MOLBIOL 3B03 Chapter 4, 6, 7 : Compilation of Transcription 1 and 2 Notes from Textbook and Lecture

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TRANSCRIPTION I
Protein production depends:
1. mRNA stability
2. Protein stability
3. Transcription rate
Transcription requires: DNA template + DNAP/RNAP polymerase + promoters + transcription factors
Cis elements- non-coding DNA that affect s the transcription of genes on the same DNA molecule
Ex. promoter region, enhancer region
Trans elements- non-coding DNA that affects the transcription of genes on another DNA molecule
Ex. polymerase, transcription factors
PROKARYOTE vs. EUKARYOTE
- Control of transcription initiation is the most impt step
- P: couple transcription and translation can occur; cannot occur in
E
- P: transcript is polycistronic
E is monocistronic
- P: transcription occurs in cytoplasm
E: transcription occur sin nucleus
PROKARYOTIC Gene Control
Promoter region contains
1. -35 region = TTGACA
a. Secures RNAP to promoter
2. Pribnow Box (-10 region) = TATAAT
a. Where transcription begins and initiates DNA melting
b. Some variation in sequence => broad specificity
RNA Polymerase
- : two subunits; interact with regulatory sequences to control initiation frequency of promoter
- +  : polymerize Ribonucleotides (catalytic)
- 70 : initiation factor; recognizes promoter region, increases RNAP affinity to promoter and decreases non-specific
binding
o Released after polymerization ~10 rNTPs
Lac Operon
REPLICATION
TRANSCRIPTION
DNA template DNA polymer
DNA template RNA polymer
Deoxyribose + Deoxyribonucleotides
(thymine)
Ribose + Ribonucleotides (uracil)
Both strands, entirely
Non-coding strand, partially
Synthesis is discontinuous; ligated
later
Synthesis is discontinuous; no ligated
Product not modified (ligated and
methylated)
Product modified: spliced, capped,
polyadenylated
Stays in nucleus
Nucleus cytoplasm
DNAP needs RNA primer
Without a primer
Template copied in all cells
Transcription depends on cell, cell
types, cell stage and genes
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Activated: presence of lactose binds to repressor = inactive, so it cannot bind to operator. RNAP can then transcribe lacI,
lacZ, lacY and lacA, LacI forms repressor that gets bound to lactose and lacZYA forming a single transcript that is later
cleaved.
- In presence of cAMP, it will bind to CAP CAP-cAMP complex. This binds to CAP site found upstream lacZYA
promoter and allows for RNAP to bind readily and allows transcription
Inactive: RNAP transcribes lacI repressor that can bind to operator of lac operon. In the absence of lactose, the
repressor remains bound to operon, preventing transcription of lacZYA
- Absence of cAMP, the CAP is inactive and cannot bind to CAP site
EUKARYOTIC Gene Control
- Three RNAP
Experiment: Column chromatography- using sea urchins/frog eggs, they
prepared a nuclear extract and eluted it at increasing [NaCl] concentrations.
Fractions of the elute were assayed for RNAP activity.
- Pink curve: RNA transcription
o Three points = 3 RNAP
- Blue curve: RNAP activity in presence of -amanitin (transcription
inhibitor: poisonous octapeptide)
o RNAPI insensitive; RNAPII highly sensitive; RNAPIII
moderately sensitive
RNAPI
- Transcribes: repetitious DNA Pre-rRNA
o DNA coding for rRNA (18S, 5.8S and 28S) are separated by
DNA coding for spacer RNA and non-transcribed spacer RNA
- Operates in the nucleolus
- No ATP hydrolysis needed
- Promoter lacks TATA box; found upstream gene
RNAPIII
- Transcribes: client genes = tRNA, 5S rRNA (<200bp), snRNA
- Operates in nucleoplasm
- No ATP hydrolysis needed
- Promoter elements: A and B Box, downstream gene
Ie. in the transcription unit
Transcription:
tRNA Initiation
1) TFIIIC binds to A and B box
2) Binding recruits TFIIIB to bind ~30bp upstream transcription start site
3) Binding recruits Pol III to bind and initiate transcription
RNAPII mRNA
- Operates in the nucleoplasm
- ATP hydrolysis required
- It’s L’ differs from L’ of RNAPI and RNAPIII
- Essential C-terminal domain: YSPTSPS hepta-peptide repeat
o Involved in 5’ capping and splicing
o Unphosphorylated = no transcription; S phosphorylated =
active transcription
Tagged P-CTD with Red and non-P-CTD with
green, and Red formed puffed regions (bc DNA
unwound) = where transcription occurs
Initiation
1) TBP binds TATA region and makes a bend
2) TFIIA binds to TBP
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