TF & TC

TF & TC

(RNAPII Transcription Cycle)

​MMBT 1153

​RNA Pol II transcription cycle

• Divided into 4 steps as seen in the figure on the right​

• 1st step - ​the assembly of the pre-initiation complex (PIC) on the promoter

• The PIC is composed of many factors, including, but not restricted to, the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNA Pol II, and Mediator

  The PIC then opens the DNA double strand allowing transcription initiation

• RNA Pol II starts transcribing, producing short nascent RNA

​Promoter-proximal pausing (RNA Pol II paused state)

• Conformational change in the RNA:DNA hybrid within the polymerase active site leads to the absence of a free DNA template base in the active site

• As a result, the progression of RNA Pol II is prevented

• The negative elongation factors NELF (Negative elongation factor) and DSIF (DRB-sensitivity inducing factor) stabilize and extend the lifetime of the paused complex​

• RNA Pol II is released from this pausing and allowed to resume transcription by the Cdk9 kinase component of the positive transcription elongation factor b (P-TEFb) complex, which phosphorylates the Spt5 subunit of DSIF, NELF, and serines 2, 5, and 7 (Ser2, Ser5, and Ser7) of the RNA Pol II carboxy terminal domain (CTD)

• ​P-TEFb is recruited to promoters through interactions with transcription factors, Mediator, and coactivators

• The P-TEFb-mediated phosphorylation events lead to both the dissociation of NELF from the RNA Pol II and the switch of Spt5 from a repressor to an activator of transcription

• Spt5 remains associated with RNA Pol II and serves as a recruitment platform for additional factors involved in RNA Pol II elongation and RNA processing

​Productive transcription elongation

• ​Transcript elongation process regulated at; (1) RNA Pol II processivity (ability of RNA Pol II to travel the entire length of the gene and thus controls the production of full-length transcripts) and (2) speed (elongation rate or the number of nucleotides synthesized per unit of time)

• ​Premature transcription termination reduces RNA Pol II processivity and can lead to the production of transcripts that depending on the context are either rapidly degraded or stabilized

• Stabilized prematurely terminated transcripts can give rise to non-coding RNAs or to proteins with different properties compared to those synthesized from full-length transcripts

Transcription termination​

• ​The termination process involves poly(A) site recognition

• The poly(A) site is recognized by the cleavage and polyadenylation complex composed of multi-subunit factors including the cleavage and polyadenylation specificity factor (CPSF), the cleavage stimulatory factor (CstF), and the cleavage factors (CF) I and II​

• Upon recognition of the poly(A) site by the CPSF30 and WDR33 components of CPSF, the nascent pre-mRNA is cleaved by the CPSF73 endonuclease

• The cleaved 3′ end of the pre-mRNA is then polyadenylated, while the unprotected 5′ end of the nascent RNA provides an entry site for the Xrn2 5′–3′ exonuclease, which co-transcriptionally degrades the RNA synthesized beyond the poly(A) site

• In addition, transcription of the poly(A) site induces allosteric changes in the elongation complex mediated by dephosphorylation of Spt5 by the PP1 phosphatase, which slow down elongation by at least two- to threefold

• This reduction of elongation speed facilitates the catch-up of RNA Pol II by the Xrn2 exonuclease, which dislodges RNA Pol II from the DNA template leading to transcription termination