Fig. 4: Target-site position and upstream single-stranded DNA determine the efficiency of nicking and TPRT.

a, The full-length L1 ORF2p RNP structure (this study) superposed with a structure of the EN domain: duplex DNA complex (Protein Data Bank (PDB): 78NS). For a cleavage site near the 5′ end of the duplex DNA, there is no steric clash with L1 ORF2p (top). A modelled, longer DNA duplex engaged with the EN domain illustrates the steric clash of upstream duplex DNA with L1 ORF2p (bottom). b,c, Denaturing gel analysis of TPRT reaction products using target DNA with a varying cleavage-site position from 7 to 26 bp from the 5′ end of the duplex DNA (b) and 5 to 13 bp (c). d,e, Denaturing gel analysis of TPRT reaction products using target DNA of varying length and sequence of upstream single-stranded DNA. A blunt duplex end and short overhangs with T-rich sequences (d), and longer overhang lengths from 9 to 27 nt (e) were used. The red arrowheads in b–e denote the expected nicked product sizes from cleavage at the consensus target site. The experiments in b–e were replicated three independent times. The relative amount of full-length cDNA was quantified as the TPRT product. The mean ± s.d. for n = 3 replicates is shown. The purple bars in b–e bar graphs indicate the common DNA target site in all panels, which was used for normalization of the relative TPRT product. The rightmost lanes in d use a T-rich overhang with an alternative sequence. AJh 25A (141 nt) was used as the template RNA across b–e. f, Model for the initial stages of template engagement, target-site identification and first-strand synthesis by L1 ORF2p. The overhang single-stranded DNA is drawn near the CTS domain for illustration purposes only.