Thermodynamics of a stable yeast 5.8S rRNA hairpin helix

Abstract

The 5. 8S ribosomal RNA of bakers yeast contains one particularly stable hairpin helix which is isolated by partial T1 ribonuclease digestion. Thermal hyperchromism analysis of the hairpin fragment showed that it dissociates cooperatively with 18% hyperchromism, with a Tm of 83 degree C at 2.7 mM sodium ion concentration, and with a hyperchromic difference spectrum indicative of over 90% G + C content. The probable secondary structure for the fragment was used to predict a helix free energy, delta G = -16.2 kcal/mole, which was the same as that determined from the melting equilibrium. The predicted enthalpy however, was 77% of the value, delta H = -114 kcal/mole, determined from the van't Hoff relationship. The effect on these data of a G.U base pair within the 9 base pair helix is discussed.

Site specific enzymatic cleavage of RNA

“…(cTUA}) or at nucleotides 136-141 (Wff These sites are located adjacent to or within a stable hairpin structure at nucleotides 116-137, observed by others (25,26), which may block cleavage by RNase H. Two other regions apparently uncleaved by calf thymus RNase H, nucleotides 24-29…”

“…Effects of RNA Secondary Structure Although the RNase H's cleave the RNA at regions of complementarity to the DNA oligomers, not all the primary sequence homologies are cut. The secondary structure of the RNA can prevent the formation of RNA-DNA hybrids or block their acessibility to RNase H. As mentioned before, in 5.8S rRNA a stable hairpin structure at nucleotides 116-137 (25,26) apparently blocks the cleavage by both RNase H's at three DNA oligomer binding sites. Furthermore, the two enzymes require buffers of different ionic strengths for optimal activity (11,16, the calf thymus RNase H reactions were done in a "high salt" buffer, 0.1 M KC1 with 25 mM MgCl2 and the E. coli RNase H digests in the absence of KC1, with 4 mM MgCl2).…”

Enzymic and chemical structure mapping of mouse 28S ribosomal RNA-contacts in 5.8S ribosomal RNA

“…nuclease digestions, and severe band compression in this region of the sequencing gels are all compatible with it. Moreover, arm IV is readily isolated from nuclease digests of 5.8S rRNA (Lightfoot, 1978; T. A. Walker, unpublished results). Thermodynamic data on the melting of the isolated arm IV from yeast have been gathered and are consistent with its proposed structure (Lightfoot, 1978).…”

Demonstration of the GC-rich common arm in yeast ribosomal 5.8S RNA via 500-MHz proton nuclear magnetic resonance and Overhauser enhancements

“…Although we are confident that we have identified most of the base pairs in the GC-rich arm of yeast 5.8S RNA, an unequivocal assignment of an extended base-pair sequence in an RNA of molecular weight >50000 is not possible without additional experiments. Future experiments on 5.8S rRNAs might be directed at comparing different biological species with (slightly) different primary nucleotide sequences and/or isolation of enzyme-cleaved fragments (Lightfoot, 1978) as aids in identifying this and other base-paired helical segments of the molecule. A kinetic and quantitative characterization of the fusion process between Sendai virus and phospholipid vesicles is presented.…”