 80psu3+su0, produces only 20% of mature suppressor tRNA as compared with phi 80psu3+. Both the mutant tRNA precursor and mature tRNA have an altered conformation. The precursor tRNA coded for by phi 80DHA61.3 is processed by RNase P more slowly than the su3+ precursor and does not form as stable an enzyme-substrate complex as does su3+ precursor. phi 80 DHA61.3 also contains a large deletion which begins in the spacer region between the su3+ gene and the su0- gene, extends through the su0- gene and includes most of the repeated region following the tRNA genes.     

A precursor to a minor species of yeast tRNASer contains an intervening sequence.

Certain tRNAs in S. cerevisiae (tRNATyr and tRNAPhe) arise via precursor molecules which are mature at the 5' and 3' termini but contain intervening sequences adjacent to the anticodon (Knapp et al., 1978; O'Farrell et al., 1978). In addition to these molecules, precursors to several other tRNAs accumulate in a temperature-sensitive mutant (ts136) at the nonpermissive temperature. We have analyzed one of these species and shown that it is a precursor to a minor species of tRNASer. This precursor is also mature at both termini and contains an intervening sequence of 19 nucleotides adjacent to the hypermodified A residue 3' to the anticodon. The sequence can be arranged in a secondary structure in which the anticodon stem is extended by additional base-pairing, and contains the sites of excision and ligation within two looped regions. Support for this structure was provided by analysis of the products of limited digestion with RNAase T1. recently Piper (1978) reported the isolation of a minor species of tRNASer which decodes UCG. He found this species to be structurally heterogeneous and determined that the less abundant form corresponds to the tRNA which is altered in the recessive lethal SUP-RL1 amber suppressor. Our data now suggest that the more abundant form may be restricted to reading UCA in vivo; thus mutation of the minor species would result in complete loss of UCG-decoding ability and explain the recessive lethality of SUP-RL1. We have shown that the precursor which accumulates in ts136 corresponds exclusively to this minor tRNASerUCG species. Our results suggest that this may be the only gene for tRNASer in yeast which contains an intervening sequence.     

Suppression of amber codons in vivo as evidence that mutants derived from Escherichia coli initiator tRNA can act at the step of elongation in protein synthesis

The absence of a Watson-Crick base pair at the end of the amino acid acceptor stem is one of the features which distinguishes prokaryotic initiator tRNAs as a class from all other tRNAs. We show that this structural feature prevents Escherichia coli initiator tRNA from acting as an elongator in protein synthesis in vivo. We generated a mutant of E. coli initiator tRNA in which the anticodon sequence is changed from CAU to CUA (the T35A36 mutant). This mutant tRNA has the potential to read the amber termination codon UAG. We then coupled this mutation to others which change the C1.A72 mismatch at the end of the acceptor stem to either a U1:A72 base pair (T1 mutant) or a C1:G72 base pair (G72 mutant). Transformation of E. coli CA274 (HfrC Su- lacZ125am trpEam) with multicopy plasmids carrying the mutant initiator tRNA genes show that mutant tRNAs carrying changes in both the anticodon sequence and the acceptor stem suppress amber codons in vivo, whereas mutant tRNA with changes in the anticodon sequence alone does not. Mutant tRNAs with the above anticodon sequence change are aminoacylated with glutamine in vitro. Measurement of kinetic parameters for aminoacylation by E. coli glutaminyl-tRNA synthetase show that both the nature of the base pair at the end of the acceptor stem and the presence or absence of a base pair at this position can affect aminoacylation kinetics. We discuss the implications of this result on recognition of tRNAs by E. coli glutaminyl-tRNA synthetase.     

The kinetics and specificity of cleavage by RNase P is mainly dependent on the structure of the amino acid acceptor stem.

