Transcription

• Synthesis of an RNA molecule on the template of DNA(template or antisense strand)

• Segment of antisense DNA that takes part in transcription-transcription unit
• May consist of one or more cistrons
• Has a promoter region and terminator region
• Takes place in nucleus
Mode of transcription
• The enzyme taking part in transcription- Rna polymerase
• 3 distinct classes of RNA polymerases:
 RNA POL I-rRNA
 RNA POL II-mRNAs, some snRNAs
 RNA POL III-tRNAs, 5S rRNA, some snRNAs
• In eukaryotes separate protein factors take place in recognition and initiation
• Called Transcription factors
Mechanism of transcription consists of three major steps
initiation
elongation
termination

Initiation
• In eukaryotes, a collection of proteins called
transcription factors mediate the binding of
RNA polymerase and the initiation of
transcription
• Transcription initiation complex is opened
and the first phosphodiester bond is formed
• recognize the promoter and unwind the DNA
double helix
• The RNA pol II is associated with 6
transcription factors: TFIIA, TFIIB, TFIID,
TFIIE, TFIIF, TFIIH
• TFIID consists of TBP and TAFs.
SEQUENCE OF TRANSCRIPTION FACTORS
• TFIID- first attached to TATA sequence- strong bending of DNA helix
• TFIIA- stabilize the TFIID-DNA complex
• TFIIB- allows binding to the initiator complex of RNA pol II and TFIIF
• TFIIE- attach to facilitate TFIIH(catalyze DNA melting) joining
• The complex performs the function of helicase and causes phosphorylation of the RNA pol II with the participation of ATP.
• DNA sites recognized by transcription factors- enhancers and promoters
• Core promoters– TATA and CAAT sequences
• Enhancers- activated after binding of activator proteins to their specific binding sites
activate their target(promoters) over variable distances
as a result, there is a dynamic regulatory network formed by regulatory chromatin loops of different size and stability
TATA box
• RNA pol II promoter – “core promoter” plus B recognition element
• Core promoter - consists of consensus sequence, TATA box
• 30 bp upstream from the transcriptional start site, consensus sequence; 5’ – TATAAA – 3’
• TATA box is recognized by TBP
• B recognition element is recognized by TFIIB
• Role of TATA box
• Define the direction of transcription
• Indicate the DNA strand to be read
• Recruit an enzyme called RNA pol II
Elongation
• Catalyzed by the RNA polymerase core enzyme
• Takes place within transcription bubble
• Continuously unwinds the DNA double helix ahead of the polymerization site and rewinds the complementary DNA strands
behind as it moves along the double helix
• Length of a transcription bubble:17-18 bp
• Grows in 5’→ 3’ direction by the addition of nucleotides to 3’ end
Termination
• Occurs when RNA polymerase encounters a termination sequence
• 2 types: rho-dependent terminators, rho-independent terminators
• Rho- large hexameric protein, helicase function
- attached to RUT, moves along newly formed RNA molecule towards 3’ end, unwind it from DNA template
• Rho-independent terminators contain a palindrome, followed by 6-8 A-T bp with adenins present on template DNA
• RNA strand synthesized on the palindrome consists of two complementary arms that easily form a hairpin-like structure
which retards the movement of RNA polymerase along the DNA and cause dissociation
Modification of primary transcript
• The RNA transcript must undergo processing before it is a functional mRNA
• Occurs in the nucleus and involves 3 steps: 5’ capping, 3’ polyadenylation, exon splicing
Capping
• Addtion of 7-methylguanosine triphosphate attached to 5’ end of primary transcript
• Enzyme- guanyl transferase
• Produces 5’ to 5’ phosphodiester bond
• 7-mG cap protects the mRNA from degradation, and is also needed to initiate ribosomal binding during translation
 Polyadenylation

• Begins as the transcription of a gene terminates

• Most important components are polyadenylate polymerase (PAP) and
two protein factors CPSF and CstF
• The 3’ end of transcript is cleaved by the enzyme CPSF to free a 3’
hydroxyl group
• CPSF recognize the polyadenylation signal sequence 5’-AAUAAA-3’
and cleaves 10-30 nucleotides downstream from that sequence
• CstF binds to a GU- rich region further downstream of CPSF’s
polyadenylation site
• After cleaving, PAP builds the poly(A) tail by adding AMPs from
ATPs.
• Poly(A) tail protects the mRNA from degradation by nucleases, plays
an important role in initiation of translation