Splicing of a yeast proline tRNA containing a novel suppressor mutation in the anticodon stem

The intron-containing proline tRNAUGG genes in Saccharomyces cerevisiae can mutate to suppress +1 frameshift mutations in proline codons via a G to U base substitution mutation at position 39. The mutation alters the 3' splice junction and disrupts the bottom base-pair of the anticodon stem which presumably allows the tRNA to read a four-base codon. In order to understand the mechanism of suppression and to study the splicing of suppressor pre-tRNA, we determined the sequences of the mature wild-type and mutant suppressor gene products in vivo and analyzed splicing of the corresponding pre-tRNAs in vitro. We show that a novel tRNA isolated from suppressor strains is the product of frameshift suppressor genes. Sequence analysis indicated that suppressor pre-tRNA is spliced at the same sites as wild-type pre-tRNA. The tRNA therefore contains a four-base anticodon stem and nine-base anticodon loop. Analysis of suppressor pre-tRNA in vitro revealed that endonuclease cleavage at the 3' splice junction occurred with reduced efficiency compared to wild-type. In addition, reduced accumulation of mature suppressor tRNA was observed in a combined cleavage and ligation reaction. These results suggest that cleavage at the 3' splice junction is inefficient but not abolished. The novel tRNA from suppressor strains was shown to be the functional agent of suppression by deleting the intron from a suppressor gene. The tRNA produced in vivo from this gene is identical to that of the product of an intron+ gene, indicating that the intron is not required for proper base modification. The product of the intron- gene is a more efficient suppressor than the product of an intron+ gene. One interpretation of this result is that inefficient splicing in vivo may be limiting the steady-state level of mature suppressor tRNA.     

Substrate structural requirements of Schizosaccharomyces pombe RNase P

RNase P from Schizosaccharomyces pombe has been purified over 2000-fold. The apparent Km for two S. pombe tRNA precursors derived from the supS1 and sup3-e tRNA(Ser) genes is 20 nM; the apparent Vmax is 2.5 nM/min (supS1) and 1.1 nM/min (sup3-e). Processing studies with precursors of other mutants show that the structures of the acceptor stem and anticodon/intron loop of tRNA are crucial for S. pombe RNase P action.     

Mutations in the anticodon stem affect removal of introns from pre-tRNA in Saccharomyces cerevisiae.

To evaluate the role of exon domains in tRNA splicing, the anti-codon stem of proline pre-tRNAUGG from Saccharomyces cerevisiae was altered by site-directed mutagenesis of the suf8 gene. Sixteen alleles were constructed that encode mutant pre-tRNAs containing all possible base combinations in the last base pair of the anticodon stem adjacent to the anticodon loop (positions 31 and 39). The altered pre-tRNAs were screened by using an in vitro endonucleolytic cleavage assay to determine whether perturbations in secondary structure affect the intron excision reaction. The pre-tRNAs were cleaved efficiently whenever secondary structure in the anticodon stem was maintained through standard base pairing or G.U interactions. However, most of the pre-tRNAs with disrupted secondary structure were poor substrates for intron excision. We also determined the extent to which the suf8 alleles produce functional products in vivo. Each allele was integrated in one to three copies into a yeast chromosome or introduced on a high-copy-number plasmid by transformation. The formation of a functional product was assayed by the ability of each allele to suppress the +1 frameshift mutation his4-713 through four-base codon reading, as shown previously for the SUF8-1 suppressor allele. We found that alleles containing any standard base pair or G.U pair at position 31/39 in the anticodon stem failed to suppress his4-713. We could not assess in vivo splicing with these alleles because the tRNA products, even if they are made, would be expected to read a normal triplet rather than a quadruplet codon. However, all of the alleles that contained a disrupted base pair at position 31/ 39 in the anticodon stem altered the structure of the tRNA in a manner that caused frameshift suppression. Suppression indicated that splicing must have occurred to some extent in vivo even though most of the suppression alleles produced pre-tRNAs that were cleaved with low efficiency or not at all in vitro. These results have important implications for the interpretation of in vitro cleavage assays in general and for the potential use of suppressors to select mutations that affects tRNA splicing.     

The Schizosaccharomyces pombe sup3-i suppressor recognizes ochre, but not amber codons in vitro and in vivo.

