The Bipartite Structure of the tRNA m\u3csup\u3e1\u3c/sup\u3eA58 Methyltransferase from \u3cem\u3eS. cerevisiae\u3c/em\u3e is Conserved in Humans

Among all types of RNA, tRNA is unique given that it possesses the largest assortment and abundance of modified nucleosides. The methylation at N1 of adenosine 58 is a conserved modification, occurring in bacterial, archaeal, and eukaryotic tRNAs. In the yeast Saccharomyces cerevisiae, the tRNA 1-methyladenosine 58 (m1A58) methyltransferase (Mtase) is a two-subunit enzyme encoded by the essential genes TRM6 (GCD10) and TRM61 (GCD14). While the significance of many tRNA modifications is poorly understood, methylation of A58 is known to be critical for maintaining the stability of initiator tRNAMet in yeast. Furthermore, all retroviruses utilize m1A58-containing tRNAs to prime reverse transcription, and it has been shown that the presence of m1A58 in human tRNA3 Lys is needed for accurate termination of plus-strand strong-stop DNA synthesis during HIV-1 replication. In this study we have identified the human homologs of the yeast m1A Mtase through amino acid sequence identity and complementation of trm6 and trm61 mutant phenotypes. When coexpressed in yeast, human Trm6p and Trm61p restored the formation of m1A in tRNA, modifying both yeast initiator tRNAMet and human tRNA3 Lys. Stable hTrm6p/hTrm61p complexes purified from yeast maintained tRNA m1A Mtase activity in vitro. The human m1A Mtase complex also exhibited substrate specificity—modifying wild-type yeast tRNAi Met but not an A58U mutant. Therefore, the human tRNA m1A Mtase shares both functional and structural homology with the yeast tRNA m1A Mtase, possessing similar enzymatic activity as well as a conserved binary composition

Conserved Amino Acids in Each Subunit of the Heteroligomeric tRNA m\u3csup\u3e1\u3c/sup\u3eA58 Mtase from \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e Contribute to tRNA Binding

In Saccharomyces cerevisiae, a two-subunit methyltransferase (Mtase) encoded by the essential genes TRM6 and TRM61 is responsible for the formation of 1-methyladenosine, a modified nucleoside found at position 58 in tRNA that is critical for the stability of . The crystal structure of the homotetrameric m1A58 tRNA Mtase from Mycobacterium tuberculosis, TrmI, has been solved and was used as a template to build a model of the yeast m1A58 tRNA Mtase heterotetramer. We altered amino acids in TRM6 and TRM61 that were predicted to be important for the stability of the heteroligomer based on this model. Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation. In addition, recombinant mutant enzymes had reduced in vitro Mtase activity. We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-l-methionine. Instead, amino acid substitutions in either Trm6p or Trm61p destroy the ability of the yeast m1A58 tRNA Mtase to bind , indicating that each subunit contributes to tRNA binding and suggesting a structural alteration of the substrate-binding pocket occurs when these mutations are present

Rex1p Deficiency Leads to Accumulation of Precursor Initiator tRNA\u3csup\u3eMet\u3c/sup\u3e and Polyadenylation of Substrate RNAs in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e

A synthetic genetic array was used to identify lethal and slow-growth phenotypes produced when a mutation in TRM6, which encodes a tRNA modification enzyme subunit, was combined with the deletion of any non-essential gene in Saccharomyces cerevisiae. We found that deletion of the REX1 gene resulted in a slow-growth phenotype in the trm6-504 strain. Previously, REX1 was shown to be involved in processing the 3′ ends of 5S rRNA and the dimeric tRNAArg-tRNAAsp. In this study, we have discovered a requirement for Rex1p in processing the 3′ end of tRNAiMet precursors and show that precursor tRNAiMet accumulates in a trm6-504 rex1Δ strain. Loss of Rex1p results in polyadenylation of its substrates, including tRNAiMet, suggesting that defects in 3′ end processing can activate the nuclear surveillance pathway. Finally, purified Rex1p displays Mg2+-dependent ribonuclease activity in vitro, and the enzyme is inactivated by mutation of two highly conserved amino acids

