Thermodynamic measurements indicate that native proteins are only marginally stable under physiological conditions. The free energy required to denature them is ∼0.4 kJ ∙ mol−1 per amino acid residue.

TLS Online TPP Program

#Id: 6501

#Unit 3. Fundamental Processes

The phosphorylation pattern on the CTD is dynamic during the elongation process, catalyzed and controlled by multiple protein kinases, including P-TEFb, and phosphatases. Most oft the basal transcription factors are released from the promoter at this stage.

TLS Online TPP Program

#Id: 6502

#Unit 3. Fundamental Processes

Sites of phosphorylation on the CTD serve as a recognition or anchor point for other proteins to dock with the polymerase. The capping enzyme (guanylyl transferase), which adds the G residue to the 5' end of newly synthesized mRNA, binds to CTD phosphorylated at serine 5, the first phosphorylation event catalyzed by TFIIH. 

P-TEFb leads to recruitment of a set of proteins called SCAFs to the CTD, and they in turn bind to splicing factors. This may be a means of coordinating transcription and splicing.

TLS Online TPP Program

#Id: 6503

#Unit 3. Fundamental Processes

These two mechanisms, (1) binding of the capping enzyme to RNA polymerase II specifically through its unique CTD phosphorylated on serine 5 of the heptapeptide repeat during transcription initiation by TFIIH, and (2) activation of the capping enzyme by the serine 5–phosphorylated CTD, result in specific capping of RNAs transcribed by RNA polymerase II.

TLS Online TPP Program

#Id: 6504

#Unit 3. Fundamental Processes

Once bound to Pol II, Ptefb phosphorylates the serine residue at position 2 of the CTD repeats. That phosphorylation event correlates with elongation. In addition, P-TEFb phosphorylates and thereby activates another protein, called SPT5, itself an elongation factor. Finally, TAT-SF1, yet another elongation factor, is recruited by P-TEFb. Thus, P-TEFb stimulates elongation in three separate ways.

TLS Online TPP Program

#Id: 6755

#Unit 3. Fundamental Processes

Most mRNAs decay stochastically (like the decay of radioactive isotopes), and as a result mRNA stability is usually expressed as a half- life (t½).

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.