Effect of Cleaving the Dihydrouridine Loop and the Ribothymidine Loop on the Amino Acid Acceptor Activity of Yeast Phenylalanine Transfer Ribonucleic Acid

Structure mapping of 5'-32P-labeled RNA with S1 nuclease

“…The RNA structure mapping approach described above has the potential for generating detailed information concerning the locations of hairpin single-stranded loop and helical stem regions in 5'-32P-labeled RNA molecules. End-labeling tRNA with polynucleotide kinase and [7-32P]ATP (Hanggi et ah, 1970;Lillenhaug & Kleppe, 1977;Silberklang et al, 1977), partial enzymatic cleavage using base-specific (Schmidt et al, 1970;Samuelson & Keller, 1972;Streeck & Zachau, 1972;Vigne & Jordan, 1977), or structure-specific (Harada & Dahlberg, 1975;Tal, 1975;Flashner & Vournakis, 1977; Rushizky & Mozejko, 1977) endoribonucleases, and the analysis of tRNA fragment molecules (Zachau et al, 1974), are combined with high resolution polyacrylamide gel electrophoresis to produce a rapid technique for mapping structure in molecules whose primary sequences are known.…”

“…nuclease, whereas it is hydrolyzed upon partial digestion with RNase T1 under different experimental conditions (Schmidt et al, 1970; Samuelson & Keller, 1972;Rich & RajBhandary, 1976). Although G20 has not been implicated in tertiary interactions, both G19 and A21 are hydrogen bonded to C56 and m7G46, respectively, in the crystal model (Jack et al, 1976;Quigley & Rich, 1976).…”

High-resolution nuclear magnetic resonance determination of transfer RNA tertiary base pairs in solution. 1. Species containing a small variable loop

“…Yeast tRNAphe. This material was purified to 95% homogeneity as described previously (Schmidt et al, 1970).…”

Regions of tRNA important for binding to the ribosomal A and P sites

“…Fractions containing the appropriate peaks (Simsek et al, 1973;Thiebe and Zachau, 1971) were pooled and the urea was removed by dialysis against water. Desalted fractions were concentrated by evaporation dissolved in 10 mM phosphate buffer, pH 7.5, containing 100 mM NaCl and applied onto a Sephadex G-100 column (3 X 100 cm), which was equilibrated and run at 50 °C with the same buffer (Schmidt et al, 1970). Fractions were analyzed by polyacrylamide gradient microelectrophoresis (Wolfrum et al, 1974) and the ones containing a uniform oligonucleotide were pooled, concentrated to onetenth of the volume by evaporation, and desalted on Bio-Gel P-2 column.…”

“…The reaction mixture was then extracted with Macaloid adsorbent (National Lead Company, Houston, Texas) three times (0.5 mg of adsorbent/mg of digest). The fragments were isolated by chromatography on DEAE-cellulose columns using buffers containing 7 M urea and by Sephadex G-100 gel filtration at 50 °C (Schmidt et al, 1970). Characterization was performed by polyacrylamide gradient microelectrophoresis.…”