Modified tRNAs for probing tRNA binding sites on the ribosome.
Unusual chemical properties of hypermodified nucleosides N6-(threoninocarbonyl)adenosine (t6 A) located at position 37 and 3-(3-amino-3-carboxypropyl)uridine (acp3U) located at position 20:1 have been utilized for the introduction of photoreactive azidonitrophenyl probes to the anticodon loop and to the dihydrouridine loop of yeast tRNA(mMet) and lupin tRNA(mMet), respectively. The very efficient and selective modification procedures involve condensat on of the carboxyl group of t6A with ethylenediamine in the presence of a water soluble carbodiimide followed by acylation of the newly introduced amino group with the respective N-hydroxysuccinimide ester, and acylation of the primary amino or up of acp3U with the respective N-hydroxysuccinimide ester. Binding and crosslinking of the modified, uncharged tRNAs to E coli ribosome have been studied in the presence and absence of poly(AUG) as a message. Both tRNAs carrying about 20 A long photoreactive probes retain their binding activity and upon irradiation with visible light crosslink to the ribosome with high yields showing their usefulness for structural studies on the tRNA-ribosome complex.
Podkowinski
J
J
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland.
Dymarek-Babs
T
T
Gornicki
P
P
D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry; D000345:Affinity Labels; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D012358:RNA, Transfer, Met / Q000737:chemistry*; D012270:Ribosomes / Q000378:metabolism*; D014529:Uridine / Q000031:analogs & derivatives* / Q000737:chemistry
D000345:Affinity Labels; D012358:RNA, Transfer, Met; C003268:N(6)-(N-threonylcarbonyl)adenosine; C006514:3-(3-amino-3-carboxypropyl)uridine; D000241:Adenosine; D014529:Uridine