TRANSCRIPTION : TRANSCRIPTION By
Brindavan college, Bangalore
www.Pridhvee.page.tl AGENDA : AGENDA Central Dogma
Processing of RNA
Measuring and detecting transcription
References CENTRAL DOGMA- Francis Crick (1958) : CENTRAL DOGMA- Francis Crick (1958) PROTEIN RNA DNA TRANSCRIPTION-PRE-INITIATION : TRANSCRIPTION-PRE-INITIATION In 2007 Roger D. Kornberg proposed the model of the PIC at promoters with TATA boxes
Does not require primers for initiation
Most common core promoter – TATA box
TFIID binds to the TATA box via TBP
five more transcription factors and RNA polymerase combine around the TATA box in a series of stages to form what is known as the preinitiation complex.
TFIIA, TFIIB, TFIIF, Pol-II TRANSCRIPTION-INITIATION : TRANSCRIPTION-INITIATION ATP dependent process
Transcription initiation is far more complex in eukaryotes, the main difference being that eukaryotic polymerases do not directly recognize their core promoter sequences.
The completed assembly of transcription factors and RNA polymerase bind to the promoter, called Transcription initiation complex.
Transcription in archaea is similar to transcription in eukaryotes Slide 6: After the first bond is synthesized the RNA polymerase must clear the promoter
During this time there is a tendency to release the RNA transcript and produce truncated transcripts. This is called abortive initiation and is common for both eukaryotes and prokaryotes.
Once the transcript reaches approximately 23 nucleotides it no longer slips and elongation can occur
TFIIE & TFIIH helps in the clearance of the Promoter TRANSCRIPTION-INITIATION TRANSCRIPTION- ELONGATION : TRANSCRIPTION- ELONGATION RNA polymerase directs the sequential binding of ribonucleotides to the growing RNA chain in the 5’ - 3’ direction.
Each ribonucleotide is inserted into the growing RNA strand following the rules of base pairing. This process is repeated till the desired RNA length is Synthesized. TRANSCRIPTION- ELONGATION : TRANSCRIPTION- ELONGATION ds DNA mRNA RNA Polymerase TRANSCRIPTION- TERMINATION : TRANSCRIPTION- TERMINATION Other regions at the end of the gene; called Terminators, signal termination. These work in conjugation with RNA polymerase to loosen the association between RNA product and DNA template. The result is that the RNA dissociate from RNA polymerase and DNA and so stop transcription.
The product is immature RNA or Pre mRNA (primary transcript). PROCESSING OF mRNA : PROCESSING OF mRNA Pre mRNA ? mRNA
Capping: synthesis of the Cap. The 5’cap is a 7-methylguanosine(m7G). The cap protects the mRNA from being degraded by enzymes; enhancement of mRNA translatability.
Splicing: Step-by-step removal of introns present in the pre-mRNA and joining of the remaining exons. The removal of introns and joining of exons takes place on a special structures called spliceosomes.
Polyadenylation: synthesis of the poly (A) tail involving cleavage of its 3’ end and then the addition of about 200 adenine residues to form a poly (A) tail; This completes the mRNA molecule (mature mRNA), which is now ready for export to the cytosol for protein synthesis. Slide 12: 5'CAPPING Polyadenylation : Polyadenylation MEASURING & DETECTING : MEASURING & DETECTING Nuclear Run-on assay
RNase protection assay and Chip-Chip of RNAP, detect active transcription sites
In situ hybridization
RNA-Sequencing REVERSE TRANSCRIPTION : REVERSE TRANSCRIPTION Transcription – www.dnai.org : Transcription – www.dnai.org REFERENCES : REFERENCES Books
Watson, Baker, Bell, Gann, Levine and Losick. Molecular biology of the gene, 5th edition, Pearson Education, Benjamin Cummings Publishing Inc, 2004. Cold spring harbor laboratory press, San-Francisco, U.S.A.,
Lodish, H. et al Molecular Cell Biology, 4th edition, Scientific American Books, 1999. Chapter 10
Alberts, B. et al. Molecular Biology of the Cell, 3rd edition, Garland Publishing Inc. 1994. Chapter 9 pp 401-453
Biotechnology by B.D.Singh
Journal on Transcription in Eukaryotes by Dr. Sheila Graham (2004) Slide 18: THANK YOU