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Gene Regulation > Transcription > Flashcards

Transcription Flashcards

1
Q

How much of the genome are actually transcribed into proteins?

A

the whole genome is 3 billion base pairs: 2% (around 21,000 genes) become proteins

98% is transcribed but are used as RNA molecules that don’t code for proteins (ribosomal RNA)

20%-40% regulatory; allowing for tissue specific expression; transcription act as regulation controlling what genes are coded for

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2
Q

What in the DNA directs transcription? Describe initation

A

A promoter (dispersed sequence) and an enhancer (closely arranged sequences) control transcription.

The promoter and the proximoter proximal sequences determine the start site and bind to MULTIPLE Transcription factors at binding sites.

Promoter DISTAL sequences have a FLEXIBLE location ages upstream or downstream from the gene

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3
Q

Describe the promoter

A

It located immediately upstream (5’) of the transcription start site at +1 fixed direction/orientation relative to gene with in 500bp of the start site.

It has short (consensus) sequences as binding sites for GTFs

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4
Q

What is gene expression regulated by?

A

TRANSCRIPTION FACTORS

1) Combination of promoter and enhancer elements and their cognate transcription factors
2) Transcription factors responsive to signals (ligands, phosphorylation; cofactor)
3) Constitutive and cell type specific transcription factors
4) Transcription factor cooperativity.

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5
Q

Describe the influence of promoter distal sequences

A

They can silence or enhance transcription through the modulation UP/DOWN of initiation.

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6
Q

What is an enhanceosome?

A

DNA looping allows for enhancer contact with the promoter region.

The role of these complexes is to facilitate the assembly of the pre-initiation complex at the start site of transcription

Activator proteins responding to infection or stimuli cooperatively bind to the upstream enhancer region. The interaction is mediated by a protein which assists in stabilizing the complex by promoting inter-protein interactions.

The assembled enhanceosome (through binding activator/TF proteins into a macromolecular complex at the enhancer sequence) recruits transcriptional machinery such as RNA polymerase to the promoter region to initiate.

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7
Q

Describe other long range method of controlling initiation.

A

(1) Insulators/Boundary Elements (BE) which Prevents enhancers on NEIGHBORING genes to affect the expression. (separates genes)
2) Matrix Attachment Regions (MAR) help attach chromatin making new domains possible.

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8
Q

List and describe the three classes of factors needed for transcription.

A

1) machinery needed for transcription: nuclear RNA polymerases: 1-3 and GTFs (general transcription factors) both needed.
2) Activators: influence assembly of machinery at promotor: TF and at distal sites (enhancers)
3) Co-activators with a bridging function/connect TF and GTFs at the machinery level. Onvolved with chromatin remodelling/alter gene structure.

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9
Q

Give details of the RNAP enzymes

A

1: rRNA (needed in every cell for ribosome biogenesis)
2: mRNA
3) tRNA

Similar conserved structures in all three domains of life (eukaryotic and archaeal very similar arrangements of subunits)

The largest subunit in RNA polymerase II has a CTD (carboxy-terminal domain) consisting of multiple heptamer.repeats.

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10
Q

Describe the process of GTFs and enzyme Pol II interaction.

A

1) The GTFs Transcription factor (TFIID) binds to TATA box on the gene
2) Transcription factor IIA which STABILISES TFIID
3) TFIIB (recruited by TFIID) allows binding of TFIID and the enzyme
4) TFIIE and TFIIH are needed for whole complex
5) Final complex is called DABPol II F.
6) The complex is recycled

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11
Q

Describe the process of TFIID binding as a result of its structure

A

The GTFs Transcription factor (TFIID) has a TATA binding protein (TBP) and TBP associated factors (TAFs). These are both specific to the promoter structure.

TBP recognises TATA through minor groove contacts, (DNA is kinked and unwound) It then binds to the TATA box in the core promoter (at about -30) and nucleates.

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12
Q

What is the role of TFIIE and TFIIH?

A

TFIIE involved in promoter opening and regulation of TFIIH.

TFIIH has multiple roles: provided kinase for phosphorylation at initiation, it also provides a REPAIR COMPLEX when elongation stage.

The C terminal of Pol II (CTD) is phosphorylated by TFIIH triggers release of Pol II (promoter clearance).

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13
Q

What can happen if TFIIH is mutated ?

A

Mutations in the XPD (Xeroderma Pigmentosum-D) component of TFIIH cause human disease: skin cancers, neurological abnormalities, cellular sensitivity to UV irradiation

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14
Q

What is the mechanism for Pol 1 initation?

A

Promoter of Pol 1 enzyme Core element has no TATA box and there is an Upstream Control Element (UCE)

The gene promoters are:
SL1: (TBP-TAF complex) binds the core promoter and stabilises UBF. It is essential for the recruitment of Pol I

UBF: an architectural 
protein (DNA bending),
activates transcription
(by anti-repression and 
stimulated promoter clearance)

POL 1 binds and elongates, dissociates

TAF1B of SL1 is similiar to TFIIB.

