Transcripts of the viroid central conserved region contain the local tertiary structural element found in full‐length viroid

Abstract

The viroid central conserved region (CCR) is highly conserved among different viroids and is thought to be involved in viroid replication. A novel tertiary structure occurs in the CCR of native circular potato spindle tuber RNAs. To permit more detailed studies of this structural clcmcnt. a sITX$ll RNA oligonucleotide containing the CCR of the viroid genomc was synthesized. The tertiary structureof these CCR transcripts wasexamined by UV-crosslinking of the RNA, followed by mapping of the crosslink using limited alkaline digestion and ciassical RNA secondary analysis. The CCR transcript was found to undergo W-crosslinking between the same two bases as in full-length viroid, indicating that the tertiary structure is the Same and that the CCR transcript will be useful for the aninity purification of host components.

Viroid processing: switch from cleavage to ligation is driven by a change from a tetraloop to a loop E conformation

“…Biochemical studies on loop B and NMR studies on model oligonucleotides have revealed a complex pattern of Watson‐Crick and non‐canonical base‐pairs (Wimberly et al ., 1993; Butcher and Burke, 1994b; Szewczak and Moore, 1995). Branch and colleagues deduced the loop E‐like motif in viroids from a characteristic UV crosslink between the two bases G 98 and U 260 located in opposite strands of the CCR (Branch et al ., 1985; Paul et al ., 1992).…”

An RNA tertiary structure of the hepatitis delta agent contains UV-sensitive bases U-712 and U-865 and can form in a bimolecular complex

“…To produce alkaline hydrolysis ladders, samples were incubated at 90°C in 0.05 M sodium bicarbonate/carbonate at pH 9.2 for either 15 min (lanes a, d and e) or 5 min (lanes b, c and f). To produce a family of RNase TI partial digestion products, non-crosslinked samples were incubated at 65°C in buffer containing RNase TI and urea for 15 min (lane g) or4 min (lane h), as described before (62). Each band in the alkaline ladder is marked by a dot, to assist in the counting of the bands.…”