Temperature dependent chemical and enzymatic probing of the tRNA-like structure of TYMV RNA

Abstract

In this paper we report on the thermal unfolding of the tRNA-like structure present at the 3' end of turnip yellow mosaic virus (TYMV) RNA. Diethyl pyrocarbonate (DEP), sodium bisulphite, nuclease S1 and ribonuclease T1 were used as structure probes at a broad range of temperatures. In this way most of the nucleotides present in the tRNA-like moiety were analysed. The melting behaviour of both secondary and tertiary interactions could be followed on the basis of the temperature dependent accessibility of the individual nucleotides or bases towards the various probes. The three-dimensional model of the tRNA-like domain (Dumas et al., J. Biomol. Struct. and Dyn. 4, 707 (1987] was supported by the results to a large extent. The interactions occurring between the T- and D-loop appear to be more complex than proposed in the latter model. Additional evidence for the presence of the RNA pseudoknot (Rietveld et al., Nucleic Acids Res. 10, 1929 (1982] was derived from the fact that the three coaxially stacked helical segments in the aminoacylacceptor arm displayed different melting transitions under certain experimental conditions. Aspects of melting behaviour and thermal stability of double helical regions within the tRNA-like structure are discussed, as well as the applicability of nucleases and modifying reagents at various temperatures in the analysis of RNA structure.

Comparisons of the genomic sequences of erysimum latent virus and other tymoviruses: a search for the molecular basis of their host specificities

“…It has been shown that several tymoviral genomic RNAs can be aminoacylated with valine (Rietveld et al, 1982), however the fact that ELV could not be acylated led van Belkum et al (1987) to suggest that ELV might lack a tRNA-like structure, and this agrees with our results. It is uncertain how essential are the 3'-terminal tRNA-like structures of tymoviruses.…”

“…The sequence of each clone was determined, in both orientations. Their 5' portions were homologous, and they terminated in -C or -C(A) like other tymoviruses (van Belkum et al, 1987). It is unlikely that the ELV genome has a longer poly(A) tail as the primer dT8G failed to assist transcription unless the genomic RNA had been polyadenylated.…”

Biochemical and biophysical studies on the folding of the core region of the origin of replication of bacteriophage M13

“…S nuclease digestions were carried out with 2 units (0°C), 1 unit (37°C) and 0.2 unit (55°C) for 60, 30 and 8 minutes, respectively (for more experimental details see also [20]). Digestion was stopped by the addition of 100 Zl ethanol, followed by centrifugation.…”

Biochemical and biophysical analysis of pseudoknot‐containing RNA fragments

“…3'-Terminally labeled transcripts were analysed by diethyl pyrocarbonate and sodium bisulphite modification and RNase T I digestion at various temperatures as described for the 112-nucleotide fragment [15]. were identified and treated as described for the 112-nucleotide fragment .…”

“…It is clear that pseudoknots are important elements in RNA structure and functioning. The pseudoknot that has been investigated most thoroughly using both biochemical [3,4,14,15] and computer graphics [16] methods, is the one present in the aminoacyl acceptor arm of the tRNA-like structure of turnip yellow mosaic virus RNA. However, the current model, which accomodates most of the data available, needs biophysical verification.…”