Identification of yeast ORFs associated with tRNA^His guanylyltransferase activity. (A) Cloverleaf structure of S. cerevisiae cytoplasmic tRNA^His. Numbers indicate standard nt numbering positions; +1, the first nt encoded by the genome; -1, the extra nt added posttranscriptionally. Note that the tRNA is 75-nt long before G_-1 addition, because nt 17 and nt 47 are not present in this tRNA, and nt 20A is present. (B) Assay of a genomic collection of pools of purified yeast GST-ORF fusion proteins for tRNA^His guanylyltransferase activity. Substrate p-tRNA^His (0.4 μM) was incubated in 40-μL reaction mixtures containing 3 mM ATP, 0.6 μM [α-³²P]GTP, and ∼0.01 μg/μL protein from 64 pools of purified GST-ORF fusion proteins, each derived from 96 yeast strains, and products were resolved on gels as described in Materials and Methods. a, S100 extract (2μg/μL); b, no extract. (C) Assay of subpools from pool 4 for activity. Substrate p-tRNA^His was incubated with pools of GST-ORFs derived from the strains in rows A-H and columns 1-12 from plate 4, as indicated. a, S100 extract; b, no extract. (D) Assay of individual purified proteins from plate 4 for activity. a, S100 extract; b, no extract; B9, F4, G3, G4, G5, and H4 each correspond to the purified GST-ORF fusion protein from the corresponding position of microtiter plate 4.

Analysis of the activity of purified GST YGR024c fusion protein. (A) tRNA substrate specificity of YGR024p. Here, 0.4 μM p-tRNA^Phe (lanes a-c) or 0.4 μM p-tRNA^His (lanes d-f) was incubated in reaction mixtures containing 100 μM ATP, 0.6 μM [α-³²P]GTP, and GST dialysis buffer (lanes a,d), 0.5 μM GST-YGR024p (lanes b,e), or 2.4 μg/μL S100 extract (lanes c,f), and tRNA was resolved by PAGE as described in Materials and Methods. Lane g, [5′-³²P] tRNA^His. (B) Phosphatase treatment of guanylylated tRNA^His. Gel-purified tRNA product from reaction with GST-YGR024p was incubated with calf intestinal phosphatase (CIP), and products were resolved by thin layer chromatography as described in Materials and Methods. a, tRNA incubated on ice; b, tRNA on ice in CIP buffer; c, tRNA in CIP buffer at 37°C; d, tRNA incubated with CIP in CIP buffer at 37°C; e, ³²P-labeled inorganic phosphate. (C) Partial RNase T1 digestion of guanylylated tRNA^His. Gel-purified guanylylated tRNA^His and labeled tRNA standards were incubated with RNase T1 under partial digestion conditions, as described in Materials and Methods, and products were resolved by 12% PAGE. a, [5′-³²P]tRNA^His 76-mer, beginning with G_-1 (p^*76mer); b, [5′-³²P]tRNA^His 75-mer, beginning with G₊₁ (p^*75mer); c,d, p-tRNA^His substrate guanylylated with GST-YGR024p and GST-YDL076p, respectively, in the presence of 100 μM ATP and [α-³²P]GTP; e, 75-mer tRNA^His 3′-labeled with ^*pCp. Numbers on the left and right indicate the length of partially digested fragments in lanes a and e, respectively.

Effect of lack of ORF YGR024c protein on cell growth and on tRNA^His guanylylation. Strain WG18 conditionally lacking YGR024c (relevant genotype: ygr024c-Δ P_GAL-YGR024c) and the corresponding wild-type control strain (WT) WG12 were grown in rich medium containing galactose and inoculated in medium containing glucose. Growth, tRNA 5′-end maturation, and guanylyltransferase activity of extracts were monitored as a function of time. (A) Growth defect of strain conditionally lacking YGR024c. (•) Growth of strain conditionally lacking YGR024c. (○) Growth of WT cells. (B) Analysis of the 5′ end of tRNA^His. Bulk RNA was purified at each time point, and analyzed by primer extension as described in Materials and Methods to determine the 5′ ends of tRNA^His. Lanes a-d, sequencing reactions using RNA purified from WG12 at 7 h; e,f, RNA purified at 7 h (e) and 20 h (f) from WG12 after switch to glucose-containing medium; g-j, RNA purified from WG18 strain at various times after switch to glucose-containing medium, as indicated; k, primer alone. Arrow at -1 indicates position of the mature 5′ end of guanylylated tRNA^His. (C) Analysis of the 5′ end of tRNA^Lys. Bulk RNA was analyzed by primer extension to determine the 5′ end of tRNA^Lys, as described in B and Materials and Methods. (D) Assay for tRNA^His guanylyltransferase activity in crude extracts. Crude extracts made at different times after switch to glucose medium were assayed for tRNA^His guanylyltransferase activity as described in Materials and Methods. a-f, crude extracts from WT strain made at different time points, each assayed with 0.2μg/μL (a,c,e) and 0.02μg/μL (b,d,f) protein; g-i, crude extracts from WG18 strain made at different time points, assayed with 0.2μg/μL (g,i,k) and 0.02μg/μL (h,j,l) protein; m, no protein.

