Introduction to Genetics
Winter semester 2014 / 2015
Seminar room 00.005 INF230
Thursdays 18:15 - 19:45
14 lectures
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Molecular biology of genes and genomes.
Key technologies: Next generation sequencing.
Bioinformatics analysis of sequencing data.
Background.
Molecular Biology of the Cell (Alberts, 5th Edition)
Genes X (Lewin)
Background reading (guided by lectures).
Talks (pdfs or powerpoints of talks posted on “Moodle” elearning platform after lectures).
Introduction to Genetics: timetable
2014
October 23
October 30
November 6
November 13
November 20
November 27
December 4
December 11
December 18
Thomas Dickmeis
Thomas Dickmeis
Clemens Grabher
Felix Loosli
Felix Loosli
Felix Loosli
Clemens Grabher
Harald Koenig
David Ibberson
Introduction / Basic transcription mechanisms I(Lecture 1)
Basic transcription mechanisms II (Lecture 2)
Control of transcription in eukaryotes (Lecture 3)
DNA replication, recombination and repair I (Lecture 4)
DNA replication, recombination and repair II (Lecture 5)
RNA machines and translation I (Lecture 6)
RNA machines and translation II (Lecture 7)
mRNA splicing and processing (Lecture 8)
Next generation sequencing (Lecture 9)
January 8
January 15
Juan Mateo
Clemens Grabher
January 22
January 29
February 5th
Rüdiger Rudolf
Rüdiger Rudolf
Jochen Wittbrodt
Data analysis of Next generation sequencing (Lecture 10)
Transposable elements, recombination, hypermutation: immune
system. (Lecture 11)
Epigenetic control of gene expression (Lecture 12)
Viruses (Lecture 13)
Genome structure, function, evolution (Lecture 14)
2015
Date to be announced. Nicholas S. Foulkes
Final Examination
Contacts:
Nick Foulkes: nicholas.foulkes@kit.edu
Jochen Wittbrodt: jochen.wittbrodt@cos.uni-heidelberg.de
Thomas Dickmeis: thomas.dickmeis@kit.edu
Clemens Grabher: clemens.grabher@kit.edu
Rüdiger Rudolf: ruediger.rudolf@kit.edu
Felix Loosli: felix.loosli@kit.edu
Harald Koenig: h.koenig@kit.edu
David Ibberson: david.ibberson@bioquant.uni-heidelberg.de
Juan Mateo: juan.mateo@cos.uni-heidelberg.de
Examination:
Mid-February
Short questions / answers
34 questions, 2.5 hours.
Resit, new set of questions (Andrea Wolk).
Announcement of date, place and time closer to the
date (January)
Questions based on information content of lectures
Course contact: Nicholas S. Foulkes
nicholas.foulkes@kit.edu
Basic transcription
mechanisms
Thomas Dickmeis
Institut für Toxikologie und Genetik,
KIT, Karlsruhe
thomas.dickmeis@kit.edu
5
Transcription:Definitions I
(DNA dependent) RNA polymerase
Transcription: 5′ to 3′ on a DNA template strand that is 3′ to 5′
non-template strand of the DNA = coding strand
6
Transcription:Definitions II
promoter – region of DNA where RNA polymerase binds to initiate transcription
transcription startpoint or start site (TSS) - position on DNA
corresponding to the first base incorporated into RNA
terminator – a sequence of DNA that causes RNA polymerase to terminate
transcription
7
Transcription:Definitions III
upstream – sequences in the opposite direction from transcription
downstream – sequences in the direction of transcription
8
Transcription: Definitions IV
transcription unit – the sequence between sites of initiation and termination by
RNA polymerase
primary transcript – the original unmodified RNA product corresponding to a
transcription unit
(a transcription unit may contain several genes, e.g. in bacteria)
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typical cartoon of a transcription unit
TSS
Stop
AUG
promoter
5‘ UTR
coding region
3‘ UTR
(UnTranslated Region)
10
The transcription „bubble“
11
Reaction catalyzed by RNA polymerase
RNA (n residues) + ribonucleotide triphosphate (NTP) ↔ RNA (n+1 residues) + PPi
PPi + H2O ↔ 2 Pi
12
Stryer 2002
Transcription bubble of bacterial RNA
polymerase
Bubble size: 12-14 bp
(DNA-RNA-hybrid within the bubble: 8-9 bp)
Speed: 40-50 nucleotides/second
(DNA replication: 800 bp/second)
13
The stages of transcription
14
Prokaryotic Transcription
15
Bacterial RNA polymerase consists of
multiple subunits
Core polymerase: α2ββ′
catalyzes transcription
Holoenzyme: α2ββ′ and σ (sigma factor)
core enzyme and σ factor together
competent for initiation
16
σ factor ensures promoter specific binding
and is required for initiation
cannot initiate
able to initiate
17
How does RNA polymerase find
promoter sequences?
too fast for simple diffusion
unspecific DNA binding, then „one-dimensional random walk“
proposed mechanisms:
intersegment
transfer
„hopping“
direct transfer
(wrong labels in the book)
18
What defines a promoter?
