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

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 ["-32P]GTP to tRNA His. 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.