The inefficient suppressor sup3-i of the fission yeast Schizosaccharomyces pombe is an ochre suppressor. Sup3-i was derived from the efficient serine inserting UGA suppressor sup3-e. The cloning and sequencing of the sup3-i gene indicate that the suppressor is different from the parent sup3-e by a C----T substitution in the sequence coding for the middle position of the anticodon. In vitro translation assays supplemented with purified sup3-i tRNA and programmed with Xenopus globin mRNAs lead to the accumulation of a readthrough product in response to UAA termination signals, but not in response to UGA termination codons. Transformation of Saccharomyces cerevisiae nonsense mutant strains with plasmid DNA carrying the S. pombe sup3-i gene, led to ochre, but not amber or UGA suppression in vivo.     

In vivo processing of an intron-containing archael tRNA

In vitro studies on the processing of halobacterial tRNA introns have led to the proposal that archaeal and eukaryotic tRNA intron endonucleases have distinctly different requirements for the recognition of pre-tRNAs. Using a Haloferax volcanii in vivo expression vector we have examined the in vivo processing of modified forms of the halobacterial intron-containing tRNA_Trp gene. As observed in vitro, changes in the exon–intron boundary structure of this pre-tRNA block processing. Intron sequences, other than those at the exon–intron boundaries, are not essential for processing in vivo. We also show that conversion of the tryptophan anticodon to an opal suppressor anticodon is tolerated when the exon-intron boundary structure is maintained.     

Plant nonsense suppressor tRNA(Tyr) genes are expressed at very low levels in vitro due to inefficient splicing of the intron-containing pre-tRNAs.

Oligonucleotide-directed mutagenesis was used to generate amber, ochre and opal suppressors from cloned Arabidopsis and Nicotiana tRNA(Tyr) genes. The nonsense suppressor tRNA(Tyr) genes were efficiently transcribed in HeLa and yeast nuclear extracts, however, intron excision from all mutant pre-tRNAs(Tyr) was severely impaired in the homologous wheat germ extract as well as in the yeast in vitro splicing system. The change of one nucleotide in the anticodon of suppressor pre-tRNAs leads to a distortion of the potential intron-anticodon interaction. In order to demonstrate that this caused the reduced splicing efficiency, we created a point mutation in the intron of Arabidopsis tRNA(Tyr) which affected the interaction with the wild-type anticodon. As expected, the resulting pre-tRNA was also inefficiently spliced. Another mutation in the intron, which restored the base-pairing between the amber anticodon and the intron of pre-tRNA(Tyr), resulted in an excellent substrate for wheat germ splicing endonuclease. This type of amber suppressor tRNA(Tyr) gene which yields high levels of mature tRNA(Tyr) should be useful for studying suppression in higher plants.     

Transfer RNA splicing in Saccharomyces cerevisiae. Secondary and tertiary structures of the substrates

Secondary and tertiary structures of four yeast tRNA precursors that contain introns have been elucidated using limited digestion with a variety of single-strand- and double-strand-specific nucleases. The pre-tRNAs, representing the variety of intron sizes and potential structures, were: pre-tRNALeuCAA, pre-tRNALeuUAG, pre-tRNAIleUAU, and pre-tRNAPro-4UGG. Conventional tRNA cloverleaf structure is maintained in these precursors except that the anticodon loop is interrupted by the intron. The intron contains a sequence which is complementary to a portion of the anticodon loop and allows the formation of a double helix often extending the anticodon stem. The 5' and 3' splicing cleavage sites are located at either end of this helix and are single-stranded. The intron is the most sensitive region to nuclease cleavage, suggesting that it is on the surface of the molecule and available for interaction with the splicing endonuclease. Absence of Mg2+ or spermidine renders the dihydrouridine and T psi C loops of these precursors highly sensitive to nuclease digestion. These ionic effects mimic those observed for tRNAPhe and suggest that the tRNA portion of these precursors has native tRNA structure. We propose consensus secondary and tertiary structures which may be of significance to eventual understanding of the mechanism of yeast tRNA splicing.     

Intron-dependent formation of pseudouridines in the anticodon of Saccharomyces cerevisiae minor tRNA(Ile).

