introduction to genetics winter semester 2014 / 2015 seminar room 00.005 inf230 thursdays 18:15 -...
TRANSCRIPT
![Page 1: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/1.jpg)
Introduction to Genetics
Winter semester 2014 / 2015
Seminar room 00.005 INF230Thursdays 18:15 - 19:45
![Page 2: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/2.jpg)
14 lectures
•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” e-learning platform after lectures).
![Page 3: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/3.jpg)
Introduction to Genetics: timetable2014
October 23 Thomas Dickmeis Introduction / Basic transcription mechanisms I(Lecture 1)October 30 Thomas Dickmeis Basic transcription mechanisms II (Lecture 2)November 6 Clemens Grabher Control of transcription in eukaryotes (Lecture 3) November 13 Felix Loosli DNA replication, recombination and repair I (Lecture 4) November 20 Felix Loosli DNA replication, recombination and repair II (Lecture 5) November 27 Felix Loosli RNA machines and translation I (Lecture 6) December 4 Clemens Grabher RNA machines and translation II (Lecture 7)December 11 Harald Koenig mRNA splicing and processing (Lecture 8)December 18 David Ibberson Next generation sequencing (Lecture 9) 2015
January 8 Juan Mateo Data analysis of Next generation sequencing (Lecture 10)January 15 Clemens Grabher Transposable elements, recombination, hypermutation: immune
system. (Lecture 11)January 22 Rüdiger Rudolf Epigenetic control of gene expression (Lecture 12)January 29 Rüdiger Rudolf Viruses (Lecture 13) February 5th Jochen Wittbrodt Genome structure, function, evolution (Lecture 14)
Date to be announced. Nicholas S. Foulkes Final Examination
Contacts:Nick Foulkes: [email protected] Wittbrodt: [email protected] Dickmeis: [email protected] Grabher: [email protected]üdiger Rudolf: [email protected] Loosli: [email protected] Koenig: [email protected] Ibberson: [email protected] Mateo: [email protected]
![Page 4: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/4.jpg)
Examination:
Mid-February
Short questions / answers34 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. [email protected]
![Page 5: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/5.jpg)
5
Basic transcription mechanisms
Thomas Dickmeis
Institut für Toxikologie und Genetik,
KIT, Karlsruhe
![Page 6: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/6.jpg)
6
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
![Page 7: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/7.jpg)
7
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
![Page 8: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/8.jpg)
8
Transcription:Definitions III
upstream – sequences in the opposite direction from transcription
downstream – sequences in the direction of transcription
![Page 9: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/9.jpg)
9
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)
![Page 10: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/10.jpg)
10
typical cartoon of a transcription unit
promoter
TSS
coding region5‘ UTR 3‘ UTR
AUG Stop
(UnTranslated Region)
![Page 11: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/11.jpg)
11
The transcription „bubble“
![Page 12: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/12.jpg)
12
Reaction catalyzed by RNA polymerase
RNA (n residues) + ribonucleotide triphosphate (NTP) ↔ RNA (n+1 residues) + PPi
PPi + H2O ↔ 2 Pi
Stryer 2002
![Page 13: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/13.jpg)
13
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)
![Page 14: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/14.jpg)
14
The stages of transcription
![Page 15: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/15.jpg)
15
Prokaryotic Transcription
![Page 16: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/16.jpg)
16
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
![Page 17: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/17.jpg)
17
σ factor ensures promoter specific bindingand is required for initiation
cannot initiate
able to initiate
![Page 18: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/18.jpg)
18
How does RNA polymerase find promoter sequences?
too fast for simple diffusion
unspecific DNA binding, then „one-dimensional random walk“
proposed mechanisms:
(wrong labels in the book)
intersegment transfer„hopping“
direct transfer
![Page 19: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/19.jpg)
19
Aligment of many promoters reveals stretches of conserved sequences -> functionally important
What defines a promoter?