SDS-PAGE analysis of GST-Thg1p purified from yeast and His₆-Thg1p purified from E. coli. THG1 (YGR024c) expressed in yeast as GST-Thg1p and in E. coli as His₆-Thg1p, was purified as described in Materials and Methods and analyzed by SDS-PAGE on an 8%-16% gel (BioRad). (A) Analysis of His₆-Thg1p purified from E. coli and mock purification. a,d, crude extracts from transformed E. coli cells and mock-transformed cells; b,c, purified His₆-Thg1p from pooled elution fractions 8-12(3 μg) and 5-7 (6 μg); e,f, corresponding mock purification fractions; g, SDS-PAGE broad range protein standards (BioRad). Samples were visualized with Coomassie staining. (B) Analysis of GST-Thg1p purified from yeast. a-c, different amounts of purified GST-Thg1p as indicated; d, SDS-PAGE broad-range protein standards. Samples were visualized with silver staining.

Analysis of guanylyltransferase activity of purified His₆-Thg1p and GST-Thg1p. (A) Analysis with standard tRNA^His substrate. Purified proteins were assayed for guanylyltransferase activity in the presence or absence of 50 μM ATP as indicated, as described in Materials and Methods. a, [5′-³²P] p-tRNA^His substrate; b,c, no protein; d-i, pooled fractions 8-12 of mock purification from E. coli (see Fig. 4A, lane e) in the presence (d-f) or absence (g-i) of ATP; j-o, pooled fractions 8-12 of purified His₆-Thg1p in the presence (j-l) or absence (m-o) of ATP; p-u, GST-Thg1p in the presence (p-r) or absence (s-u) of ATP. Triangles, fourfold serial dilutions of protein (0.5 μM, 0.125 μM, and 0.032 μM). (B) Analysis of His₆-Thg1p with defined p-tRNA and ppp-tRNA substrates. Defined p-tRNA^His and ppp-tRNA^His substrates, prepared as described in Materials and Methods, were assayed for activity with His₆-Thg1p in the presence or absence of 50 μM ATP, and products were resolved by PAGE as described above. a, [5′-³²P] tRNA^His substrate; b-k, assay of p-tRNA^His substrate in the presence (b-f) or absence (g-k) of ATP. l-u, assay of ppp-tRNA^His substrate in the presence (l-p) or absence (q-u) of ATP; b,g,l,q, buffer controls; c,h,m,r, mock purification controls; d-f, i-k, n-p, s-u, fourfold serial titrations of His₆-Thg1p from 0.5 μM to 0.032μM.

Analysis of reaction of His₆-Thg1p with p-tRNA and ATP. (A) End analysis of reaction. p-tRNA^His substrate was incubated with [α-³²P]ATP and His₆-Thg1p, as described in Materials and Methods; labeled product tRNA was gel purified, then analyzed on thin-layer plates after digestion. a, no enzyme; b, nuclease P1; c, nuclease P1 followed by CIP; d, nuclease P1 followed by snake venom pyrophosphatase (SVP); e, nuclease P1 followed by CIP and SVP treatment. (B) Adenylylation specificity of His₆-Thg1p. p-tRNA was prepared from ppp-tRNA by dephosphorylation and rephosphorylation, as described in Materials and Methods, and then assayed for adenylylation activity. a, p-tRNA^His (75 nt); b, p-tRNA^Phe; c, p-tRNA^Leu; d, p-tRNA^His (93 nt); e, labeled tRNA^His standard. (C) Proposed mechanism for tRNA^His guanylylation.

CLUSTALX alignment of full-length Thg1p homologs. Accession numbers and related information are compiled in Supplementary Tables 1 and 2. The archaeal Thg1p homologs are set between horizontal lines. In a few cases, putative N-terminal mitochondrial targeting peptides were identified; numbers in parentheses preceding individual sequences indicate the length of these sequences. For clarity the variable C-terminal portion of the alignment has been omitted; numbers in parentheses at end of each sequence refer to the number of residues thus excluded. The rice and Arabidopsis genes represent continuous ORFs, each encoding a tandem duplication (to be described elsewhere) of the Thg1p homolog, with no identifiable internal methionine for initiation of the second copy. Only the 5′ complete copy of each of the plant genes is included in the alignment. Consensus motifs are shown at the bottom of the alignment. Using BioEdit, shading is based on a 60% consensus, with identical residues depicted on a black background and similar residues on a gray background.