The yeast tRNATyr gene intron is essential for correct modification of its tRNA product

Precise deletion of the intervening sequence of a yeast tRNATyr ochre suppressor gene (SUP6) significantly reduced its suppressor activity relative to that of the unaltered gene. This is probably the result of the absence of the pseudouridine modification, normally present at the middle anticodon position of mature suppressor tRNA, in tRNA synthesized in vivo from the deleted gene.     

Uridine-33 in yeast tRNA not essential for amber suppression

The nucleotide at position 33 on the 5' side of the anticodon of almost all tRNAs is a uridine. Crystallographic studies of different tRNAs reveal that although the precise orientation of uridine-33 is not always the same, it connects the anticodon stacked along the 3' side of the loop with the pyrimidine-32 stacked on the 5' side of the loop. The remarkably conserved nature of uridine-33 and its unique position in the anticodon loop structure has led to suggestions that this nucleotide has an essential role in the translational mechanism. We have developed a biochemical procedure to replace nucleotides 33-35 in yeast tRNATyr with any desired sequence and used it to construct amber suppressor tRNAs having different nucleotides at position 33. As all of these synthetic amber suppressor tRNAs functioned well in eukaryotic in vitro suppression assays, we conclude that uridine-33 does not have an obligatory role in the translation mechanism.     

A test for intron function in the yeast actin gene

Many eukaryotic genes contain intervening sequences (IVS), but the rationale for their existence remains a mystery. Previous studies done in our laboratory demonstrated that the intron in a yeast tRNATyr gene, SUP6, does have a function. We used the same approach to determine the role of introns in nuclear genes encoding messenger RNAs. A single actin gene with one intron exists in Saccharomyces cerevisiae. The level of actin in yeast appears to be crucial to viability: either too much or too little actin inhibits growth. Therefore, small effects on synthesis of actin protein resulting from the removal of the actin gene intron would be expected to cause measurable changes in cell growth. In the present study, an intron-deleted actin gene was constructed in vitro and was used to replace the single resident actin gene in a haploid strain. Analysis of the cells carrying the intron-deleted actin gene shows that the intervening sequence is not essential for actin gene expression.     

A model of synthetase/transfer RNA interaction as deduced by protein engineering

The recognition of transfer-RNA by their cognate aminoacyl-tRNA synthetases is the crucial step in the translation of the genetic code. In order to construct a structural model of the complex between the tyrosyl-tRNA synthetase (TyrTS) from Bacillus stearothermophilus and tRNATyr, 40 basic residues at the surface of the TyrTS dimer have been mutated by site-directed mutagenesis and heterodimers created in vitro by recombining subunits derived from different mutants. As reported here a cluster of basic residues (Arg 207-Lys 208) in the N-terminal domain of one TyrTS subunit interacts with the acceptor stem of tRNATyr and two separated clusters of basic residues (Arg 368-Arg 371; Arg 407-Arg 408-Lys 410-Lys 411) in the C-terminal domain of the other subunit interact with the anticodon arm. The TyrTS would thus clamp the tRNA in a fixed orientation. The precise alignment of the flexible... ACCA 3' end of the tRNA for attack on the tyrosyl adenylate is made by contacts closer to the catalytic groups of the enzyme, such as with Lys 151.     

Analysis of cDNA for human erythrocyte ankyrin indicates a repeated structure with homology to tissue-differentiation and cell-cycle control proteins

Analysis of complementary DNA for human erythroid ankyrin indicates that the mature protein contains 1,880 amino acids comprising an N-terminal domain binding integral membrane proteins and tubulin, a central domain binding spectrin and vimentin, and an acidic C-terminal 'regulatory' domain containing an alternatively spliced sequence missing from ankyrin variant 2.2. The N-terminal domain is almost entirely composed of 22 tandem 33-amino-acid repeats. Similar repeats are found in yeast and invertebrate proteins involved in cell-cycle control and tissue differentiation.     

Structural basis of anticodon loop recognition by glutaminyl-tRNA synthetase

The refined crystal structure of Escherichia coli glutaminyl transfer RNA synthetase complexed with transfer RNA(Gln) and ATP reveals that the structure of the anticodon loop of the enzyme-bound tRNA(Gln) differs extensively from that of the known crystal structures of uncomplexed tRNA molecules. The anticodon stem is extended by two non-Watson-Crick base pairs, leaving the three anti-codon bases unpaired and splayed out to bind snugly into three separate complementary pockets in the protein. These interactions suggest that the entire anticodon loop provides essential sites for glutaminyl tRNA synthetase discrimination among tRNA molecules.     