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15
Q

What is the mechanism for Pol 3 initation?

A

Pol III GTFs are:
1) TFIIIC (assembly)

2) TFIIIA (assembly)
3) TFIIIB (recruitment of enzyme)

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16
Q

Understand the significance of the CTD of Pol II in coordinating transcription with RNA processing.

A

1) DNA damage repair (DNA helicase subunits of TFIIH have a role in
nucleotide excision repair; transcription-coupled
repair)

2)RNA processing (complexes involved in mRNA splicing and 3’ end formation are associated with CTD of pol II)

3) DNA replication (TF binding sites near origins
of replication; actively transcribed genes are replicated early in S phase)

4)Nuclear architecture (nuclear matrix; chromosomal domains;
transcription foci in the nucleus;
heterochromatin; nucleolus)

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17
Q

After initiation, there is a process of transcription elongation, what are elongation factors and their effects?

A

Elongation factors help to overcome stalling and premature termination (in the context of chromatin); they help escape from the promoter.

1) increase the rate of elongation
2) assist Pol II through pause sites
3) facilitate transcription through chromatin
4) polymerase associated factors provide a platform for chromatin remodelling activities

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18
Q

What is stalling and how does NELF have a role?

A

If the Pol 2 is STALLED at the promoter and proximal sequence, it means GENE EXPRESSION can be regulated. This is found across whole genome and means genes can be POISED ready for activation

NELF is a negative elongation factor important in maintaining polymerase stalled near a large number of promoters.

NELF and elongation factor DSIF promote the stalling of Pol 1.

19
Q

Which elongation factors will help increase the rate of elongation?

A

ELLs increase the catalytic rate of Pol II.

Elongin A, B and C (required for proper heat shock gene expression; alleviation of pausing at developmentally regulated genes)

DSIF (DRB Sensitivity Inducing Factor) can either negatively or positively affect transcription by Pol II.

20
Q

Which elongation factors will help elongation through pause sites?

A

TFIIS counteracts transcriptional arrest by binding to the transcript RNA; cleaves RNA so 3’ end can align again.

P-TEFb (Positive transcription elongation factor b): a CDK9/Cyclin T1 complex

21
Q

Which elongation factors will help elongation through CHROMATIN?

A

FACT (Facilitates chromatin transcription): a SSRP1/hSPt16 complex; it can displace a dimer from the nucleosome.

Paf1 complex modifies histone activity

22
Q

How do transcriptional activators locate at the promoter ?

A

Sequence specific DNA-binding domain

23
Q

Generally How do transcriptional activators function ?

A

Activation domain: to interact, directly or indirectly, with components of the transcription apparatus

Different functions:DNA-binding activity and transcription activation are carried by SEPARATE domains of an activator.

There are variations in DNA binding domains which classify the activator.

In C-terminal, of the activator has a helix. The helix binds one turn of the MAJOR groove of DNA. leucine zippers on the helix dimerize with other identical zippers (homo) or different zippers (hetero).

The domains interact with the transcription machinery such as the TF of the PIC complex of pol II.

24
Q

What are the domains of activators?

A

1) Proline rich
2) Glutamine rich
3) Acidic

Hydrophobic residues are essential.

Domains can transform structurally.

25
Q

How does the activator stimulate transcription?

A

It helps the promoter open by stimulating remodeling of chromatin where it unfolds (dencondensation) via remodeling complexes

It also helps recruit GTF to the promoter (therefore causing transcription activation)
It stabilises GTF at the promoter. Conformational change helps to stabilise the GTPcomplex.

Example: herpes Gal4-VP16- induces conformational change in TFIIB, which facilitates the binding of TFIIF and pol II

Example: VP16 after initation induced covalent modification of complex on the CTP domain of the enzyme.

Example: HSF stimulates promoter clearance, and elongation to begin.

26
Q

What is a co-activator?

A

A mediator complex of about 30 sunubits that helps promotoe opening through ATP DEPENDANT chromatin remodeling

27
Q

What are the functions of co-activators?

A

On recruitment by activators they arrive at the promoter site where they act as a physical and functional bridge between activators and GTFs.

They stimulate PIC assembly and stabilises re-initiation scaffold (for paused transcription)

It also binds hypophosphorylated Pol II

28
Q

How can activators be regulated?

A

1) the GENES that code for TF are regulated via feedback control and destruction of mRNA ect.
2) Regulation of transcription factor LOCALIZATION
3) Regulation of transcription factor ACTIVITY via phosphorylation
4) Regulation of transcription factor DIMERIZATION(depending of the TF concentration)
5) Regulation of transcription factor PROTEOLYSIS
6) use of LIGAND BINDING like hormones

There is a network of TF that regulates other TF.

29
Q

How do you switch on gene transcription?

