Codon-stop | EPFL Graph Search

220px|vignette|Les trois codons qui arrêtent la traduction d'une séquence codant une protéine|alt= Les trois codons qui arrêtent la traduction de l'ARN messager Un codon-stop, codon de terminaison ou encore codon non-sens est l'un des trois codons (parmi les 64 codons du code génétique) qui marquent la fin de la traduction d'un ARNm en protéine. Il n'est en général jamais traduit car il n'existe pas d'ARN de transfert correspondant. Le codon UGA peut cependant, dans les cas exceptionnels des sélénoprotéines, coder une sélénocystéine ; cet acide aminé rare est alors incorporé à l'aide de signaux supplémentaires présents sur l'ARN messager et d'un ARN de transfert particulier. De la même manière, il a été montré que chez certaines archées méthanogènes, le codon UAG peut coder un acide aminé, la pyrrolysine. Une mutation ponctuelle qui donne naissance à un codon d'arrêt est appelée une mutation non-sens. Les trois codons-stop sont :

UAA (ocre)
UAG (ambre)
UGA (opale)

Lorsque le r

Isolation and Analysis of Regulators Coordinating the Nuclear Cycle with Cytokinesis in Schizosaccharomyces Pombe

Evelyn Lattmann

During the division of a mother cell into two daughter cells the genetic material of the mother cell is replicated and segregated to the daughter cells before the cytoplasm divides into two portions in a process called cytokinesis. In order to maintain the genetic stability of the daughter cells it is crucial that the bisection of the cells only occurs once the genetic material has been faithfully partitioned. In the model organism S. pombe the timely coordination of cytokinesis is regulated by a cellular signaling cascade called the Septation Initiation Network (SIN). The SIN is required for the formation and contraction of the contractile actomyosin ring (CAR) that assembles in the cell centre and guides the centripetal deposition of septum material during its contraction. Most of the regulators of the SIN are located to the SPB at some stage of the cell cycle. Interestingly, in anaphase B, when the SIN is supposed to be fully active, positive and negative regulators of the SIN localize to opposite SPBs. This asymmetric configuration of the SIN is lost at the end of cytokinesis, when the SIN is turned off; the positive regulators dissociate from the new SPB and the negative regulators assemble on this SPB. In order to understand this process in more detail we analyzed the localization of Cdc7p, a positive regulator of the SIN. We found that the disappearance of Cdc7p from the new SPB at the end of cytokinesis correlated with the separation of the two halves of the cytoplasm. Moreover, our data suggested that the separation of the two SPBs resulting from the completion of septum formation is important for the resetting of the SIN. Interestingly, we observed that Sid2p, the downstream effector of the SIN, was important for the maintenance of asymmetry in anaphase B and for the timing of the resetting of the SIN by acting in a negative feedback. Taken together, we suggested a model where the downstream effector of the SIN may promote the loading of the negative regulators to the SPBs; in anaphase to the old SPB (due to higher affinity of the negative regulators for this SPB) and upon cell separation to both SPBs, because the old SPB cannot buffer the negative feedback. In a separate study we designed a screen to isolate new regulators of the SIN. Mutagenesis of a strain that over-expressed spg1, the core activator of the SIN, led to the isolation of 28 mutants that are dependent on high levels of Spg1p for viability. The characterization of the nine heat-sensitive mutants among them revealed that they are all new alleles of known SIN genes. Interestingly, two mutants that mapped to the SIN effector sid2, lysed at the time of cytokinesis, when shifted to the restrictive temperature. This observation points to a possible role of the SIN in regulating cell separation. Surprisingly, one of the mutants that mapped to the essential positive SIN regulator sid1 contained a premature STOP codon after 243 nucleotides. The analysis of this mutant revealed that translational read-through level of the STOP codon is sufficient for successful cytokinesis. Similarly, high levels of sid1 expression did not interfere with cytokinesis. This indicated that the regulation of the SIN by Sid1p does not depend on its levels but on a precise control of its action. Since over expression of sid1 led to the accumulation of the protein in the nucleus this may hint that Sid1p recruitment to the SPB is regulated by nuclear proteins.

EPFL2012

Monitoring mis-acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery

Erwin Ilegems, Horst Pick, Horst Vogel

A reporter assay was developed to detect and quantify nonsense codon suppression by chem. aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site-specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run-off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amts. of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately det. suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser-scanning confocal microscopy. Control expts. showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl-tRNA synthetases. This reporter assay opens the way for the optimization of essential exptl. parameters for expanding the scope of the suppressor tRNA technol. to different cell types. [on SciFinder (R)]

Oxford University Press2002

Isolation and Analysis of Regulators Coordinating the Nuclear Cycle with Cytokinesis in Schizosaccharomyces Pombe

Evelyn Lattmann

EPFL2012