Rex1p deficiency leads to accumulation of precursor initiator tRNAMet and polyadenylation of substrate RNAs in Saccharomyces cerevisiae.

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 tRNA(Arg)-tRNA(Asp). In this study, we have discovered a requirement for Rex1p in processing the 3' end of tRNA(i)(Met) precursors and show that precursor tRNA(i)(Met) accumulates in a trm6-504 rex1Delta strain. Loss of Rex1p results in polyadenylation of its substrates, including tRNA(i)(Met), suggesting that defects in 3' end processing can activate the nuclear surveillance pathway. Finally, purified Rex1p displays Mg(2+)-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 tRNA(Met)i 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 tRNA(Met)i, indicating that each subunit contributes to tRNA binding and suggesting a structural alteration of the substrate-binding pocket occurs when these mutations are present.

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.