Psoralen crosslinking between human immunodeficiency virus type 1 RNA and primer tRNA3(Lys).
Initiation of reverse transcription is a crucial step of retroviral infection. In HIV-1, it involves hybridization of the 18 3'-terminal nucleotides of the primer tRNA3(Lys) to the primer binding site (PBS) of the viral RNA. Moreover, additional interactions between the two RNAs were recently evidenced [Isel et al. (1995) J. Mol. Biol. 247, 25269-25272]. To get further information on the topology of the viral RNA/tRNA3(Lys) complex, we used psoralen to induce RNA-RNA crosslinking. A defined intermolecular crosslinked complex was obtained. The crosslinked regions were characterized by RNase T1 digestion followed by bi-dimensional gel electrophoresis. The crosslinked residues (nucleotide mcm5S2U34 and U35 in the anticodon loop of tRNA3(Lys) and UCU154 in the viral RNA upstream of the PBS) were mapped using a retardation method coupled with random hydrolysis. The formation of this crosslink depends on the same elements that are required for the formation of the extended interactions between primer and template RNAs, i.e., the modified bases of the tRNA and a conserved A-rich loop located upstream of the PBS in the genomic RNA. Therefore, the present crosslinking data provide relevant information on the topology of the template/primer binary complex.
Skripkin
E
E
Unité Propre de Recherche, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France.
Isel
C
C
Marquet
R
R
Ehresmann
B
B
Ehresmann
C
C
D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000378:metabolism*; D005363:Ficusin / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D012357:RNA, Transfer, Lys / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism
D003432:Cross-Linking Reagents; D012357:RNA, Transfer, Lys; D012367:RNA, Viral; D005363:Ficusin