Ln all retroviruses, reverse transcription is primed by a cellular tRNA, which is base-paired through its 3′-terminal 18 nucleotides to a complementary sequence on the viral RNA genome termed the primer binding site (PBS). Evidence for specific primer-template interactions in addition to this standard interaction has recently been demonstrated for several retroviruses. Here, we used chemical and enzymatic probing to investigate the interactions between Moloney murine leukemia virus (M-MuLV) RNA and its natural primer tRNA(Pro). The existence of extended interactions was further tested by comparing the viral RNA/tRNAP(Pro) complex with simplified complexes in which viral RNA or tRNA were reduced to the 18 nt of the PBS or to the complementary tRNA sequence. These data, combined with computer modeling, provide important clues on the secondary structure and three-dimensional folding of the M-MuLV RNA/tRNA(Pro) complex. In contrast with other retroviruses, we found that the interaction between tRNA(Pro) and the M-MuLV RNA template is restricted to the standard PBS interaction. Ln this binary complex, the viral RNA is highly constrained and the rest of tRNA(Pro) is rearranged, with the exception of the anticodon arm, leading to a very compact structure. Unexpectedly, when a synthetic tRNA(Pro) lacking the post-transcriptional modifications is substituted for the natural tRNA primer, the interactions between the primer and the viral RNA are extended. Hence, our data suggest that the post-transcriptional modifications of natural tRNA(Pro) prevent additional contacts between tRNA(Pro) and the U5 region of M-MuLV RNA. (C) 1998 Academic Press Limited.
Modified nucleotides of tRNA(Pro) restrict interactions in the binary primer/template complex of M-MuLV
Fosse, P.; Mougel, M.; Keith, G.; Westhof, E.; Ehresmann, B.; Ehresmann, C.
Journal of molecular biology
1998-02-06 / vol 275 / pages 731-746
human-immunodeficiency-virus; conformation; hiv-1 reverse transcription; lead-catalyzed cleavage; m-mulv retrovirus; murine leukemia-virus; primer-binding-site; primer/template interactions; reverse transcription; secondary structures; tertiary structure; transfer rnaasp; transfer-rnas; trna primer; yeast transfer rnaphe