Genetics

Front 1

How many RNAP do eukaryotes have? What do they do?

Back 1

Three

RNAPI - synthesises RNA precursors (18s, 28s and 5.8s rRNA)

RNAPII - makes mRNA

RNAPIII - synthesises small non coding RNA (tRNA anf 5s RNA)

Front 2

How are yeast and human RNAP similar?

Back 2

Yeast RNAPI, II and III have 14, 12 and 16 subunits respectively.

Human RNAPII also has 12 subunits.

Yeast and human subunits interchangeable.

Front 3

Similarities between bacteria (E. coli) and human RNAP?

Back 3

Homology exists.

Largest human subunit, RPB1 is similar to beta'

RPB2 similar to second largest beta subunit.

RPB3 and 11 similar to alpha subunits.

RPB6 similar to omega subunit

Front 4

What is the CTD?

Back 4

Carboxy terminal domain found on biggest subunit of RNAPII.

Tandem repeats of consensus sequence:

Tyr-ser-pro-thr-ser-pro-ser

Yeast has 26 repeats, drosophila 42 and humans 52.

It is relatively unstructured.

Front 5

CTD modifications?

Back 5

Can be post transationally modified e.g. phosphorylation (usually of serine) and proline isomerisation.

These affect transcription stages.

CTD is landing site for many nuclear factors.

Front 6

CTD role in initiation?

Back 6

Umodified CTD is vital for PIC formation. High affinity for mediator.

Cdk7 of TFIIH enables promoter release/elongation start by phosphorylating ser 5 and 7.

Front 7

CTD role in elongation?

Back 7

Proximal promoter pausing is a refulatory event.

Caused by RNAPII binding to NELF and DSIF.

P-TEFb (cdk9 and cyclin T) phosphorylates NELF to disassociate it. Also phosphorylates ser 2 AND DSIF to recruit chromatin modifying and transcription factors.

Front 8

CTD role in RNA processing?

Back 8

mRNA capping - high levels of ser5P found in early transcription, recruits mRNA capping complex.

Splicing - phosphorylation of CTD (ser2 thought to recruit spliceosome.

Chromatin modification - ser5P recruits proteins associated to set1 to 5'end to methylate H3K4.

Front 9

CTD role in termination?

Back 9

Phosphorylation of residues important.

Ser7P does 3' processing of small non coding RNA.

Thr4 does 3' processing of histones in vertebrates.

Front 10

What are sequence elements? Examples?

Back 10

Control transcriptional activity.

Core - RNAPII and GTFs bind

Enhancer and PPE - activator and repressor proteins bind (PPE must be close to core enhancer can be anywhere in seq e.g. in genes/introns)

Insulator - binds to nuclead factors in order to compartmentalise DNA into transcriptional domains so enhancers can't act on wrong genes

Front 11

What is a focused core promoter?

Back 11

- 1 transcriptional start site.

- regulated (on/off switch)

- high levels if trans

Front 12

What is a dispersed promoter?

Back 12

- most are dispersed

- multiple TSS within 100 bases

- constitutive (on all the time)

- houskeeping genes

- low levels of trans

Front 13

Sequence elements in focused promoter?

Back 13

TATA box - AT rich (many promoters don't have this)

BRE - B recognition elements

Inr - initiator seq (most common even dispersed have these)

DPE - downstream promoter element

Front 14

CpG population?

Back 14

Under represented but evolutionary pressure to prevent loss of CpG in important places leads to CpG islands.

CpG islands coincide with promoters of known genes.

Oxidative deamination: c to u and cm to t.

Front 15

What sequences do CpG islands contain?

Back 15

- lack TATA and DPE

- have several weak Inr

- binding sites for TFs e.g. E2F and Sp1

Front 16

Type of promoter at CpG islands?

Back 16

- multiple start sites

- constitutive

BUT

- can be regulated by:

1. Methylation of C: long term repression as methyl binding domain proteins recruit HDAC

2. Polycomb group proteins: dynamic repression (switched on and off) and leads to repressive epigenetic marks.

Front 17

What does RNAPII need for in vivo trans?

Back 17

- GTFs (DABFEH - all multiprotein except B)

- correct core

- unwound DNA

- RNAP in correct orientation

Front 18

TFIID structure?

Back 18

Has TBP and 13-15 TBP associated factors.

TAFs make sequence specific contacts with DNA.

TBP-SAGA complex can replace TFIID at some promoters.

Front 19

Role of TAFS?

Back 19

- contact with DNA

- recognised modified histones

- target for activators

Front 20

What are TATA less promoters?

Back 20

Prevelant in housekeeping genes.

Sequence is that of TATA box but differs by 2 or more bases.

Front 21

Role of TAFs in TATAless promoters?

Back 21

Interact with DNA downstream TATA box to compensate for weak TBP binding via additional contacts.

Also is upstream 1st nucleosome so prevents this from interrupting promoter thus gene is expressed.

Front 22

Issue with TATA promoters?

Back 22

TSS and initiation complex overkap with 1st nucleosome. Nucleosome represses gene transcription.

Rely on SAGA protein to help unravel DNA and unblock promoter.

Front 23

TFIIA

Back 23

3 subunits.

Stabilises TBP-TATA complex by

- preventing TBP dimer formation

- preventing non productive TAF1 and TBP interaction

Is a coactivator.

Front 24

TFIIB

Back 24

Contact made with DNA via BRE

Recruits RNAP/TFIIF complex. Acts as bridge between them and TFIID.

Interacts with activators

Stabilises TFIID/promoter complex

Transcription start site selection influenced by this and RNAPII

Front 25

TFIIF

Back 25

Heterotetramer

Recruited with RNAPII and stays bound to RNAPII during elongation.

