A Study of the Thermal Unfolding of Escherichia coli Phenylalanine Transfer RNA by Chemical Modification at Elevated Temperatures

Abstract

Escherichiu coli tRNAPh' was modified by 3 M sodium bisulphite pH 6.0 for 24 h in the temperature range 25 "C ( x 5 "C) to 55 "C and in the absence of added magnesium ions. The sites and extents of conversion of cytidines to uridine occurring at each temperature were determined by fingerprinting. The new sites of cytidine modification found at higher reaction temperatures were assumed to arise from breakage of secondary and tertiary structure hydrogen bonds involving cytidine residues. From these data, we conclude that hydrogen bonds within the 'complex core' of the tRNA (including the base pairs G-10 . C-25, C-1 1 G-24 and C-13 . G-21 within the dihydrouridine stem and the tertiary structure base pair G-15 . C-48 melt at a lower temperature than the tertiary structure hydrogen bonds between G-19 in the dihydrouridine loop and C-56 in the TYC loop.Conformational changes in tRNA have been postulated on aminoacylation [l -31 and on enzymatic binding of aminoacyl-tRNA to the ribosome-mRNA complex [4,5]. Certain tRNAs may exist in two different conformations, one active and one inactive in aminoacylation. It is therefore of interest to examine some of the conformations in which tRNA molecules may exist.The detailed X-ray crystallographic analysis of yeast tRNAPh ' [6,7] and accumulated primary sequence data [8] have vastly increased our understanding of the static conformation of tRNAs. However much remains unclear about the nature of the conformational changes which tRNAs could undergo in biochemical processes.The denaturation of tRNA is not a single cooperative process but a sequential one and its study provides a means of assessing the conformational stability of a tRNA. Several such studies have recently been made by physical techniques [9 -171. Chemical modification studies have proved to be an effective technique in the study of the static tertiary structure of tRNAs in solution [18]. In particular, sodium bisulphite has been widely used to convert accessible cytidine residues in tRNAs to uridine residues I19 -251. Here we report an extension of this technique, viz the analysis of E. coli tRNA;he modified with bisulphite at a series of elevated temperatures. We interpret our results in terms of the sequential unfolding of the tRNA and relate our interpretation to those of comparable physical studies. MATERIALS AND METHODS MaterialsSoluble 32P-labelled ribonucleic acid from E. coli K12 CA265 and all other radiochemicals were obtained from the Radiochemical Centre (Arnersham, Bucks). Mixed transfer RNA from E. coli K 12 CA265 was supplied by the Microbiological Research Establishment (Porton, Salisbury, Wilts.). Purified tRNAP'Ie (from E. coli MRE 600) and benzoylated DEAEcellulose were obtained from the Boehringer Corporation, poly(C) was from P-L Biochemicals.E. coli phenylalanyl-tRNA synthetase was prepared from E. coli MRE 600 cells, obtained from the Microbiological Research Establishment, by the method of Stulberg [26], the hydroxyapatite chromatography stages being omitted. Pancreatic ribonuclease, snake...

Elucidating the influence of RNA modifications and Magnesium ions on tRNAPheconformational dynamics inS. cerevisiae: Insights from Replica Exchange Molecular Dynamics simulations

“…We observed that at high Mg 2+ concentrations (20mM and 40mM), the tRNA with no modifications maintains its native L-shaped structure even at elevated temperatures. This aligns with the previous notion that magnesium ions play a central role in stabilizing tRNA’s secondary and tertiary structure (15, 54, 55).…”

The structure of cruciforms in supercoiled DNA: probing the single-stranded character of nucleotide bases with bisulphite.

“…It seems feasible that reversible formation of 5,6-dihydrocytosine-6-sulphonate may occur at such a location, but the second-stage conversion of deoxyuracil appears improbable. Comparison with studies on tRNA (Furuichi et al, 1970;Kucan et al, 1971;Goddard and Schulman, 1972;Chambers et al, 1973;Singhal, 1974;Lowdon and Goddard, 1976;Goddard and Lowdon, 1978) is useful, where there is a known secondary and tertiary structure (Kimn et al, 1974;Robertus et al, 1974). Goddard and Lowdon (1978) studied the reaction of cytosines in E. coli tRNAPhe as a function of temperature.…”

“…Comparison with studies on tRNA (Furuichi et al, 1970;Kucan et al, 1971;Goddard and Schulman, 1972;Chambers et al, 1973;Singhal, 1974;Lowdon and Goddard, 1976;Goddard and Lowdon, 1978) is useful, where there is a known secondary and tertiary structure (Kimn et al, 1974;Robertus et al, 1974). Goddard and Lowdon (1978) studied the reaction of cytosines in E. coli tRNAPhe as a function of temperature. At 25°C they observed high-frequency modification of cytosines in the loop regions, and no detectable reaction with base-paired cytosine, even when present at the ends of helices.…”

“…The reaction is not self-propagating, and modification of a single base in one molecule is readily detected. Bisulphite has been used extensively as a probe of secondary structure in RNA molecules (Furuichi et al, 1970;Kucan et al, 1971;Chambers et al, 1973;Singhal, 1974;Lowdon and Goddard, 1976;Goddard and Lowdon, 1978), but application to DNA structure has not been tried before to our knowledge. However, the method is similar in principle, if not in purpose, to that of D-loop and gap mutagenesis (Shortle and Nathans, 1978;Peden and Nathans, 1982).…”