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

Abstract In contrast to all other known tRNAs, mammalian tRNAVal1 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 tRNAVal1 fragment does not necessarily mean that this is also true for intact tRNA. However, we showed that U54 is far less available for enzymatic methylation in mammalian tRNAVal1 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.