Conserved amino acids in each subunit of the heteroligomeric tRNA m1A58 Mtase from Saccharomyces cerevisiae contribute to tRNA binding

In Saccharomyces cerevisiae, a two-subunit methyltransferase (Mtase) encoded by the essential genes TRM6 and TRM61 is responsible for the formation of 1-methyladenosine, a modified nucleoside found at position 58 in tRNA that is critical for the stability of tRNAiMet. The crystal structure of the homotetrameric m1A58 tRNA Mtase from Mycobacterium tuberculosis, TrmI, has been solved and was used as a template to build a model of the yeast m1A58 tRNA Mtase heterotetramer. We altered amino acids in TRM6 and TRM61 that were predicted to be important for the stability of the heteroligomer based on this model. Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation. In addition, recombinant mutant enzymes had reduced in vitro Mtase activity. We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-l-methionine. Instead, amino acid substitutions in either Trm6p or Trm61p destroy the ability of the yeast m1A58 tRNA Mtase to bind tRNAiMet, indicating that each subunit contributes to tRNA binding and suggesting a structural alteration of the substrate-binding pocket occurs when these mutations are present

Studies of a Methyltransferase and an Exonuclease Involved in Initiator tRNAmet Maturation in Saccharomyces Cerevisiae

More than 95% of the nucleic acid found in yeast cells is non-coding ribonucleic acid (RNA), which is RNA that specifies a RNA product, rather than a protein product (1). Noncoding RNA in eukaryotes includes transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and ribosomal RNA (rRNA), which is the most abundant noncoding RNA (2). Formation of a functional noncoding RNA does not result from the transcription of the gene alone---a number of processing and modification reactions occur before maturation. For example, in the case ofrRNA, maturation involves cleavage of polycistronic transcripts to release individual rRNAs, trimming of 3\u27 ends, and modification of nucleotides (3). Likewise, tRNAs are transcribed as precursors with extra nucleotides at the 5\u27 and 3\u27 ends that need to be eliminated (4) (Fig. 1.1). In addition, many nucleotides are modified, introns are removed, and the sequence CCA is added to the 3\u27 end in preparation for aminoacylation (Fig. 1.1). In general, synthesis of noncoding RNAs is complex and requires that cells not only transcribe the RNAs, but also manufacture additional RNAs, proteins, and cofactors that are involved in producing the mature RNA. I have used the yeast Saccharomyces cerevisiae to study processing and modification events in the production of tRNA, the noncoding RNA that is essential for messenger RNA to be translated and proteins produced. tRNA was discovered in the late 1950\u27s when it was found that radiolabeled leucine could be transferred to an RNA and then into a protein (5). The first tRNAs to be purified in significant amounts were yeast tRNAs, and, as a result, the first tRNA to be sequenced was yeast tRNA Ala (6).g.

Deciphering Tuberactinomycin Biosynthesis: Isolation, Sequencing, and Annotation of the Viomycin Biosynthetic Gene Cluster

The tuberactinomycin antibiotics are essential components in the drug arsenal against Mycobacterium tuberculosis infections and are specifically used for the treatment of multidrug-resistant tuberculosis. These antibiotics are also being investigated for their targeting of the catalytic RNAs involved in viral replication and for the treatment of bacterial infections caused by methicillin-resistant Staphylococcus aureus strains and vancomycin-resistant enterococci. We report on the isolation, sequencing, and annotation of the biosynthetic gene cluster for one member of this antibiotic family, viomycin, from Streptomyces sp. strain ATCC 11861. This is the first gene cluster for a member of the tuberactinomycin family of antibiotics sequenced, and the information gained can be extrapolated to all members of this family. The gene cluster covers 36.3 kb of DNA and encodes 20 open reading frames that we propose are involved in the biosynthesis, regulation, export, and activation of viomycin, in addition to self-resistance to the antibiotic. These results enable us to predict the metabolic logic of tuberactinomycin production and begin steps toward the combinatorial biosynthesis of these antibiotics to complement existing chemical modification techniques to produce novel tuberactinomycin derivatives