• cis-acting element:
recognized and specifically bound by proteins
• consensus sequence
Aligment of many
promoters reveals
stretches of conserved
sequences -> functionally
important
Sequence logo: illustrates conservation and frequency
of bases in each position
19
adapted from Stryer 2007
consensus sequence: the most conserved bases in each position
The bacterial promoter
consensus sequence
TTGACA
TATAAT
• main elements: -35 box and -10 box (or Pribnow box)
• additional elements (UP, Ext, Dis...) can affect promoter efficiency
• individual promoters usually differ from the consensus
also: not all elements have to be present: modularity
• distance between -35 and -10 boxes: 16-18 bp in 90% of promoters
-> Important! (Why?)
20
The bacterial promoter
consensus sequence
TTGACA
TATAAT
• several regions of s factor and the a subunit C-Terminal-Domains
bind at the consensus elements
• seen in crystal structure of the bacterial holoenzyme in bound
to promoter DNA
21
X ray crystallography in a nutshell
atomic model
ribbon diagram
electron density map
22
Stryer 2002
Crystal structure model of
holoenzyme-DNA complex
Sigma factor is
extended, with short
alpha-helical domains
connected by flexible
linkers
s
(Detailed view – but you cannot
always determine a crystal structure
each time you want to map proteinDNA interactions)
23
Illustration adapted from D. G. Vassylyev, et al., Nature 417 (2002): 712-719
footprinting
(How do you get labelling just at one end?)
24
Stryer 2007
Preparing a footprinting probe I
putative putative
binding binding
site 2
site 1
5‘
3‘
3‘
5‘
genomic DNA
PCR-amplification
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Preparing a footprinting probe II
„asymmetric“ digestion
(blunt)
putative putative
binding binding
site 2
site 1
(5‘ overhang)
Nature Protocols 3, 900 - 914 (2008)
(How must the nucleotide be labelled?)
(What if no suitable restriction sites in the sequence?)
Klenow enzyme can add radioactive
nucleotide (*) at this end
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(„Klenow fill-in reaction“)
Real world examples
footprint
experiment
sequencing
reaction
Nucl. Acids Res. (2000) 28 (18): 3551-3557.
Nature Protocols 3, 900 - 914 (2008)
Increasing protein concentration
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Footprinting achieves single nucleotide resolution!
(Sanger Sequencing – original method)
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(Sanger Sequencing – the standard today)
Now even faster methods with higher throughput become available
29 –
„next generation sequencing“ – see lecture by David Ibberson
(another new method to map protein binding
down to single bp resolution: ChIP-exo)
Rhee et al., Cell Volume 147, Issue 6, 9 December 2011, Pages 1408–1419
ChIP =
Chromatin
Immuno
Precipitation
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Footprinting reveals polymerase shape
changes during the stages of transcription
s factor
closed binary complex
core enzyme
Initiation
open complex
ternary complex
general elongation
complex
Elongation
core enzyme
s factor
Knippers 1997
31
Detection of unwinding
s factor
core enzyme
Initiation
Unwound bases
become accesible for
reagents that cannot
reach them within the
double helix
e.g. KMnO4
Elongation
core enzyme
s factor
Knippers 1997
32
Functional promoter analysis by mutation
• „down“ mutations – decrease in efficiency
• „up“ mutations – increase (e.g. mutation towards consensus)
• not all promoters match the consensus – the „perfect“ example above
doesn‘t exist in nature!
• „maximal“ activity not necessarily „optimal“ activity
• „down“ in -35:
closed complex formation rate ↓
open complex conversion ↔
• „down“ in -10:
either closed complex formation rate ↓
or open complex conversion ↓
or both
• AT-rich sequence around -10 helps melting – why?
33
Summary promoter
1.
2.
3.
4.
