The kinetics of bisulphite modification of reactive residues in E.coli tRNAFormula

Abstract

E coli tRNA2Phe was modified at 25 degrees C with 3M sodium bisulphite, pH6.0, for periods of up to 48 hours, Three cytadinine residues, at position 17, 74 and 75 from the 5' end were each deaminated to uridine. The 2-methylthio-N6-isopentenyl adenosine at position 37 formed a 1:1 bi-sulphite addition product which was stable to alkaii. No other residues were permanently modified. The rate of modification of each residue was first order with respect to remaining unmodified nucleotide, the time of half reaction, t1/2, being different for each residue. C17 reaction reacted at twice the rate of cytidine in PolyC, indicating that it occupied a very exposed position in the tRNA.

The effect upon aminoacylation of bisulphite addition to 2‐methylthio‐N6‐isopentenyl adenosine of Escherichia coli phenylalanine tRNA

“…Purification of tRNAPhe from mixed tRNAs of E. coli K12CA265 (Microbiological Research Establishment, Porton, Salisbury, England) was performed as previously described [2].…”

Evidence on the conformation of HeLa-cell 5.8S ribosomal ribonucleic acid from the reaction of specific cytidine residues with sodium bisulphite

“…The HeLa-cell 5.8 S rRNA sequence differs from that of rat hepatoma in possessing C-U-U instead of C-U at the 3'-end. Some points about the molar recoveries of some of our digestion products are mentioned in the legends to Tables 1 and 2. Bisulphite reaction: choice ofconditions On the basis of experience gained during studies with other RNA species (Goddard & Schulman, 1972;Lowdon & Goddard, 1976;Goddard & Maden, 1976) we studied the reaction of 5.8S rRNA with NaHSO3 by using 3 M-NaHSO3, pH 6, at 25°C with 10mM-Mg2+ present. Fig.…”

“…As will become apparent below, it would be difficult to examine the initial reactivities of all the cytidines in such detail as in . Therefore, on the basis of these experiments and earlier experience with tRNA (Lowdon & Goddard, 1976), a 24h reaction period in 3 M-NaHSO3, pH 6, at 25°C was chosen for examining the relative reactivities of most of the cytidine residues within 5.8S rRNA. Although this single reaction period does not permit estimation of initial rates, it offers the advantages that the more reactive cytidine residues undergo extensive reaction during the period, and are therefore easily identified, and that relatively low degrees of reactivity of other cytidine residues are also detectable.…”

“…Possibly tertiary interactions may underly the differences between the two sets of results. For example in tRNA the dihydrouracil loop and the T-P-C loop interact in the tertiary structure (Jack et al, 1976), so that the phosphodiester backbone is inaccessible to S, nuclease (Harada & Dahlberg, 1975), whereas several nucleotide bases project and are accessible to chemical modifying agents (Rhodes, 1975), including NaHSO3 (Lowdon & Goddard, 1976). [This is discussed more fully by Goddard (1977).…”

“…NaHSO3 was removed by two dialyses (each of2h) against 0.15M-NaCI/0.0I M-Tris/ HCI, pH 7.5, and the adducts were removed by dialysis against 0.1 M-Tris/HCI, pH 9.0, at 37°C, for 9-lOh. The samples were then neutralized and desalted by dialysis (Goddard & Schulman, 1972;Lowdon & Goddard, 1976 (20,ug) was added as carrier to each portion and the samples were freeze-dried. They were digested with T1 ribonuclease (Calbiochem, London W.1, U.K.) or pancreatic ribonuclease (Sigma, London S.W.6, U.K.) in 0.01 M-Tris/HCI/0.0l M-EDTA (pH7.4) for 30-45min, at a 1: 10 (w/w) enzyme: substrate ratio.…”