We have isolated and sequenced the minor species of tRNA(Ile) from Saccharomyces cerevisiae. This tRNA contains two unusual pseudouridines (psi s) in the first and third positions of the anticodon. As shown earlier by others, this tRNA derives from two genes having an identical 60 nt intron. We used in vitro procedures to study the structural requirements for the conversion of the anticodon uridines to psi 34 and psi 36. We show here that psi 34/psi 36 modifications require the presence of the pre-tRNA(Ile) intron but are not dependent upon the particular base at any single position of the anticodon. The conversion of U34 to psi 34 occurs independently from psi 36 synthesis and vice versa. However, psi 34 is not formed when the middle and the third anticodon bases of pre-tRNA(Ile) are both substituted to yield ochre anticodon UUA. This ochre pre-tRNA(Ile) mutant has the central anticodon uridine modified to psi 35 as is the case for S.cerevisiae SUP6 tyrosine-inserting ochre suppressor tRNA. In contrast, neither the first nor the third anticodon pseudouridine is formed, when the ochre (UUA) anticodon in the pre-tRNA(Tyr) is substituted with the isoleucine UAU anticodon. A synthetic mini-substrate consisting of the anticodon stem and loop and the wild-type intron of pre-tRNA(Ile) is sufficient to fully modify the anticodon U34 and U36 into psi s. This is the first example of the tRNA intron sequence, rather than the whole tRNA or pre-tRNA domain, being the main determinant of nucleoside modification.     

Conversion of a methionine initiator tRNA into a tryptophan-inserting elongator tRNA in vivo.

The role of the anticodon and discriminator base in aminoacylation of tRNAs with tryptophan has been explored using a recently developed in vivo assay based on initiation of protein synthesis by mischarged mutants of the Escherichia coli initiator tRNA. Substitution of the methionine anticodon CAU with the tryptophan anticodon CCA caused tRNA(fMet) to be aminoacylated with both methionine and tryptophan in vivo, as determined by analysis of the amino acids inserted by the mutant tRNA at the translational start site of a reporter protein containing a tryptophan initiation codon. Conversion of the discriminator base of tRNA(CCA)fMet from A73 to G73, the base present in tRNA(Trp), eliminated the in vivo methionine acceptor activity of the tRNA and resulted in complete charging with tryptophan. Single base changes in the anticodon of tRNA(CCA)fMet containing G73 from CCA to UCA, GCA, CAA, and CCG (changes underlined) essentially abolished tryptophan insertion, showing that all three anticodon bases specify the tryptophan identity of the tRNA. The important role of G73 in tryptophan identity was confirmed using mutants of an opal suppressor derivative of tRNA(Trp). Substitution of G73 with A73, C73, or U73 resulted in a large loss of the ability of the tRNA to suppress an opal stop codon in a reporter protein. Base pair substitutions at the first three positions of the acceptor stem of the suppressor tRNA caused 2-12-fold reductions in the efficiency of suppression without loss of specificity for aminoacylation of the tRNA with tryptophan.(ABSTRACT TRUNCATED AT 250 WORDS)     

Testing for intron function in the essential Saccharomyces cerevisiae tRNA(SerUCG) gene

The gene sup61+, which codes for the essential Saccharomyces cerevisiae tRNA(SerUCG), is the only single-copy tRNA gene in this organism know to contain an intron. To assess the role of this intron in tRNA gene expression, an intron-deleted sup61+ gene was constructed in vitro and introduced into the yeast genome. Isogenic intron- and intron+ strains were found to be indistinguishable by criteria that include growth rates, ability to undergo meiosis, levels of mature tRNA(SerUCG) transcribed in vivo, and the suppressor efficiency of amber- and ochre-specific alleles of this gene.     

The nucleotide sequence of a UGA suppressor serine tRNA from Schizosaccharomyces pombe.

The UGA suppressor tRNA produced by Schizosaccharomyces pombe strain sup3-e was purified to homogeneity. It can be aminoacylated with a serine by a crude aminoacyl-tRNA synthetase preparation from S. pombe cells. By combining post-labeling fingerprinting and gel sequencing methods the nucleotide sequence of this tRNA was determined to be: pG-U-C-A-C-U-A-U-G-U-C-ac4C-G-A-G-D-G-G-D-D-A-A-G-G-A-m2G2-psi-U-A-G-A-N-U-U-C-A-i6A-A-psi-C-U-A-A-U-G-G-G-C-U-U-U-G-C-C-C-G-m5C-G-G-C-A-G-G-T-psi-C-A-m1A-A-U-C-C-U-G-C-U-G-G-U-G-A-C-G-C-C-A OH. The anticodon sequence u ca is complementary to the UGA codon.