Modular
Consensus sequence
Most important: -35 and -10 box
Mutations may affect:
s factor and polymerase binding
DNA unwinding
34
The stages of initiation
s factor
closed binary complex
core enzyme
Initiation
open complex
ternary complex
general elongation
complex
Elongation
core enzyme
s factor
Knippers 1997
35
crystal structure models of initiation snapshots of a molecular machine
closed binary complex
most contacts on non-template strand
36
Nature Reviews Microbiology 6, 507-519 (July 2008)
crystal structure models of initiation snapshots of a molecular machine
closed binary complex
open complex
Conformational changes:
DNA bends
opens between -11 and +3
moves into the enzyme („jaws close“)
37
Nature Reviews Microbiology 6, 507-519 (July 2008)
crystal structure models of initiation snapshots of a molecular machine
closed binary complex
open complex
ternary complex
„ternary“ – RNA polymerase, DNA and first RNA nucleotides
abortive initiation:
Nature Reviews Microbiology 6, 507-519 (July 2008)
short RNAs formed and released
RNA polymerase stays on promoter
„DNA scrunching“
38
Transition to elongation – promoter escape
Two problems:
1) Initiation requires tight binding to specific sequences
Elongation requires binding to all sequences encountered
2) s occupies exit channel for the RNA:
s mediates specific binding and blocks RNA exit
→ get rid of it!
→ TEC = Transcription Elongation Complex
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Nature Reviews Microbiology 6, 507-519 (July 2008)
the sigma factor cycle
40
The elongation complex
s factor
closed binary complex
core enzyme
Initiation
open complex
ternary complex
general elongation
complex
Elongation
core enzyme
s factor
Knippers 1997
41
The catalytic mechanism
Groove lined with positively charged
amino acid residues, why?
42
The catalytic mechanism
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The catalytic mechanism
44
The catalytic mechanism
Mg2+
• facilitates attack of 3‘ OH
• stabilizes negative charges
of transition state
45
Nature Reviews Microbiology 6, 507-519 (July 2008) and Stryer 2007
The mechanism of elongation
RNA
Volume 19, Issue 6, December 2009
Pages 708-714
nucleotide
in catalytic site
non-template DNA
„trigger loop“
template DNA
„bridge helix“
46
Mg2+
Brownian ratchet model
47
Nat Struct Mol Biol. 2008 August ; 15(8): 777–779
Direct observation of base-pair stepping of
single RNA polymerase molecules
laser beam
bead
Nature 426:684–87 (2003)
RNA polymerase
„optical tweezers“:
small beads can be trapped in highly focused laser beams,
position of the beads can be monitored with high precision:
= ~ 1 bp
48
Nature. 2005 November 24; 438(7067): 460–465
Summary initiation and elongation
s factor
Closed binary complex:
promoter recognition
core enzyme
Open complex:
melting of DNA
„jaws close“
Initiation
Ternary complex:
RNAP, DNA, RNA
abortive transcription
Elongation
Knippers 1997
Elongation complex:
s factor off
core enzyme
catalysis: transition state
stabilized
s factor
elongation movement:
Brownian ratchet
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model
What happens if transcription is blocked?
• Transcription can be transiently blocked e.g. by hairpin
structures in the RNA or misincorporation of NTPs
(transitory)
Annu. Rev. Biochem. 2008. 77:149–76
• RNA polymerase can cleave the RNA to generate new 3´-OH end
(cleavage activity intrinsic to RNA Pol, stimulated by accessory factors)
50
Transcriptional termination
Often difficult to find the termination site:
1) in vivo, the primary transcript gets cleaved or partially degraded
2) in vitro, experimental conditions influence termination capacity
-> if both approaches find the same, probably the true site...
Two classes of terminators:
1) intrinsic terminators (no other
factors required)
2) rho (r) dependent terminators
Annu. Rev. Biochem. 2008. 77:149–76
51
Intrinsic termination
Why?
Interaction of hairpin with RNA Pol. or
forces created by its formation lead to
misalignment of 3‘ end of the mRNA
with the active centre -> destabilisation
52
Rho termination
rut = rho utilisation
recognition site and effect site of rho are different
pausing gives time for the other necessary events to occur
What else binds to the nascent mRNA?
53
Summary termination
1. transient pausing – backtracking, hairpins, misincorporation
2. RNA can be cleaved by polymerase to give new free 3‘ -OH
3. termination: intrinsic (e.g. hairpin) or extrinsic (rho factor)
54
Polycistronic transcripts
TSS
Stop
AUG
promoter
5‘ UTR
coding region
the general
cartoon
3‘ UTR
a polycistronic transcript
55
Transcription and translation
(in prokaryotes!)
56
The cycle of bacterial mRNA
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Transcription in prokaryotes vs. eukaryotes
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Stryer 2002
three important differences
• Chromatin is the template (bacteria: „naked“ DNA)
• Polymerase needs general transcription factors
(GTFs) for promoter binding and initiation
(bacteria: holoenzyme binds directly)
•
three polymerases (bacteria: one):
– RNA pol I: 18S/28S rRNA
– RNA pol II: mRNA, few small RNAs
– RNA pol III: tRNA, 5S rRNA, other small RNAs
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