Human U2 snRNA can function in pre-mRNA splicing in yeast

The removal of introns from messenger RNA precursors requires five small nuclear RNAs (snRNAs), contained within ribonucleoprotein particles (snRNPs), which complex with the pre-mRNA and other associated factors to form the spliceosome. In both yeast and mammals, the U2 snRNA base pairs with sequences surrounding the site of lariat formation. Binding of U2 snRNP to the highly degenerate branchpoint sequence in mammalian introns is absolutely dependent on an auxiliary protein, U2AF, which recognizes a polypyrimidine stretch adjacent to the 3' splice site. The absence of this sequence motif in yeast introns has strengthened arguments that the two systems are fundamentally different. Deletion analyses of the yeast U2 gene have confirmed that the highly conserved 5' domain is essential, although the adjacent approximately 950 nucleotides can be deleted without any phenotypic consequence. A 3'-terminal domain of approximately 100 nucleotides is also required for wild-type growth rates; the highly conserved terminal loop within this domain (loop IV) may provide specific binding contacts for two U2-specific snRNP proteins. We have replaced the single copy yeast U2 (yU2) gene with human U2 (hU2), expecting that weak or no complementation would provide an assay for cloning additional splicing factors, such as U2AF. We report here that hU2 can complement the yeast deletion with surprising efficiency. The interactions governing spliceosome assembly and intron recognition are thus more conserved than previously suspected. Paradoxically, the conserved loop IV sequence is dispensable in yeast.     

Nucleotide sequence of the gene coding for the major protein of hepatitis B virus surface antigen.

DNA extracted from hepatitis B virus Dane particles has been cloned in bacteria using a plasmid vector. A full-length clone has been examined by restriction endonuclease analysis, and the nucleotide sequence of an 892-base pair fragment from cloned hepatitis B viral DNA encoding the surface antigen gene is reported. The amino acid sequence deduced from the DNA indicates that the surface antigens is a protein consisting of 226 amino acids and with a molecular weight of 25,398. The portion of the gene coding for this protein apparently contains no intervening sequences.     

The transorientation hypothesis for codon recognition during protein synthesis

During decoding, a codon of messenger RNA is matched with its cognate aminoacyl-transfer RNA and the amino acid carried by the tRNA is added to the growing protein chain. Here we propose a molecular mechanism for the decoding phase of translation: the transorientation hypothesis. The model incorporates a newly identified tRNA binding site and utilizes a flip between two tRNA anticodon loop structures, the 5'-stacked and the 3'-stacked conformations. The anticodon loop acts as a three-dimensional hinge permitting rotation of the tRNA about a relatively fixed codon-anticodon pair. This rotation, driven by a conformational change in elongation factor Tu involving GTP hydrolysis, transorients the incoming tRNA into the A site from the D site of initial binding and decoding, where it can be proofread and accommodated. The proposed mechanisms are compatible with the known structures, conformations and functions of the ribosome and its component parts including tRNAs and EF-Tu, in both the GTP and GDP states.     

The yeast ubiquitin gene: head-to-tail repeats encoding a polyubiquitin precursor protein

Ubiquitin, a 76-residue protein, occurs in cells either free or covalently joined to a variety of protein species, from chromosomal histones to cytoplasmic proteins. Conjugation of ubiquitin to proteolytic substrates is essential for the selective degradation of intracellular proteins in higher eukaryotes. We show here that a protein homologous to human ubiquitin exists in the yeast Saccharomyces cerevisiae, and that yeast extracts conjugate human ubiquitin to a variety of endogenous proteins in an ATP-dependent reaction. We have isolated the S. cerevisiae ubiquitin gene and found it to contain six consecutive ubiquitin-coding repeats in a found it to contain six consecutive ubiquitin-coding repeats in a head-to-tail arrangement. This apparently unique gene organization suggests that yeast ubiquitin is generated by processing of a precursor protein in which several exact repeats of the ubiquitin amino acid sequence are joined directly via Gly-Met peptide bonds between the last and first residues of mature ubiquitin, respectively. Ubiquitin-coding yeast DNA repeats are restricted to a single genomic locus; although the sequenced repeats differ in up to 27 of 228 bases per repeat, they encode identical amino acid sequences. As this predicted amino acid sequence differs in only 3 of 76 residues from that of ubiquitin in higher eukaryotes, ubiquitin is apparently the most conserved of known proteins.     

Nucleotide sequence of the yeast plasmid

The nucleotide sequence of the yeast DNA plasmid (2 mu circle) from Saccharomyces cerevisiae strain A364A D5 has been determined. The plasmid contains 6,318 base pairs, including two identical inverted repeats of 599 base pairs. Possible functions are suggested, and attributes of an improved vector for cloning foreign DNAs in yeast are discussed.