REVIEW | doi:10.20944/preprints202002.0458.v1
Subject: Life Sciences, Biophysics Keywords: LacI; Lac repressor; TetR; TAL-Effector; transcription activator-like effector; Cas9d; dissociation rate constant
Online: 29 February 2020 (09:13:12 CET)
The recent developments in the delivery and design of transcription factors put their therapeutic applications within reach, exemplified by cell replacement, cancer differentiation and T-cell based cancer therapies. The success of such applications depends on the efficacy and precision in the action of transcription factors. The biophysical and genetic characterization of the paradigmatic prokaryotic repressors, LacI and TetR and the designer transcription factors, TALE and CRISPR-Cas9 revealed common rules, which can help the optimization of activators and repressors. Further studies will be required to analyze the linkage between dissociation constants and enzymatic activity, the role of phase separation and squelching in activation and repression, and the long-range interaction of transcription factors with epigenetic regulators in the context of the chromosomes. Understanding these mechanisms will help to tailor systematically optimized designer transcription factors to the needs of specific applications.
REVIEW | doi:10.20944/preprints201711.0057.v1
Subject: Life Sciences, Molecular Biology Keywords: posttranscriptional regulation; Saccharomyces cerevisiae; nonsense mediated decay; NMD; splicing; 4-thiouracil; 4sU; rpb1-1; exponential decay
Online: 9 November 2017 (03:24:17 CET)
The turnover of the RNA molecules is determined by the rates of transcription and RNA degradation. Several methods have been developed to study mRNA turnover since the beginnings of molecular biology. Here we summarize the main methods to measure RNA half-life: transcription inhibition, gene control and metabolic labelling. These methods were used to detect the cellular activity of the mRNAs degradation machinery, including the exo-ribonuclease Xrn1 and the exosome. Less progress has been made in the study of the differential stability of mature RNAs because the different methods have often yielded inconsistent results so that an mRNA considered to be stable can be classified as unstable by another method. Recent advances in the systematic comparison of different method variants in yeast have permitted the identification of the least invasive methodologies that reflect half-lives the most faithfully, which is expected to open the way for a consistent quantitative analysis of the determinants of mRNA stability.