Rabbit liver tRNA1Val: II. Unusual secondary structure of TΨC stem and loop due to a U54:A60 base pair

摘要

In contrast to all other known tRNAs, mammalian contains two adenosines A59 and A60, opposite to U₅₄ and Ψ₅₅ in the UΨCG sequence of the TΨC loop, which could form unusual A:U (or A:Ψ) pairs in addition to the five “normal” G:C pairs. In order to measure the number of G:C and A:U (A:Ψ ) pairs in the TΨC stem, we prepared the 30 nucleotide long 3-terminal fragment of this tRNA by “m7G-cleavage”. From differentiated melting curves and temperature jump experiments it was concluded that the TΨC stem in this fragment is in fact extended by an additional A₆₀:U₅₄ pair. A dimer of this fragment with 14 base pairs was characterized by gel electrophoresis and by the same physical methods. An additional A:U pair in the fragment does not necessarily mean that this is also true for intact tRNA. However, we showed that U₅₄ is far less available for enzymatic methylatlon in mammalian compared to tRNA from T- E. coli. This clear difference in U₅₄ reactivity, together with the identification of an extra A₆₀:U₅₄ pair in the UΨCG containing fragment suggests the presence of a 6 base pair TΨC stem and a 5 nucleotide TΨC loop in this tRNA.