-transparent.png){kind=logo}
We have constructed eight anticodon-modified Escherichia coli initiator methionine (fMet) tRNAs by insertion of synthetic ribotrinucleotides between two fragments ('half molecules') derived from the initiator tRNA. The trinucleotides, namely CAU (the normal anticodon), CAA, CAC, CAG, GAA, GAC, GAG and GAU, were joined to the 5' and 3' tRNA fragments with T4 RNA ligase. The strategy of reconstruction permitted the insertion of radioactive 32P label between nucleotides 36 and 37. tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes, and the following properties were evaluated: the stability of these eubacterial tRNA variants in the eukaryotic oocytes; the enzymatic modification of the adenosine at position 37 (3' adjacent to the anticodon) and aminoacylation of the chimeric tRNAs by endogenous oocyte aminoacyl-tRNA synthetases. In contrast to other variants, the two RNAs having CAU and GAU anticodons were stable and underwent quantitative modification at A-37. These results show that the enzyme responsible for the modification of A-37 to N-[N-(9-beta-D-ribofuranosylpurine-6-yl)carbamoyl]threonine (t6A) is present in the cytoplasm of oocytes and is very sensitive to the anticodon environment of the tRNA. Also, these same GAU and CAU anticodon-containing tRNAs are fully aminoacylated with the heterologous oocyte aminoacyl-tRNA synthetases in vivo. During the course of this work we developed a generally applicable assay for the aminoacylation of femtomole amounts of labelled tRNAs.
H Grosjean, and
S De Henau, and
T Doi, and
A Yamane, and
E Ohtsuka, and
M Ikehara, and
N Beauchemin, and
K Nicoghosian, and
R Cedergren
September 1977,
The Journal of biological chemistry,
H Grosjean, and
S De Henau, and
T Doi, and
A Yamane, and
E Ohtsuka, and
M Ikehara, and
N Beauchemin, and
K Nicoghosian, and
R Cedergren
April 1997,
Journal of molecular biology,
H Grosjean, and
S De Henau, and
T Doi, and
A Yamane, and
E Ohtsuka, and
M Ikehara, and
N Beauchemin, and
K Nicoghosian, and
R Cedergren
August 2012,
Proceedings of the National Academy of Sciences of the United States of America,
H Grosjean, and
S De Henau, and
T Doi, and
A Yamane, and
E Ohtsuka, and
M Ikehara, and
N Beauchemin, and
K Nicoghosian, and
R Cedergren
February 1976,
Proceedings of the National Academy of Sciences of the United States of America,
H Grosjean, and
S De Henau, and
T Doi, and
A Yamane, and
E Ohtsuka, and
M Ikehara, and
N Beauchemin, and
K Nicoghosian, and
R Cedergren
December 1991,
The Journal of biological chemistry,
H Grosjean, and
S De Henau, and
T Doi, and
A Yamane, and
E Ohtsuka, and
M Ikehara, and
N Beauchemin, and
K Nicoghosian, and
R Cedergren
July 1979,
Proceedings of the National Academy of Sciences of the United States of America,
H Grosjean, and
S De Henau, and
T Doi, and
A Yamane, and
E Ohtsuka, and
M Ikehara, and
N Beauchemin, and
K Nicoghosian, and
R Cedergren
January 1987,
Proceedings of the National Academy of Sciences of the United States of America,
H Grosjean, and
S De Henau, and
T Doi, and
A Yamane, and
E Ohtsuka, and
M Ikehara, and
N Beauchemin, and
K Nicoghosian, and
R Cedergren
March 1972,
Biochemical and biophysical research communications,
H Grosjean, and
S De Henau, and
T Doi, and
A Yamane, and
E Ohtsuka, and
M Ikehara, and
N Beauchemin, and
K Nicoghosian, and
R Cedergren
January 1993,
FASEB journal : official publication of the Federation of American Societies for Experimental Biology,
H Grosjean, and
S De Henau, and
T Doi, and
A Yamane, and
E Ohtsuka, and
M Ikehara, and
N Beauchemin, and
K Nicoghosian, and
R Cedergren
February 1970,
Biochimica et biophysica acta,
