Question

1. Thoroughly explain transcriptional initiation and elongation in eukaryotic cell

Answer 1

Initiation of Transcription:

Formation of pre-initiation complex (PIC) requires

· RNA Polymerase II (RNAPII),

· Transcription Factors (TFIID, TFIIA, TFIIB, TFIIF, TFIIH, TFIIE),

· TATA Box binding protein (TBP).

RNAPII is commonly used for the synthesis of mRNA from DNA, whereas RNAPI is used for rRNAs and RNAPIII is used for 5S rRNA and tRNAs.

Most of the eukaryotic promoters have a TATA box at -27 position. TBP, a subunit of TFIID, first binds to this TATA region of the promoter followed by recruitment of additional subunits to form TFIID, a 770 kDa multi-subunit complex.

Other TFs including TFIIB, IIA, IIF, IIE, and IIH are then recruited to the region along with RNAPII in a stepwise manner as shown in the picture. N-terminal domain of TFIIB (TFIIBN) interacts with RNAPII, while its C-terminal domain (TFIIBC) binds DNA. TFIIA also binds to DNA but not on the same site of TFIIBC. TFIIF then recruits RNAPII to the site followed by the recruitment of TFIIE and TFIIH forming the PIC.

Upon assembly of this PIC, TFIIH unwinds the DNA using its ATP-dependent helicase activity so that RNA synthesis can begin.

Elongation of Transcription:

Once the RNAPII initiates transcription, the PIC undergoes modifications including phosphorylation of C-terminal domain of RNAPII’s Rpb1 subunit. This phosphorylation releases some TFs (TFIID) and advance the transcription forward via elongation. Now, RNAPII complex with TFIIH and TFIIF can continue transcribing the DNA into RNA until termination in 5’-3’ direction. The RNA chain forms a transient DNA-RNA hybrid with template DNA (antisense strand) while synthesizing RNA.