The synthesis and functional evaluation of RNA and DNA polymers having the sequence of Escherichia coli tRNA(fMet).
Stepwise, solid-phase chemical synthesis has provided long RNA and DNA polymers related to the sequence of Escherichia coli tRNA(fMet). The 34-ribonucleotide oligomer corresponding to the sequence of the 5'-half tRNA molecule has been synthesized and then characterized by gel purification, terminal nucleotide determinations and sequence analysis. This 34-nucleotide oligomer serves as an acceptor in the RNA-ligase-catalyzed reaction with a phosphorylated 43-ribonucleotide oligomer corresponding to the sequence of the 3'-half molecule of tRNA(fMet). The DNA molecule having the sequence of tRNA(fMet) is a 76-deoxyribonucleotide oligomer with a 3'-terminal riboadenosine residue and all U residues replaced by T. These polymers have been compared with an oligodeoxyribonucleotide lacking all 2'-hydroxyl groups except for the 3'-terminal 2'-OH, an oligoribonucleotide lacking modified nucleosides and E. coli tRNA(fMet). The all-RNA 77-nucleotide oligomer can be aminoacylated by E. coli methionyl-tRNA synthetase preparation from E. coli with methionine and threonylated in the A37 position using a yeast extract. In agreement with work by Khan and Roe using tDNA(Phe) and tDNA(Lys), the rA77-DNA(fMet) can be aminoacylated, and preliminary evidence suggests that it can be threonylated to a small extent. Kinetic data support the notion that aminoacylation of tRNA(fMet) does not depend on the presence of 2'-hydroxyl groups with the exception of that in the 3'-terminal nucleotide.
Perreault
J P
JP
Département de Biochimie, Université de Montréal, Canada.
Pon
R T
RT
Jiang
M Y
MY
Usman
N
N
Pika
J
J
Ogilvie
K K
KK
Cedergren
R
R
D000215:Acylation; D001483:Base Sequence; D004269:DNA, Bacterial / Q000138:chemical synthesis* / Q000502:physiology / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics*; D012329:RNA, Bacterial / Q000138:chemical synthesis* / Q000502:physiology / Q000648:ultrastructure; D012345:RNA, Transfer, Amino Acid-Specific / Q000648:ultrastructure*; D012358:RNA, Transfer, Met / Q000648:ultrastructure*; D012689:Sequence Homology, Nucleic Acid; D013698:Templates, Genetic
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012345:RNA, Transfer, Amino Acid-Specific; D012358:RNA, Transfer, Met