Transcription espyr1 S9 Transcription

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LSM1401 Summary 9 © Lim Fang Jeng

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Transcription

 What is needed?

- Template (NO PRIMERS!!!!!!) - free NTP (NOT dNTPs!!!!!!) - Enzymes/Proteins

 Steps:

(1) Binding of RNA polymerase to the PROMOTER SITE (2) Initiation (Pribnow box)

(3) Elongation (4) Termination

Prokaryotes

 Two consensus sequences

- -35 region – TTGACA (σ subunit binds here) - -10 region – Pribnow box (TATA box)

o Ideal unwinding region due to weak hydrogen bonds between A=T - Act as a regulatory region of DNA

- mRNA has the similar code with the non-template/sense strand - Synthesize from 53 (read from 3 5 )

Chain Termination

- Termination sequence occurs when there is two inverted repeats in the template strand - A hairpin structure will form with the complimentary base pairing between the two inverted

sequences

- The structure will end in a series of U residues

- This enables the bond to be broken as A=U is weak, hence ending the sequence

σ subunit binds here

first transcriptional sequence

NOT the first translational sequence

non-template strand

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Regulation of Transcription in Prokaryotes

- Genes for enzymes for certain pathways are grouped together on the chromosome – operons - A regulatory sequence adjacent to such a unit determines whether it is transcribed ( operator ) - Regulatory Proteins work with operators to control transcription of the genes

- Example, lac operon and Catabolite Activator Protein (CAP) to regulate the transcription of lactose lac_Operon

 When lactose is absent, the protein which metabolises lactose will NOT form!

 The structural genes in lac operon are controlled by negative regulation

 lacI gene forms repressor protein – which is a tetramer

 In the presence of lactose, the inducer (lac) binds to the repressor and causes it not binding the DNA, hence, transcription can proceed

When CAP is taken into account…

 Glucose is easier to metabolise, so if glucose is present, protein which metabolize lactose will not produce

 In the presence of glucose, cAMP level is low.

 CAP-cAMP complex will NOT form, it will NOT bind to the DNA

 lac operon OFF

Dual control of the lac operon: ON only when lactose is present and glucose is absent

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Eukaryotes

3 RNA polymerases (I, II and III) are involved to transcribe rRNA, mRNA and tRNA gene

respectively.

 RNA Pol III transcribes a few other RNAs as well

 All 3 are big, multimeric proteins

 Interact with promoters via transcription factors (=σ-factors)

 Transcription factors recognize and initiate transcription at specific promoter sequences

 6 transcription factors

Regulation in Eukaryotes

 Chromatin limits access of regulatory proteins to promoters

 Factors must reorganize the chromatin

 Enhancers – upstream activation sequence

 DNA must form loop in order for the protein to bind to multiple DNA sequences

 Example: Activation of transcription via CREB(TF) and CBP (Enhancer) through DNA looping - Unphosphorylated CREB will not cause DNA looping

- NO transcription

 If CREB is phosphorylated, it will cause DNA looping and hence bind with CBP then Basal Complex, activating transcription

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Post-Transcriptional Modification of mRNA

 In prokaryotes, translation closely follows transcription, or even overlaps, so no modification required.

 In eukaryotes, the two processes are different o Transcription: Nucleus

o Translation: Cytoplasm

 Modification from transcripts to mature mRNA

(1) Capping of G residue and methylation at the 5 end ( protected ) a. Resistant to 5’-exonucleolytic degradation

(2) Adding of polyA tail

a. Protect mRNA from rapid degeneration b. Increasing mRNA lifespan

(3) Splicing of coding sequences

a. Permits single gene to encode different proteins

 There will be NO introns after modification

 Splicing only occurs in nucleus

 In higher eukaryotes: o 5 end – GU o 3 end – AG

 All introns have branch site 18 to 40 nucleotides from 3 - splice site

 branch site in eukaryotes is PyNPyPuAPy

 The splice sites and branch sites are recognized by small ribonucleoprotein particles (snRNPs) to facilitate removal of intron

NOTES:

 More introns, more exons

 Larger exons does not mean to have larger protein

Alternative Splicing

 Alternative splicing (post- transcriptional modification)

 Allow a single gene with multiple exons to code for different proteins

 Introns can act as a region for buffering mutation