Carboxyfluorescein succinimidyl ester (CFSE) is more correctly named carboxyfluorescein diacetate succinimidyl ester (CFDASE) very commonly used to detect state of cell 

TLS Online TPP Program

#Id: 6756

#Unit 3. Fundamental Processes

Method for determining mRNA half-lives.

RNA polymerase II transcription is shut down, either by a drug or a temperature shift in strains with a temperature sensitive mutation in a Pol II gene. The levels of specific mRNAs are determined by northern blot or RT-PCR at various times following shutdown.

TLS Online TPP Program

#Id: 6757

#Unit 3. Fundamental Processes

In E. coli the typical mRNA half-life is about 3 minutes, but half-lives of individual mRNAs may be as short as 20 seconds or as long as 90 minutes.  In budding yeast, mRNA half-lives range from 3 to 100 minutes, whereas in metazoans, half-lives range from minutes to hours, and in rare cases, even days.

TLS Online TPP Program

#Id: 6758

#Unit 3. Fundamental Processes

Degradation of bacterial mRNAs is initiated by removal of a pyrophosphate from the 5’ terminus. The monophosphorylated form stimulates the catalytic activity of an endonuclease RNase E

TLS Online TPP Program

#Id: 6759

#Unit 3. Fundamental Processes

PNPase a 3’ to 5’ exonucleases in E. coli, are unable to progress through double-stranded regions. Thus, the stem-loop structure at the 3’ end of many bacterial mRNAs protects the mRNA from direct 3’ attack.

TLS Online TPP Program

#Id: 6760

#Unit 3. Fundamental Processes

RNase E and PNPase, along with a helicase and another accessory enzyme, form a multiprotein complex called the degradosome.

TLS Online TPP Program

#Id: 6761

#Unit 3. Fundamental Processes

Cytoplasmic mRNAs are degraded by one of the three pathways. For most mRNAs, the deadenylation-dependent pathway is followed These dense regions of cytoplasm contain the decapping enzyme (DCP1/DCP2), activators of decapping (DHH, PAT1, LSM1-7), and the major 5′→3′ exoribonuclease XRN1, as well as densely associated mRNAs