Abstract
We showed previously that introduction of two of the three unique features of Escherichia coli initiator tRNA onto an elongator methionine tRNA conferred significant activity in initiation. Surprisingly, introduction also of the third unique feature, the A11:U24 base pair in the D stem, resulted in total lack of accumulation of the mutant Mi:3 tRNA. We show here that the Mi:3 tRNA gene is transcribed efficiently in vitro. Processing of the Mi:3 precursor transcript shows, however, that both the precursor and the mature Mi:3 tRNA are unstable in E. coli extracts. To understand the basis of instability caused by the A11:U24 base pair in the elongator methionine tRNA background, we have isolated and characterized intragenic suppressor mutations in the tRNA that restore its function in translation initiation. Sequence changes in the T stem that convert the existing A51 x C63 mismatch to a base pair in the Mi:3 tRNA result in accumulation of the tRNAs in vivo. The initiation activity and in vivo levels of accumulation of these suppressors are in the order Mi:3/G51:C63 > Mi:3/A51:U63 >> Mi:3/G51.U63. These results show that the in vivo accumulation of a tRNA with A11:U24 base pair in the D stem depends upon a base pair between positions 51 and 63 in the T stem. Structural analysis in vitro of the Mi:3 and Mi:3/G51:C63 transcripts suggests that the Mi:3 tRNA is unable to adopt a stable tRNA-like conformation. Various considerations suggest that this is most likely due to a high entropic barrier to tertiary interactions, between the D and the T loops necessary for the formation of a stable tRNA structure.
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