Introduction to Genetics

Winter semester 2014 / 2015

Seminar room 00.005 INF230Thursdays 18:15 - 19:45

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).

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: nicholas.foulkes@kit.eduJochen Wittbrodt: jochen.wittbrodt@cos.uni-heidelberg.deThomas Dickmeis: thomas.dickmeis@kit.eduClemens Grabher: clemens.grabher@kit.eduRüdiger Rudolf: ruediger.rudolf@kit.eduFelix Loosli: felix.loosli@kit.eduHarald Koenig: h.koenig@kit.eduDavid Ibberson: david.ibberson@bioquant.uni-heidelberg.deJuan Mateo: juan.mateo@cos.uni-heidelberg.de

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. Foulkesnicholas.foulkes@kit.edu

5

Basic transcription mechanisms

Thomas Dickmeis

Institut für Toxikologie und Genetik,

KIT, Karlsruhe

thomas.dickmeis@kit.edu

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

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

8

Transcription:Definitions III

upstream – sequences in the opposite direction from transcription

downstream – sequences in the direction of transcription

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)

10

typical cartoon of a transcription unit

promoter

TSS

coding region5‘ UTR 3‘ UTR

AUG Stop

(UnTranslated Region)

11

The transcription „bubble“

12

Reaction catalyzed by RNA polymerase

RNA (n residues) + ribonucleotide triphosphate (NTP) ↔ RNA (n+1 residues) + PPi

PPi + H2O ↔ 2 Pi

Stryer 2002

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)

14

The stages of transcription

15

Prokaryotic Transcription

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

17

σ factor ensures promoter specific bindingand is required for initiation

cannot initiate

able to initiate

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

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

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?)

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

22

X ray crystallography in a nutshell

Stryer 2002

electron density map

atomic model

ribbon diagram

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

24

footprinting

Stryer 2007

(How do you get labelling just at one end?)

25

Preparing a footprinting probe I

putativebindingsite 1

putativebindingsite 2

PCR-amplification

5‘

5‘3‘

3‘

genomic DNA

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?)

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.

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(Sanger Sequencing – original method)

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(Sanger Sequencing – the standard today)

Now even faster methods with higher throughput become available – „next generation sequencing“ – see lecture by David Ibberson

30

(another new method to map protein binding down to single bp resolution: ChIP-exo)

ChIP =Chromatin ImmunoPrecipitation

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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

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

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?

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

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

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)

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)

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)

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)

40

the sigma factor cycle

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

42

The catalytic mechanism

Groove lined with positively chargedamino acid residues, why?

43

The catalytic mechanism

44

The catalytic mechanism

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

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

47

Brownian ratchet model

Nat Struct Mol Biol. 2008 August ; 15(8): 777–779

Mg2+

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

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

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)

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...

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?

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?

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)

55

Polycistronic transcripts

promoter

TSS

coding region5‘ UTR 3‘ UTR

AUG Stop the general cartoon

a polycistronic transcript

56

Transcription and translation

(in prokaryotes!)

57

The cycle of bacterial mRNA

58

Transcription in prokaryotes vs. eukaryotes

Stryer 2002

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