A

1) Activator binds
2) binding is ENHANCED by co activator recruitment and unwinding of the chromatin (remodeling)
3) a PREINITATION complex of enzyme and GTF is formed.
4) initation is followed by PROMOTER CLEARANCE where the enzyme is released from the promoter but still on the target gene (for mRNA production)
5) Net step stalling or ELONGATION

30
Q

Describe unusual elements in prokaryotic transcription that vary from eukaryotic cells.

A

Ribosomes and DNA exist in the same compartment so transcription can be INSTANT.

RNA synthesis occurs in the
transcription BUBBLE which is about 14 bp long and covers the length of the RNA-DNA complex, DNA rewinds outside of bubble.

31
Q

What are the basic mechanisms of prokaryotic transcription?

A

RNA Polymerase binds, used to separate strands; with 3-5 as template.

Elongation where RNA is made

Termination

32
Q

Describe prokaryotic RNAP structure.

A

In E.coli, it is same size as eukaryotic enzyme and interacts with the SIGMA factor.

The core part of the enzyme can bind to ANY bit of DNA in its catalytic central binding cleft but it does NOT know which bit it has bound to; there is a channel for DNA processing to take place.

In the cleft/active site the aspartic acid loop is HIGHLY CONSERVED

T7 virus is smaller than most it is only 100kDA

33
Q

Describe the mechanism of prokaryotic RNAP.

A

A protein bridge changes conformation to control the entry of nucleotides to the active site; changes conformation very rapidly as each nucleotide is added to the chain.

34
Q

What is a holo RNAP enzyme complex? What effect does it have?

A

where the core enzyme and sigma factor combine; the sigma factor is needed for INITATION; it can alter the DNA-binding properties of RNAP so its affinity for general DNA is reduced and its affinity for promoters is increased which allows the promoter to be RECOGNIZED.

It does this by constantly probing the random DNA (briefly loosely binding to it) and “hops” along until it finds the promoter and tightly binds.

35
Q

Describe the mechanism of prokaryotic RNAP binding conformations

A

RNAP binds to the promoter as a closed complex (DNA double-stranded) which it then separates to form an open complex

There may be a abortive initiations before the enzyme moves to the next phase where the Sigma factor may be released from RNAP (once the new RNA chain reaches eight to nine bases in length )

A change in association between sigma factor and holoenzyme changes binding affinity for DNA so that core enzyme can move along DNA and synthesis RNA

When in CLOSED state, DNA outside of enzyme, When in open state DNA runs through middle channel.

36
Q

What is the significance of the sigma factor?

A

It is an additional subunit required for specificity of initiation of different promoters that regulates initiation and therefore GENE EXPRESSION

One sigma factor can have two corresponding sequences to compensate for slightly varied promoter sequences.

37
Q

What are Promoter consensus sequences? What is promoter strength?

A

Individual promoters usually differ from the consensus at one or more positions (due to mutation)

Variation here can mean effects on binding, opening complex up.

Strength of most promoters correlates with fit to consensus sequences/how close it is to the perfect sequence; determined by the amount of energy needed to separate the strands.

38
Q

What is supercoiling and its significance?

A

DNA strand is supercoiled.

DNA topoisomerases remove supercoils when they are processed so DNA is not over-wound.

This is Important as it can increase the efficiency of promoters as it helps in the unwinding/separation of strands.

39
Q

What is the difference between the sigma 70 and 54 factors?

A

sigma70 isimmediately able to initiate, 54 needs ATP from an activator for melting and initiation.

40
Q

In what ways can transcription be controlled in prokaryotes?

A

Repression caused by a Repressor that binds to operator and obstructs promoter such as Lac repressor.

Activation by an Activator that binds UPSTREAMof promoter and causes initiation of transcription LIKE eg CAP.

CAP activates the alpha subunit of the core RNAP enzyme, specifically the a-CTD subunit. (it does not recruit but stabilises)

41
Q

In what way is elgongation in prokaryotes discontinuous?

A

Movement of the enzyme is not uniform as it pauses through two methods

1) Hairpin loop formation of mRNA
2) Backtrack caused by backward enzyme motion

42
Q

What are the characteristics of a termination sequence in prokaryotes?

A

G-C-rich hairpin in the mRNA followed by a U-rich region at which point the enzyme is released.

43
Q

What is Rho? In what way is bacterial transcription Rho dependant?

A

Rho is a site-specific termination factor (46 kDa hexameric RNA-dependent ATPase) which binds to a site on the new RNA (rut)

Rho then caches up withthe RNAP at the TERMINATION SEQUENCE where it UNWINDS the RNA and DNA in the enzyme.

RNAP and RNA released.

44
Q

What is anti-termination?

A

Regulation function in which termination can be prevented when antitermination proteins act on RNAP befor the termination site and cause it to CARRY ON THROUGH a specific terminator or terminators

Gene Regulation (5 decks)

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