Cleavage by RNase P of the tRNA(His precursor yields a mature tRNA with an 8 base pair amino acid acceptor stem instead of the usual 7 base pair stem. Here we show, both in vivo and in vitro, that this is mainly dependent on the primary structure and length of the acceptor stem in the precursor. Furthermore, the tRNA(His) precursor used in this study was processed with a change in both kinetic constants, Km and kcat, in comparison to the kinetics of cleavage of the precursor to tRNA(Tyr)Su3. Cleavage of a chimeric tRNA precursor showed that these altered kinetics were due to a difference in the primary structure and in the length of the acceptor stems of these two tRNA precursors. We also studied the cleavage reaction as a function of base substitutions at positions -1 and/or +73 in the precursor to tRNA(His). Our results suggest that the nucleotide at position +73 in tRNA(His) plays a significant role in the kinetics of cleavage of its precursor, possibly in product release. In addition, it appears that the C5 protein of RNase P is involved in the interaction between the enzyme and its substrate in a substrate-dependent manner, as previously suggested.     

The nucleotide sequence of a precursor to the glycine- and threonine-specific transfer ribonucleic acids of Escherichia coli.

The primary nucleotide sequence of an Escherichia coli tRNA precursor molecule has been determined. This precursor RNA, specified by the transducing phage lambdah80dglyTsuA36 thrT tyrT, accumulates in a mutant strain temperature-sensitive for RNase P activity. The 170-nucleotide precursor RNA is processed by E. coli extracts to form mature tRNA Gly 2 suA36 and tRNA Thr ACU/C. The sequence of the precursor is pG-U-U-C-C-A-G-G-A-U-G-C-G-G-G-C-A-U-C-G-U-A-U-A-A-U-G-G-C-U-A-U-U-A-C-C-U-C-A-G-C-C-U-N-C-U-A-A-G-C-U-G-A-U-G-A-U-G-C-G-G-G-T-psi-C-G-A-U-U-C-C-C-G-C-U-G-C-C-C-G-C-U-C-C-A-A-G-A-U-G-U-G-C-U-G-A-U-A-U-A-G-C-U-C-A-G-D-D-G-G-D-A-G-A-G-C-G-C-A-C-C-C-U-U-G-G-U-mt6A-A-G-G-G-U-G-A-G-m7G-U-C-G-G-C-A-G-T-psi-C-G-A-A-U-C-U-G-C-C-U-A-U-C-A-G-C-A-C-C-A-C-U-UOH(tRNA sequences are italicized). It contains the entire primary nucleotide sequences of tRNA Gly2 suA36 and tRNA Thr ACU/C, including the common 3'-terminal sequence, CCA. Nineteen additional nucleotides are present, with 10 at the 5' end, 3 at the 3' end, and the remaining 6 in the inter-tRNA spacer region. RNase P cleaves the precursor specifically at the 5' ends of the mature tRNA sequences.     

Transfer RNA intron processing in the halophilic archaebacteria

An in vitro assay system has been developed for the Halobacterium volcanii tRNA intron endonuclease using in vitro generated precursor RNAs. A partially purified enzyme preparation is capable of precise and accurate excision of the intron from the halobacterial tRNA(Trp) precursor. The cleavage reaction produces products having 5' hydroxyl and 2',3' cyclic phosphate termini. Processing of precursor molecules containing deletions within the exon regions indicates that the halobacterial endonuclease does not require intact mature tRNA structure in the substrate; this is in contrast to the eukaryotic endonuclease enzyme that has an absolute requirement for these structures. The large halobacterial tRNA(Trp) intron does not appear to be a primary site for recognition by the endonuclease, however, its removal affects cleavage efficiency. Through a comparison of the structural and sequence features of the halobacterial substrates and the precursors of other archaebacterial intron-containing precursors, a common element is proposed for the recognition of substrates by intron endonuclease.     

Control of the position of RNase P-mediated transfer RNA precursor processing

Two Bacillus subtilis tRNA(His) precursors (Green, C. J., and Vold, B. S. (1988) J. Biol. Chem. 263, 652-657) were processed by Escherichia coli RNase P in the presence of varying [Mg2+]. The wild type precursor was processed under all conditions to afford a single tRNA product containing 8 base pairs in the acceptor stem. In contrast, the position of processing of a mutant tRNA(His) precursor (containing a G27----A27 alteration) was shown to be condition-dependent. Processing occurred at A27 under conditions consistent with formation of an A27-C100 base pair in the acceptor stem but at G28 under conditions that disfavored base pair formation. The ability to control the site of RNase P-mediated tRNA precursor processing is unprecedented and permits analysis of the chemical factors that promote processing.     