• cis-acting element:
recognized and specifically bound by proteins
• consensus sequence
Sequence logo: illustrates conservation and frequencyof bases in each position
consensus sequence: the most conserved bases in each positionadapted from Stryer 2007
![Page 20: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/20.jpg)
20
The bacterial promoter consensus sequence
TATAATTTGACA
• 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 consensusalso: not all elements have to be present: modularity
• distance between -35 and -10 boxes: 16-18 bp in 90% of promoters-> Important! (Why?)
![Page 21: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/21.jpg)
21
The bacterial promoter consensus sequence
TATAATTTGACA
• 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
![Page 22: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/22.jpg)
22
X ray crystallography in a nutshell
Stryer 2002
electron density map
atomic model
ribbon diagram
![Page 23: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/23.jpg)
23
Crystal structure model of holoenzyme-DNA complex
Illustration adapted from D. G. Vassylyev, et al., Nature 417 (2002): 712-719
s
(Detailed view – but you cannot always determine a crystal structure each time you want to map protein-DNA interactions)
Sigma factor is extended, with short alpha-helical domains connected by flexible linkers
![Page 24: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/24.jpg)
24
footprinting
Stryer 2007
(How do you get labelling just at one end?)
![Page 25: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/25.jpg)
25
Preparing a footprinting probe I
putativebindingsite 1
putativebindingsite 2
PCR-amplification
5‘
5‘3‘
3‘
genomic DNA
![Page 26: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/26.jpg)
26
putativebindingsite 1
putativebindingsite 2
(blunt) (5‘ overhang)
Nature Protocols 3, 900 - 914 (2008)
Preparing a footprinting probe II
„asymmetric“ digestion
Klenow enzyme can add radioactive nucleotide (*) at this end(„Klenow fill-in reaction“)(What if no suitable restriction sites in the sequence?)
(How must the nucleotide be labelled?)
![Page 27: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/27.jpg)
27
Real world examples
Increasing protein concentration
Nature P
rotocols 3, 900 - 914 (2008)
sequencingreaction
footprintexperiment
Footprinting achieves single nucleotide resolution!
Nucl. A
cids Res. (2000) 28 (18): 3551-3557.
![Page 28: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/28.jpg)
28
(Sanger Sequencing – original method)
![Page 29: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/29.jpg)
29
(Sanger Sequencing – the standard today)
Now even faster methods with higher throughput become available – „next generation sequencing“ – see lecture by David Ibberson
![Page 30: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/30.jpg)
30
(another new method to map protein binding down to single bp resolution: ChIP-exo)
ChIP =Chromatin ImmunoPrecipitation
Rhe
e e
t al
., C
ell V
olu
me
14
7,
Issu
e 6
, 9 D
ece
mbe
r 2
011
, P
ag
es
14
08
–14
19
![Page 31: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/31.jpg)
31
Footprinting reveals polymerase shape changes during the stages of transcription
s factor
core enzyme
core enzyme
s factor
Knippers 1997
closed binary complex
open complex
ternary complex
general elongationcomplex
Initiation
Elongation
![Page 32: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/32.jpg)
32
Detection of unwinding
s factor
core enzyme
core enzyme
s factor
Knippers 1997
Initiation
Elongation
Unwound bases become accesible for reagents that cannot reach them within the double helix
e.g. KMnO4
![Page 33: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/33.jpg)
33
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?
