tRNA tertiary structure in solution as probed by the photochemically induced 8-13 cross-link

Abstract

The conformation of ten purified tRNAs from Escherichia Coli has been investigated by means of the photo-induced cross-linking of 4Srd8 and Cyd13, which is sensitive to the juxtaposition of the two bases. Three tRNAs photo-react abnormally slowly; tRNAPhe, tRNAMet/m a and tRNAVal/2; a comparison with normally reacting species suggests that base 47 (Urd or modified Urd) is involved in a tertiary interaction in Class I tRNAs with the triplet 8, 14, 28. The UGA suppressor tRNATrp photoreacts significantly slower than the wild type. Thus the single base change Gua 24 to Ade induces a conformational change that alters the rate constant for the cross-linking reaction.

Conformational energy and structure in canonical and noncanonical forms of tRNA determined by temperature analysis of the rate of s4U8–C13 photocrosslinking

“…In addition, the photocrosslinking rate for tRNA Phe was reduced by more than twofold when it was irradiated in a complex with tRNA synthetase (Favre et al 1979). These studies indicate that the photocrosslinking reaction is very sensitive to conformational differences in the tRNA (Favre et al 1975).…”

“…Favre and coworkers (1975) used UVA irradiation (l>324 nm) of various Escherichia coli tRNAs to activate the 4-thiouridine at position 8 (s 4 U8) and showed that it specifically crosslinks to nucleotide C13. The rate for the photocrosslinking reaction at 25°C is different for different tRNAs and depends on the buffer conditions (Favre et al 1975(Favre et al , 1979. It was also found that a mutation in the D stem of (sus + ) tRNA Trp caused a decrease the rate of photocrosslinking compared to value seen in the (wildtype) tRNA Trp ; this was taken as evidence for a conformational difference due to the alteration of the tRNA structure in the D stem (Favre et al 1975;Favre and Thomas 1981).…”

“…The rate for the photocrosslinking reaction at 25°C is different for different tRNAs and depends on the buffer conditions (Favre et al 1975(Favre et al , 1979. It was also found that a mutation in the D stem of (sus + ) tRNA Trp caused a decrease the rate of photocrosslinking compared to value seen in the (wildtype) tRNA Trp ; this was taken as evidence for a conformational difference due to the alteration of the tRNA structure in the D stem (Favre et al 1975;Favre and Thomas 1981). In addition, the photocrosslinking rate for tRNA Phe was reduced by more than twofold when it was irradiated in a complex with tRNA synthetase (Favre et al 1979).…”

Substitution of uridine in vivo by the intrinsic photoactivable probe 4‐thiouridine in Escherichia coli RNA

“…Photoactivation of this residue by 335-nm or 366-nm light led to 8 -13-link formation in all native tRNAs containing a cytidine in position 13 in addition to the 4-thiouridine 8, i.e. more than 50% of E. coli tRNA species [3]. This cross-linking reaction has unravelled a salient feature of tRNA native three-dimensional structure long before it was seen by crystallography [4].…”

“…The particles were separated on a preparative gradient. The RNA from the 70s peak migrates at the 16s (2) and (3) refer to three cultures from which RNA was extracted and 23s control RNA positions. The 50s peak contains mainly 23s-like RNA plus a small percentage of a longer 28.58 molecule (not visible on Fig.…”