tRNAHis maturation: An essential yeast protein catalyzes addition of a guanine nucleotide to the 5′ end of tRNAHis (original) (raw)

  1. Weifeng Gu1,
  2. Jane E. Jackman1,
  3. Amanda J. Lohan2,
  4. Michael W. Gray2, and
  5. Eric M. Phizicky1,3
  6. 1 Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA
  7. 2 Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax NS, B3H 1X5, Canada

Abstract

All tRNAHis molecules are unusual in having an extra 5′ GMP residue (G-1) that, in eukaryotes, is added after transcription and RNase P cleavage. Incorporation of this G-1 residue is a rare example of nucleotide addition occurring at an RNA 5′ end in a normal phosphodiester linkage. We show here that the essential Saccharomyces cerevisiae ORF YGR024c (THG1) is responsible for this guanylyltransferase reaction. Thg1p was identified by survey of a genomic collection of yeast GST-ORF fusion proteins for addition of [α-32P]GTP to tRNAHis. End analysis confirms the presence of G-1. Thg1p is required for tRNAHis guanylylation in vivo, because cells depleted of Thg1p lack G-1 in their tRNAHis.His6-Thg1p purified from Escherichia coli catalyzes the guanylyltransferase step of G-1 addition using a ppp-tRNAHis substrate, and appears to catalyze the activation step using p-tRNAHis and ATP. Thg1p is highly conserved in eukaryotes, where G-1 addition is necessary, and is not found in eubacteria, where G-1 is genome-encoded. Thus, Thg1p is the first member of a new family of enzymes that can catalyze phosphodiester bond formation at the 5′ end of RNAs, formally in a 3′-5′ direction. Surprisingly, despite its varied activities, Thg1p contains no recognizable catalytic or functional domains.

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