Transcription
Lecture 11
Remember the Central Dogma
A copy of the DNA
DNA
RNA
Protein
Information in
sequence of
bases
Intermediate
‘messenger’ mRNA
Sequence of
amino acids
Only certain bits of the DNA copied
The things that do
the work in a cell.
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?
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
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!
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
Drawings
5’
PPP
OH
3’
OH
1’
N
5’
PPP
P
P
P
P
P
P
P
OH
3’
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’
5’
PPP
3’
OH
PPP
OH 3’
5’
PPP
P
OH 3’
+
PP
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
UPSTREAM
The gene – the bit that will be copied into mRNA
-1 +1
DOWNSTREAM
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
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!)
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
σ
RNA pol
-55
+20
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!!
Elongation
• Elongation rate about 40 nucleotides/sec
– 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
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
Factor Independent Termination
mRNA can form intra-molecular base-pairs
-----------GGGGGGGGG-----CCCCCCCC-------------UUUUUUUUU-3’OH
G
G
G
G
G
G
G
C
C
C
C
C
C
C
As the hairpin loop forms,
the mRNA is pulled off
the DNA
UUUUUUUUU-3’OH
This rather weak tail helps!
Extras… and Cistrons
• If the transcription bubble gets out of the way quickly, reinitiation 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
Textbook Refs
• Chapter 8
– All the introductory blurb
– All the sections on the Enzymatic Synthesis of RNA (p147-149)
– 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
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
Fragment of double stranded DNA
Labeled with radioactivity at one end
In the original paper, done with lac repressor
Run the fragments on a gel
Reveal the radioactively labeled
strands using x-ray film
Thousands of copies
of the fragment
Incubate with
DNAaseI
Each copy cut in a different place
Every possible length represented
Ladder produced. Smaller
fragments run faster
Denature the double strands
Now do the same with the
digestion step in the presence
of the DNA binding protein.