Determination of intracellular RNA folding rates using self-cleaving RNAs.
We have developed a system that relies on RNA self-cleavage to report quantitatively on assembly of RNA structures in vivo. Self-cleaving RNA sequences are inserted into mRNAs or snoRNAs and expressed in yeast under the control of a regulated promoter. Chimeric RNAs that contain self-cleaving ribozymes turn over faster than chimeric RNAs that contain a mutationally inactivated ribozyme by an amount that reflects the rate at which the ribozyme folds and self-cleaves. A key feature of this system is the choice of assay conditions that selectively monitor intracellular assembly and self-cleavage by suppressing further ribozyme activity during the analysis.
Watson
Peter Y
PY
Department of Chemical Physiology, The Skaggs Institute for Chemical Biology,The Scripps Research Institute, La Jolla, California, USA.
Fedor
Martha J
MJ
D007700:Kinetics; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry*; D014158:Transcription, Genetic / Q000235:genetics
D016337:RNA, Catalytic; D012313:RNA