tRNAHis maturation: an essential yeast protein catalyzes addition of a guanine nucleotide to the 5' end of tRNAHis.
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 [alpha-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 highlye 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.
Gu
Weifeng
W
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA.
Jackman
Jane E
JE
Lohan
Amanda J
AJ
Gray
Michael W
MW
Phizicky
Eric M
EM
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D006150:Guanine Nucleotides / Q000378:metabolism*; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames; D012323:RNA Processing, Post-Transcriptional; D012354:RNA, Transfer, His / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D006150:Guanine Nucleotides; D012354:RNA, Transfer, His; D029701:Saccharomyces cerevisiae Proteins; D009713:Nucleotidyltransferases; C020842:guanylyltransferase