Novel one-step mechanism for tRNA 3'-end maturation by the exoribonuclease RNase R of Mycoplasma genitalium

Mycoplasma genitalium is expected to metabolize RNA using unique pathways because its minimal genome encodes very few ribonucleases. In this work, we report that the only exoribonuclease identified in M. genitalium, RNase R, is able to remove tRNA 3'-trailers and generate mature 3'-ends. Several sequence and structural features of a tRNA precursor determine its precise processing at the 3'-end by RNase R in a purified system. The aminoacyl-acceptor stem plays a major role in stopping RNase R digestion at the mature 3'-end. Disruption of the stem causes partial or complete degradation of the pre-tRNA by RNase R, whereas extension of the stem results in the formation of a product terminating downstream at the new mature 3'-end. In addition, the 3'-terminal CCA sequence and the discriminator residue influence the ability of RNase R to stop at the mature 3'-end. RNase R-mediated generation of the mature 3'-end prefers a sequence of RCCN at the 3' terminus of tRNA. Variations of this sequence may cause RNase R to trim further and remove terminal CA residues from the mature 3'-end. Therefore, M. genitalium RNase R can precisely remove the 3'-trailer of a tRNA precursor by recognizing features in the terminal domains of tRNA, a process requiring multiple RNases in most bacteria.     

The effects of matrices of paired substitutions in mid-acceptor stem on Drosophila tRNA(His) structure and end-processing

End-maturation reactions, in which the 5' end leader and 3' end trailer of precursor tRNA are removed by RNase P and 3'-tRNase, respectively, are early, essential steps in eukaryotic precursor tRNA processing. End-processing enzymes may be expected to contact the acceptor stem of tRNA due to its proximity to both cleavage sites. We constructed matrices of pair-wise substitutions in mid-acceptor stem at nt 3/70 and 4/69 of Drosophila tRNA(His) and analyzed their ability to be processed by Drosophila RNase P and 3'-tRNase. In accord with our earlier study of D/T loop processing matrices, we find that tRNA end processing enzymes respond to sequence changes differently. More processing defects were observed with 3'-tRNase than with RNase P, and substitutions at 4/69 reduced processing more than those at 3/70. We evaluated tRNA folding using structure probing nucleases and investigated the contribution of K(M) and V(Max) to the processing efficiency of selected variants. In one substitution (C3A), mis-folding correlates with processing defects. In another (C69A), a disruption of structure appears to be transmitted laterally to both ends of the acceptor stem. Poor processing of C69A by RNase P is due entirely to a reduction in V(Max), but for 3'-tRNase, it is due to an increase in K(M).     

Sequence and structure requirements for the biosynthesis of pseudouridine (psi 35) in plant pre-tRNA(Tyr).

All eukaryotic cytoplasmic tRNAs(Tyr) contain pseudouridine in the centre of the anticodon (psi 35). Recently, it has been shown that the formation of psi 35 is dependent on the presence of introns in tRNA(Tyr) genes. Furthermore, we have investigated the structural and sequence requirements for the biosynthesis of psi 35. A number of mutant genes were constructed by oligonucleotide-directed mutagenesis of a cloned Arabidopsis tRNA(Tyr) gene. Nucleotide exchanges were produced in the first and third positions of the anticodon and at positions adjacent to the anticodon. Moreover, insertion and deletion mutations were made in the anticodon stem and in the intron. The mutant genes were transcribed in HeLa cell extract and the pre-tRNAs(Tyr) were used for studying psi 35 biosynthesis in HeLa cell and wheat germ extracts. We have made the following observations about the specificity of plant and vertebrate psi 35 syntheses: (i) insertion or deletion of one base pair in the anticodon stem does not influence the efficiency and accuracy of the psi 35 synthase; (ii) the presence of U35 in a stable double-stranded region prevents its modification to psi 35; and (iii) the consensus sequence U33N34U35A36Pu37 in the anticodon loop is an absolute requirement for psi 35 synthesis. Thus, psi 35 synthases recognize both tRNA tertiary structure and specific sequences surrounding the nucleotide to be modified.