The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae.

The modified nucleoside 1-methyladenosine (m(1)A) is found at position 58 in the TPsiC loop of many eukaryotic tRNAs. The absence of m(1)A from all tRNAs in Saccharomyces cerevisiae mutants lacking Gcd10p elicits severe defects in processing and stability of initiator methionine tRNA (tRNA(i)(Met)). Gcd10p is found in a complex with Gcd14p, which contains conserved motifs for binding S-adenosylmethionine (AdoMet). These facts, plus our demonstration that gcd14Delta cells lacked m(1)A, strongly suggested that Gcd10p/Gcd14p complex is the yeast tRNA(m(1)A)methyltransferase [(m(1)A)MTase]. Supporting this prediction, affinity-purified Gcd10p/Gcd14p complexes used AdoMet as a methyl donor to synthesize m(1)A in either total tRNA or purified tRNA(i)(Met) lacking only this modification. Kinetic analysis of the purified complex revealed K(M) values for AdoMet or tRNA(i)(Met) of 5.0 microM and 2.5 nM, respectively. Mutations in the predicted AdoMet-binding domain destroyed GCD14 function in vivo and (m(1)A)MTase activity in vitro. Purified Flag-tagged Gcd14p alone had no enzymatic activity and was severely impaired for tRNA-binding compared with the wild-type complex, suggesting that Gcd10p is required for tight binding of the tRNA substrate. Our results provide a demonstration of a two-component tRNA MTase and suggest that binding of AdoMet and tRNA substrates depends on different subunits of the complex.

[1]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations. , 1984, Journal of bacteriology.

[2]  A. Byström,et al.  Rit1, a tRNA backbone-modifying enzyme that mediates initiator and elongator tRNA discrimination , 1994, Cell.

[3]  S. Shuman,et al.  Mutational analysis of the Saccharomyces cerevisiae ABD1 gene: cap methyltransferase activity is essential for cell growth , 1996, Molecular and cellular biology.

[4]  S. Clarke,et al.  S-Adenosylmethionine-dependent Methylation in Saccharomyces cerevisiae , 1999, The Journal of Biological Chemistry.

[5]  J. Yon,et al.  Precise gene fusion by PCR. , 1989, Nucleic acids research.

[6]  Katsura Asano,et al.  Identification of a Translation Initiation Factor 3 (eIF3) Core Complex, Conserved in Yeast and Mammals, That Interacts with eIF5 , 1998, Molecular and Cellular Biology.

[7]  M. Mann,et al.  The yeast protein Arc1p binds to tRNA and functions as a cofactor for the methionyl‐ and glutamyl‐tRNA synthetases. , 1996, The EMBO journal.

[8]  A. Hinnebusch,et al.  GCD10, a translational repressor of GCN4, is the RNA-binding subunit of eukaryotic translation initiation factor-3. , 1995, Genes & development.

[9]  D. Söll,et al.  Aminoacyl-tRNA synthetases: general features and recognition of transfer RNAs. , 1979, Annual review of biochemistry.

[10]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[11]  E. Hurt,et al.  A conserved domain within Arc1p delivers tRNA to aminoacyl-tRNA synthetases. , 1998, Molecular cell.

[12]  A. Nedospasov,et al.  Purification and characterization of tRNA (adenine-1-)-methyltransferase from Thermus flavus strain 71. , 1982, European journal of biochemistry.

[13]  E. Cundliffe,et al.  Site-specific methylation of 16S rRNA caused by pct, a pactamycin resistance determinant from the producing organism, Streptomyces pactum , 1991, Journal of bacteriology.

[14]  A. Hinnebusch,et al.  The essential Gcd10p-Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA. , 1998, Genes & development.

[15]  P. Leboy,et al.  Purification and properties of tRNA(adenine-1)-methyltransferase from rat liver. , 1977, The Journal of biological chemistry.

[16]  E. Cundliffe,et al.  Sites of action of two ribosomal RNA methylases responsible for resistance to aminoglycosides. , 1987, Journal of molecular biology.

[17]  P. Sigler,et al.  The 3 A crystal structure of yeast initiator tRNA: functional implications in initiator/elongator discrimination. , 1991, The EMBO journal.

[18]  S. Wolin,et al.  The trials and travels of tRNA. , 1999, Genes & development.

[19]  A. Hinnebusch,et al.  GCD14p, a Repressor of GCN4 Translation, Cooperates with Gcd10p and Lhp1p in the Maturation of Initiator Methionyl-tRNA in Saccharomyces cerevisiae , 1999, Molecular and Cellular Biology.

[20]  Dieter Söll,et al.  Trna: Structure, Biosynthesis, and Function , 1995 .

[21]  G. Björk,et al.  Transfer RNA modification. , 1987, Annual review of biochemistry.

[22]  G. Björk,et al.  The Methyl Group of theN6-Methyl-N6-Threonylcarbamoyladenosine in tRNA of Escherichia coli Modestly Improves the Efficiency of the tRNA , 1998, Journal of bacteriology.

[23]  R. Kagan,et al.  Widespread occurrence of three sequence motifs in diverse S-adenosylmethionine-dependent methyltransferases suggests a common structure for these enzymes. , 1994, Archives of biochemistry and biophysics.

[24]  N. Martin,et al.  Location of N2,N2-dimethylguanosine-specific tRNA methyltransferase. , 1995, Biochimie.

[25]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .