1. Transcription
Lecture 11

2. Remember the Central Dogma
A copy of the DNA
DNA
Information in sequence of bases
RNA
Intermediate ‘messenger’ - mRNA
Protein
Sequence of amino acids
The things that do the work in a cell.
Only certain bits of the DNA copied
Multiple copies made
Allows temporal and physical separation between DNA and protein manufacture
Why don’t we just make the proteins directly from the DNA?

3. Procaryotic vs Eucaryotic
We’ll start with Procaryotic
Simpler, no nucleus
Also control much simpler
Control is the most important and exciting thing
Each of the cells in your body contains all the information to make you
But only certain genes are transcribed and translated in specific cells – We call this EXPRESSION.
Muscle cells make the proteins that make them muscle cells (eg, the contractile filaments)
Kidney cells make the proteins that make them kidney cells (their shape and function is completely different)
Yet every cell has the DNA (the ‘genes’) required for these cells
The cells will also express many genes in common
Metabolic pathways, cell receptors, membrane assembly, etc

4. Genotype and Phenotype
Your DNA determines your GENOTYPE
What’s expressed determines your PHENOTYPE
The proteins that are made
GENOME vs PROTEOME
The time-course of expression is also important
During development and differentiation of cells
eg, Embryonic development
Genes must be switched on and off in the correct order
The problem for Jurassic Park was not getting the dinosaur sequence but expressing the genes in the right order!

5. What is mRNA? A polynucleotide
Like DNA
but using ribonucleotides instead of deoxyribonucleotides
Linked by phosphoester bonds between the 5’ residue of one nucleotide and the 3’ residue of another
Using uracil in place of thymine
Made using instruction from DNA template
Using RNA Polymerase

6. Drawings 5’
PPP OH 3’ OH 1’ N 5’ 3’
PPP P P P P P P P OH

7. Basic Reaction Then new triphosphonucleotide comes in
Phosphoester bonds formed
Release of pyrophosphate
Spontaneous hydrolysis of PP pulls reaction to completion
Note the directionality
5’ to 3’
And the lack of primer
But does need the DNA template
New chain is anti-parallel to the DNA
So the DNA template is read 3’ to 5’ 5’ 3’
PPP OH 5’ 5’ 3’ 3’
PPP
PPP P OH OH + PP

8. Starting Transcription
How does RNA pol know where to start?
DNA is long!
Specific sequences that signify the beginning of a gene
The PROMOTER region
Promoter region
The gene – the bit that will be copied into mRNA
UPSTREAM -1 +1
DOWNSTREAM

9. The Promoter Contains a consensus sequence
Not all promoters have this exact sequence
But the nearer they are to it, the more strongly the gene is initiated
It can be on either DNA strand
Whichever one it is on, the opposite one that is transcribed
The TEMPLATE strand
Both strands of DNA can act as the template in different sections
Both strands contain genes
Promoter region specifies the site and direction of mRNA synthesis
------TTGACA TATAAT |------
-35
-10
UPSTREAM -1 +1
DOWNSTREAM

10. RNA Polymerase Four main subunits in the CORE enzyme
α2β’β
Beta catalyses the polymerisation
Beta prime keeps the enzyme on track
Alphas can associate with other proteins
To initiate RNA pol has to have a partner
σ, the sigma subunit
Can find the promoter sequence, even though the DNA is double stranded
With sigma, RNA pol is called the HOLOENZYME
Sigma binds to promoter regions with 10 million times the affinity than random DNA
Specific for ds DNA (whereas core likes ss DNA better!)

11. Transcription Bubble Sigma not only finds the right spot
Also helps ‘melt’ open the DNA around the promoter
Open area initially about 80 bases
Between -55 and +20
Assisted by high A=T content in this region
Negative super-coiling also helps unwind the DNA
Beta subunit catalyses the first nucleotide entry
Usually a purine (G or A)
A triphosphonucleotide
RNA pol σ
-55
+20

12. Elongation As each nucleotide comes in
The bubble opens ahead (four nucleotides)
And closes behind (ten nucleotides open)
Once >6 nucleotides have been laid down
Sigma falls off
Remember it likes ds DNA, not ss DNA or DNA/RNA hybrid
Sigma now free to do more initiating
RNA starts to peel off template strand
NusA binds to RNA pol
Helps keep it on track
It doesn’t take much to put RNA pol off track!!

13. Elongation Elongation rate about 40 nucleotides/sec NO PROOF READING
Average gene takes about 20 seconds to transcribe
NO PROOF READING
Unlike DNA pol, RNA pol has no 3’ to 5’ exonuclease activity
1 mistake in 10,000 nucleotides

14. Termination We only want a small portion of DNA copied!
Two ways of stopping
Factor independent
Depends on the shape of the mRNA that’s formed
RNA pol pauses when these structures formed
Remember how easy RNA pol is to knock off its tracks
Factor dependent
Requires a protein factor that chases RNA pol
Rho, ρ
A circular hexamer of six identical subunits that encloses the single stranded RNA and hydrolyses ATP as it zooms in towards the transcription bubble
Unwinds the DNA-RNA hybrid and kicks off RNA pol
Especially when the latter has paused

15. Factor Independent Termination
mRNA can form intra-molecular base-pairs
GGGGGGGGG-----CCCCCCCC UUUUUUUUU-3’OH G C
UUUUUUUUU-3’OH
As the hairpin loop forms, the mRNA is pulled off the DNA
This rather weak tail helps!

16. Extras… and Cistrons
If the transcription bubble gets out of the way quickly, re-initiation occurs rapidly
PROMOTER CLEARANCE
Thousands of copies can be made after initiation
So the odd mistake doesn’t matter
RNA is VERY labile
Cistrons
A stretch of mRNA that contains structural information
Often bacterial messages are POLYCISTRONIC
Each mRNA contains multiple stop/start sites for multiple genes
3’ and 5’ untranslated regions (UTRs)
Translation does not start or finish right at the ends of the mRNA
Contain information relevant to gene stability, etc

17. Textbook Refs Chapter 8 Clarke Chapter 6 All the introductory blurb
All the sections on the Enzymatic Synthesis of RNA (p )
Transcription signals on p150
But NOT the intimate structure of the sigma subunit
Although figure 8-4 is beautiful
The figure showing different promoter regions obviously doesn’t need to be memorised
All of p151
Except the details of nucelotide binding sites in RNA pol
All of p152 and 153
Although the diagram on p153 makes things look complicated
p154
But not the stuff on the classes of RNA
Clarke Chapter 6
Everything from p133 to p140

18. Advanced Only DNA footprinting
To study the interaction between protein and DNA
Specifically the sequences on the DNA that bind to the protein
Find out which sequences are protected from digestion
Original reference
DNAase footprinting: Galas & Schmitz (1978) Nucleic Acids Research 5 (9) 3157

19. In the original paper, done with lac repressor
Fragment of double stranded DNA
Labeled with radioactivity at one end
Run the fragments on a gel
Reveal the radioactively labeled strands using x-ray film
Ladder produced. Smaller fragments run faster
Thousands of copies of the fragment
Denature the double strands
Incubate with DNAaseI
Now do the same with the digestion step in the presence of the DNA binding protein.
Each copy cut in a different place
Every possible length represented