Characterization of the Native and Denatured Conformations of tRNA^Ser and tRNA^Phe from Yeast

Abstract

Previous work on the partial digestion of the native and denatured forms of tRNASer and tRNAPhe from yeast with TI-RNAase was extended. Detailed studies with pancreatic and T2-RNAase and a few experiments with sheep kidney nuclease and acid RNAase from hog spleen revealed characteristically different fragmentation patterns for the native and denatured forms of the tRNAs. Apparently it is mainly the miniloop and dihydrouridine regions which are involved in the conformational changes responsible for denaturation.The rate constants, activation enthalpies, and activation entropies of the renaturation process were calculated from amino acid acceptance data.When the changes in hyperchromicity a t 260 nm of the denatured tRNAs were followed as a function of time, a process was detected which in tRNAPhe is 5-10 times and in tRNAser 10-20 times faster than the renaturation as measured by acceptor activity. The whole net change in absorption, possibly representing the total net change in base stacking, occurs during the fast, process. The activation enthalpies and activation entropies are different for the fast and t,he over-all processes.

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.…”

Alternative secondary structures of leader RNAs and the regulation of the trp, phe, his, thr, and leu operons.

“…(iv) Within each leader RNA, translational start and stop signals are positioned so that a peptide of [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] amino acids might be synthesized (4,5,9,(11)(12)(13). In case of the trp operon, there is indirect evidence that the leader is translated (16,17).…”

“…As soon as the distal half of a pairing region is released from the polymerase, the region will fold up very rapidly [the time required for an intramolecular helix to develop is less than 50 lisec (25)]. The time scale over which attenuation operates is in the range of 1 to 5 sec [polymerase travels at 45 nucleotides per sec at 37"C (26)], whereas .the half-time for disruption of the paired regions considered here is in the range of minutes (27,28). Therefore we believe the important consideration is which pairing region forms first and not which pairing region is strongest.…”

Effects of the ionic environment on modification of yeast tyrosine transfer ribonucleic acid with N-acetoxy-2-acetylaminofluorene

“…AcNH-Fln modification of the former is most likely to occur because: (1) the latter sequence is in a double-stranded region, and (2) in a low ionic environment, partial unfolding of the tRNA molecule would be associated with a loss of the G-15 to C-58 base pair, thus rendering G-15 susceptible to chemical modification. Streeck and Zachau (1972) found that with denatured yeast tRNAPhe T2 RNase produced a split in the dihydrouridine loop at G-15 which did not occur with native tRNAPhe, suggesting that denaturation does selectively unfold this region of the tRNA molecule.…”