Translational control negative

Prokaryotes and eukaryotes differ in their usage of translational control mechanisms. In addition to variations in Shine-Dal-gamo sequences, prokaryotes also use negative translational... 

The major differences between prokaryotic and eukaryotic translation control mechanisms are related to the complexity of eukaryotic gene expression. Features that distinguish eukaryotic translation include mRNA export (spatial separation of transcription and translation), mRNA stability (the half-lives of mRNA can be modulated), negative translational control (the translation of certain mRNAs can be blocked by the binding of specific repressor proteins), initiation factor phosphorylation (mRNA translation rates are altered by certain circumstances when eIF-2 is phosphorylated), and translational frame-shifting (certain mRNAs can be frame-shifted so that a different polypeptide is synthesized). 

A general flowchart is presented in Fig. 13.5B that we followed for identifying and sorting inhibitors of translation. Shown below is an in vitro translation protocol tailored for ten 96-well assay plates (800 compounds), which can be scaled up or down as required. Negative and positive controls are present in wells A1 to D1 and El to HI, respectively. Compounds are added to wells A2 to Hll. Column 12 is left blank and could be used for additional controls, if desired. 

In contrast to Gram-negatives, many Gram-positive bacteria employ post-translationally modified peptides processed from larger precursors as QS signal molecules. In Staphylococcus aureus, for example, a family of peptide (7-9 amino acid residues) thiolactones which vary in the primary amino acid sequence but contain a conserved cysteine at position 5 control the expression of cell wall colonization factors and exotoxins [24-26]. 

Fig. 3. Detection of a synthesized protein by fluorescent labeling. Cell-free protein synthesis was carried out with or without the use of mRNA transcribed from a linearized expression done containing the p-gaiactosidase gene, and the synthesized protein was labeled by FluoroTect. The translational reaction mixtures were resolved by 12.5% SDS-PAGE. Detection of labeled protein was performed using a laser-based fluorescent scanner (FX pro, Bio-Rad, Hercules, CA). Lanes 1 and 2 represent negative control (absence of mRNA) and p-galactosidase, respectively.

Fig. 1.54 Principle of negative control of translation initiation by protein binding to mRNA. Proteins can negatively effect translation by binding to the sequences in the 5 non-translated region of their own or other mRNAs. The participating proteins are sequence-specific RNA binding proteins and recognize RNA sequences in hairpin structures or other secondary structures of RNA. The protein binding interferes with the scanning of ribosomes and prevents the translation of mRNA.

Translated into statistics, this implies that for safety pharmacology the risk of Type 2 errors (false negatives) should be decreased as much as possible, even if there is an increase in the risk of Type 1 errors (false positives). In other words, the statistical tests employed in safety pharmacology should err in the direction of oversensitivity rather than the reverse. A test substance found not to have significant safety risks based on preclinical studies, even after the use of oversensitive statistics, is more likely to be truly devoid of risk. As a consequence, the statistical analyses proposed for the CNS safety procedures described below (mainly two-by-two comparisons with control using Student s t tests) have been selected for maximal sensitivity to possible effects per dose at the acknowledged risk of making more Type 1 errors. 

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