tRNA fluorescent labeling at 3' end inducing an aminoacyl-tRNA-like behavior.
A fluorescent tRNA derivative labeled at 3'-O position of the ultimate adenosine residue by reaction, under mild conditions, of tRNA with isatoic anhydride [3,1-benzoxazine-2,4(1H)-dione] was obtained. The labeling selectivity was determined by several criteria: digestion with RNase, followed by HPLC of the digest, produces only one labeled nucleoside, identified as 3'-O-anthraniloyladenosine; the ratio of the absorbance at 260 nm to 332 nm also suggests a 1:1 molar ratio between the nucleic acid and the fluorophore; finally, the incapacity of the labeled tRNA to be charged by the specific aminoacyltransferase further demonstrates the engagement of the 3'-O position. Although the 3'-O-anthraniloyl-labeled tRNA does not seem to be functionally active, as far as the aminoacyl charging activity is concerned, surprisingly we found that it is able to form the ternary complex with elongation factor Tu (EF-Tu) and GTP with an affinity consistently higher than uncharged tRNA. From fluorescence anisotropy measurements the ternary complex dissociation constant was estimated as 73 nM for Escherichia coli and 140 nM for yeast anthraniloyl-tRNA(Phe). These results may be interpreted in terms of the particular structure of the anthraniloyl group that makes the labeled tRNA similar to an aminoacyl-tRNA.
Servillo
L
L
Department of Biochemistry and Biophysics, University of Naples, Italy.
Balestrieri
C
C
Quagliuolo
L
L
Iorio
E L
EL
Giovane
A
A
D004926:Escherichia coli / Q000235:genetics; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry; D010078:Oxazines / Q000737:chemistry*; D010444:Peptide Elongation Factor Tu / Q000737:chemistry; D012329:RNA, Bacterial / Q000737:chemistry; D012331:RNA, Fungal / Q000737:chemistry; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry*; D012360:RNA, Transfer, Phe / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D005456:Fluorescent Dyes; D010078:Oxazines; D012329:RNA, Bacterial; D012331:RNA, Fungal; D012346:RNA, Transfer, Amino Acyl; D012360:RNA, Transfer, Phe; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu; C037902:isatoic anhydride