![Page 34: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/34.jpg)
34
Summary promoter
1. Modular
2. Consensus sequence
3. Most important: -35 and -10 box
4. Mutations may affect:
s factor and polymerase binding
DNA unwinding
![Page 35: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/35.jpg)
35
The stages of initiation
s factor
core enzyme
core enzyme
s factor
Knippers 1997
closed binary complex
open complex
ternary complex
general elongationcomplex
Initiation
Elongation
![Page 36: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/36.jpg)
36
crystal structure models of initiation - snapshots of a molecular machine
closed binary complex
most contacts on non-template strand
Nature Reviews Microbiology 6, 507-519 (July 2008)
![Page 37: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/37.jpg)
37
crystal structure models of initiation - snapshots of a molecular machine
closed binary complex open complex
Conformational changes:DNA bendsopens between -11 and +3moves into the enzyme („jaws close“)
Nature Reviews Microbiology 6, 507-519 (July 2008)
![Page 38: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/38.jpg)
38
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: short RNAs formed and releasedRNA polymerase stays on promoter„DNA scrunching“
Nature Reviews Microbiology 6, 507-519 (July 2008)
![Page 39: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/39.jpg)
39
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
Nature Reviews Microbiology 6, 507-519 (July 2008)
![Page 40: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/40.jpg)
40
the sigma factor cycle
![Page 41: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/41.jpg)
41
The elongation complex
s factor
core enzyme
core enzyme
s factor
Knippers 1997
closed binary complex
open complex
ternary complex
general elongationcomplex
Initiation
Elongation
![Page 42: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/42.jpg)
42
The catalytic mechanism
Groove lined with positively chargedamino acid residues, why?
![Page 43: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/43.jpg)
43
The catalytic mechanism
![Page 44: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/44.jpg)
44
The catalytic mechanism
![Page 45: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/45.jpg)
45
The catalytic mechanism
Mg2+
• facilitates attack of 3‘ OH• stabilizes negative charges
of transition stateNature Reviews Microbiology 6, 507-519 (July 2008) and Stryer 2007
![Page 46: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/46.jpg)
46
The mechanism of elongation
template DNA
non-template DNA
RNA
„trigger loop“
Volume 19, Issue 6, December 2009Pages 708-714
„bridge helix“
nucleotidein catalytic site
![Page 47: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/47.jpg)
47
Brownian ratchet model
Nat Struct Mol Biol. 2008 August ; 15(8): 777–779
Mg2+
![Page 48: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/48.jpg)
48
Direct observation of base-pair stepping of single RNA polymerase molecules
„optical tweezers“:small beads can be trapped in highly focused laser beams,position of the beads can be monitored with high precision: = ~ 1 bp
RNA polymerase
laser beam
bead
Nature 426:684–87 (2003)
Nature. 2005 November 24; 438(7067): 460–465
![Page 49: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/49.jpg)
49
Summary initiation and elongation
s factor
core enzyme
core enzyme
s factor
Knippers 1997
Initiation
Elongation
Closed binary complex:promoter recognition
Open complex:melting of DNA„jaws close“
Ternary complex:RNAP, DNA, RNAabortive transcription
Elongation complex:s factor offcatalysis: transition state
stabilizedelongation movement:
Brownian ratchetmodel
![Page 50: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/50.jpg)
50
What happens if transcription is blocked?
• Transcription can be transiently blocked e.g. by hairpin structures in the RNA or misincorporation of NTPs
Annu. Rev. Biochem. 2008. 77:149–76
(transitory)
• RNA polymerase can cleave the RNA to generate new 3´-OH end(cleavage activity intrinsic to RNA Pol, stimulated by accessory factors)
![Page 51: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/51.jpg)
51
Transcriptional termination
Two classes of terminators:1) intrinsic terminators (no other
factors required)2) rho ( )r dependent terminators
Annu. Rev. Biochem. 2008. 77:149–76
Often difficult to find the termination site:1) in vivo, the primary transcript gets cleaved or partially degraded2) in vitro, experimental conditions influence termination capacity -> if both approaches find the same, probably the true site...
![Page 52: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/52.jpg)
52
Intrinsic termination
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
Why?
![Page 53: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/53.jpg)
53
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?
![Page 54: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/54.jpg)
54
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)
![Page 55: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/55.jpg)
55
Polycistronic transcripts
promoter
TSS
coding region5‘ UTR 3‘ UTR
AUG Stop the general cartoon
a polycistronic transcript
![Page 56: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/56.jpg)
56
Transcription and translation
(in prokaryotes!)
![Page 57: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/57.jpg)
57
The cycle of bacterial mRNA
![Page 58: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/58.jpg)
58
Transcription in prokaryotes vs. eukaryotes
Stryer 2002
![Page 59: Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9e5503460f94a87bce/html5/thumbnails/59.jpg)
59
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