Stimulates elongation via suppressing pausing.

Other roles:

- helps RNAPII recruitment

- recruits TFIIE and TFIIH

- TSS selection and promoter escape

Front 26

TFIIF

Back 26

Heterotetramer

Recruited with RNAPII and stays bound to RNAPII during elongation.

Stimulates elongation via suppressing pausing.

Other roles:

- helps RNAPII recruitment

- recruits TFIIE and TFIIH

- TSS selection and promoter escape

Front 27

TFIIE

Back 27

Heterotetramer

Direct interaction with RNAPII, TFIIF, TFIIB and promoter.

Recruits TFIIH - Stimulates CDK kinase, ATPase and helicase activity of TFIIH.

Front 28

TFIIH

Back 28

Most complex and largest GTF (almost as large as RNAPII)

Has around 10 subunits, some with enzymatic activity (p52 and p8)

Cdk7 phosphorylates ser5 before primoter escape

ATPase stimulates transcription initiation and escape

Helicase involved in promoter melting

Plays role in transcription couples nucleotide excision repair

Front 29

What is PIC?

Back 29

For transcription to initiate the GTFs and RNAPII must assemble as pre initiation complex at core promoter.

Front 30

What is basal transcription?

Back 30

In vitro when GTFs and RNAPII bind to core causing low levels of transcription.

Front 31

Holoenzyme model?

Back 31

Different to stepwise.

TFIID interacts first. Followed by recruitment of of preassembled complex (holoenzyme)

Contains mediator and coactivators

Stepwise and holoenzyme used at diff promoters? Or same promoters at diff times?

Front 32

What is a mediator?

Back 32

A super coactivator needed for transcription.

Bridge between transcription machinery and activators.

Scaffold site for PIC assembly.

Conserved structure. Yeast and human subunits also conserved.

Front 33

Transcription elongation?

Back 33

After PIC assembly there is conformational change wherein DNA melts and 2 template strand passes through RNAP active site.

Initially get short abortive transcription with 3-10 bp products as elongation complex not stable.

Promoter contacts released when 30 bp nascent transcript made.

Front 34

GTFs in transcription elongation?

Back 34

EF compete with GTFs to cause promoter release.

TFIID, A, E, H and mediator stay bound to promoter (reinitiation complex)

RNAPII, TFIIF and B released.

Front 35

Experiment proving TFIID is bound to promoter?

Back 35

TFIID/TBP preincubated with DNA template that makes 68 bp transcript.

RNAPII and GTFs (except D) preincubated with 112 template.

Reactions mixed, nucleotides added and several rounds of transcription occurs.

Only products made are 68bp long.

Front 36

Experiment proving TFIIB leaves promoter?

Back 36

TBP, TFIIA and B preincubated with 68bp template.

TBP and TFIIA preincubated with 112 bp template.

Mixed. RNAPII, GTFs and nucleotides added.

5 mins: only 68bp products

15 mins: both 68 and 112

Front 37

Alternative GTFs?

Back 37

Tissues or genes have specific alternative GTFs.

TBP related factors replace TBP. E.g. TRF2 binds to altered TATA box.

Some cells have TAFs that aid TRFs.

Allows complex pattern of gene expression for multicellular organisms.

Front 38

Alternative GTFs in histone transcription?

Back 38

Around 100 copies of histone genes.

H1 is has different expression levels to H2A, H2B, H3 and H4 .

H1 uses TRF2 instead of TBP.

Front 39

Alternative GTFs in myogenesis?

Back 39

Differentiation from myoblasts to myotubes.

TFIID downregulated and replaced with TRF3-TAF3 complex.

Front 40

TRF1

Back 40

- insects

- binds to TATA

- mediates RNAPIII trans of tRNA using BRF1

Front 41

TRF2

Back 41

- cell dev, differentiation and cycle progression in fish and frogs

- doesn't bind to TATA as it lacks 3 conserved phenylalanines

- often used to regulate promoters containing DPE

Front 42

TRF3

Back 42

- vertebrates

- homologous to TBP

- mediates embryonic gene exp

Front 43

XR: TFIID

Back 43

1. TBP saddle shaped mol binds to minor groove of DNA causing 90° bend

2. N terminal of TAF1 can bind to TBP inhibiting TBP-TATA interaction

3. Core of histone folds enable TAF-TAF interactions

Front 44

XR: TFIIA

Back 44

1. Binds to underside of TBP

Front 45

XR: TFIIB

Back 45

1. Thought binding to BRE enables assembly of PIC in correct orientation

2. Helix-turn-helix motif at c terminus of TFIIB binds to BRE

Front 46

XR: TFIIF

Back 46

1. 2 subunits: alpha and beta

2. Prevents non specific interaction of RNAPII to DNA

3. Helps minor opening of RNAPII clamp

Front 47

XR: TFIIE

Back 47

1. 2 subunits. Alpha has acidic c terminal that binds strongly to TFIIH and beta c terminak binds to single stranded DNA - stabilises open promoter complex.

Front 48

XR: Inr

Back 48

Initiator seq recognised by TAF1 and 2.

Higher TATA occurrence in absence of Inr or Inr-like sequences.

Front 49

XR: TCT motif.

Back 49

Rare motif.

Encodes proteins involved in translation.

Single T to A conversion turns it into Inr.

Front 50

XR: DPE

Back 50

Found in drosophila but rare in humans.

Dependant upon Inr.

Position to Inr important, one nucleotide diff means it might not work.

Front 51

XR: chromatin modification

Back 51

TAF3 binds to H3K4me3 to facilitate PIC by recruiting TFIID.

H3K4me3 found downstream TSS and in active promoters.