DNA replication and transcription similarities
- Uses DNA as template
- initiation, termination, elongation steps
- 5’ to 3’
DNA replication and transcription differences
- rNTPs used (or just NTPs)
- selective (environmentally responsive)
- No primers
- Only one strand used as template
- 5’ to 3’ coding stand (nontemplate)
- 3’ to 5’ template strand
Transcriptional and translational start sites
- space between the start sites for transcription and translation (AUG) called the 5’ UTR
- Prokaryotes: 10-30 nucleotides
- Eukaryotes: >100 nucleotides
- UTR typically has regulatory purpose for initiation of transcription/translation
Bacterial RNA Pol.
- clamp structure wraps around DNA
- structure conserved for eukaryotic RNA Pol. I, II, III
- Holoenzyme= 5 subunit core enzyme plus sigma factor co-enzyme subunit
Mechanics of RNA synthesis
- three Asp (D) residues in RNA pol active site, capture and coordinate two Mg++ ions
- 1st Mg++ deprotonates 3’-OH
- one Mg++ interacts with phosphate groups of rNTP and the other Mg++ acts to bring the 3’OH of the last nucleotide close enough to incoming rNTP for a nucleophilic attack on alpha-phosphate
- this releases PPi (pyrophosphate), 2nd Mg++ facilitates departure
- assisted by H-bonding between incoming rNTP and template DNA, allowing precise alignment of active site
RNA Pol kinetic proofreading
-speed: 50-90 nucleotides/s
-Error rate: 1 in 10^4-10^5 (DNA pol ~10^6)
-error rate is acceptable b/c RNA is short lived
if incorrect nucleotide added:
-incorrect H-bonding, frays DNA-RNA duplex
-enzyme stalls
-pyrophosphate moves in, strips the incorrect NTP (called pyrophosphorolysis)
RNA Pol nucleolytic proofreading
- occurs if RNA Pol does not stall after mismatch
- RNA-DNA strand will begin to fray @ point of mismatch
- RNA Pol with freeze and backtrack
- peels fraying end into rNTP entry channel
- water hydrolyzes the phophodiester bond, which cleaves it
Steps of transcription
- binding of RNA pol. core to the DNA promoter
- Formation of transcription bubble
- Initiation
- Elongation (promoter clearance)
- termination + recycling
Sigma factors
- RNA Pol is generic enzyme
- sigma factors allow for selective, context-specific transcription
- ie. sigma-70 is most common in E.coli, activates ‘housekeeping’ genes, the consensus sequence allows for sequence ‘promiscuity’ (doesn’t have to be perfect)
Sigma factors: consensus sequences
- changing certain base pairs in the promoter to match the consensus sequence can increase transcription
- some differences from consensus may be better than consensus (more transcription) - called superconsensus
UP sequences, -10/-35 and spacer
Determines efficiency RNA Pol. binds to DNA:
- UP, -10, -35 sequences
- the spacing between -10/-35 and the UP element
- the distance of the UP element from the +1 site
- spacer between -10 and -35 is 17-20 nucleotides, depending on the factor (roughly 2 turns of helix)
- changes in spacer length with reduce transcription
Transcription Initiation Steps (4)
- primer independent
1. Start at closed complex - holoenzyme binds a certain area of DNA but DNA strands still together
- N-term of sigma-factor blocks the DNA entry channel
2. Open complex (ATP independent) - form 17 nucleotide bubble as DNA opens
- N-term of sigma-factor moves
- Pol clamps around DNA
3. must bind and hold two nucleotides in place for long enough to catalyze phosphodiester bond (unstable for first 8-10 bonds)
4. Promoter clearance - Pol clears promoter
- RNA is exiting out RNA exit channel
- sigma-factor falls off
- Pol. moves until termination
Abortive initiation
- within first 8-10 nucleotides, the RNA oligomer is highly unstable
- if the Pol releases transcript without extending it further than the <8-10 nucleotides, called abortive initiation
Different RNA pol. functions in eukaryotes and inhibitor
Pol I: produces rRNA
Pol II: produces mRNA, microRNA, noncodingRNA
Pol III: tRNA, some rRNA
-Pol II is inhibited by alpha-amanitin
Eukaryotic transcription
- more sophisticated genome, so transcriptional regulation is more sophisticated (than prokaryotes)
- TATA box used as a promoter for some, but not all promoters (all Pol. still require the TATA binding protein (TTB))
- Bacterial RNA Pol. and Euk RNA Pol. very similar at core
Typical eukaryotic promoter layout
- Binding sites can be found upstream, downstream, within genes (transcription factors=sigma factors in Euk)
- General transcription factors bind the core promoter region
- Transcription activators and co-activators bind regulatory sequences (more gene specific)
- core promoter includes TATA box 25% of the time (5’-TATAAA-3’)
TATA Binding protein
- Essential to transcription of ALL genes, including those lacking TATA box in core promoter
- binds minor groove by inserting 2 Phe residues between BP, bends DNA, opens up minor groove
- binds to A-T b/c easier to distort
- enables sequence-specific H-bonding
Naming transcription factors
- for general TF, it indicates the RNA Pol. that is involved
- ie. TFII indicates transcription factor for RNA Pol. II
- individual factors distinguished by letter afterwards
- ie. TFIIA
Typical layout for Eukaryotic RNA Pol. II core promoter
- TATA box (-30) sometimes
- TFIIB recognition element (BRE) 5’ of TATA box
- Initiation sequence (INR) (+1)
- Downstream promoter element (+30)
- these all allow for recruitment of RNA Pol. II
Regulatory sequences in complex eukaryotes
- DNA looping allows contact between the core promoter and distant regulatory elements
- even if thousands of BP away, packaging could have right next to each other
Nuclear Reprogramming
-Pluripotent and embryonic stem cells can become any cell in body
-as cell differentiates, you get certain gene expression profile that designates it as specific cell (ie. heart, neuron)
Induced pluripotency: return differentiated cell to embryonic stem cell-like state
Direct reprogramming: Convert to another differentiated cell type by introducing different transcription factor (iPSC induced pluripotent stem cells)
EMSA technique
- fragments of DNA of known sequence are incubated with protein of interest, then analyzed on non-denaturing (protein not denatured) polyacrylamide gel
- DNA used in experiment visualized by staining with dye or attaching radioactive phosphate group to end
- free DNA fragments migrate through gel more quickly than protein bound DNA
Ways to amplify only cDNA with PCR/qPCR
- at least one primer spans an exon-exon boundary
- OR the PCR product spans the boundary (if introns are too long to fully replicate during the PCR cycle
DNA Footprinting
-used to map the exact nucleotide bases in contact with the bound protein
