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Flashcards in RNA Transcription In Eu Genes Deck (24):
1

What are the 6 stages of regulation of eukaryotic gene expression?

1) transcription (most important regulatory step)
2) RNA processing
3) mRNA transport
4) mRNA translation
5) mRNA degradation
6) protein degradation

Steps 2-6 are dependent on transcription having taken place correctly

2

When is gene transcription regulated?

1) during differentiation and development (highly ordered)

2) as a response to the environment (food, hormones, toxins)

3) depending on the function of the cell (tooth cells will need different proteins to kidney cells)

3

What type of diseases do problems in gene transcription result in?

1) cancer: absence of T off switch leads to wrong genes being expressed and cell division.

Oncogenes and proto-oncogenes = mutated or missing:
Cells continue to divide and can’t differentiate because T has not been switched off.

So absence of an off switch can lead to cancer.


2) developmental diseases: mistakes in transcription, early in embryogenesis can lead to serious organ developmental problems.

Wilms’ tumour (kidney cancer affecting young children): caused by mutation on ch 11 (WT1 gene). It is a transcriptional regulator, so a mutation means the kidney doesn’t differentiate properly (parts keep growing)

3) chronic diseases: eg obesity.
Abnormal GE as a result of the wrong signals in the cell cause inflammation and increased risk of other disease eg cardiovascular disease

4

What is needed for transcription?

1) DNA template with correct signals
2) enzymes which can copy DNA into RNAs

The output of transcription is RNAs which can be used by the cell to make proteins

5

What are the 3 main types of RNA produced by T in Eu cells?

1) mRNA - code for proteins (3% of total RNA)

2) ribosomal rRNA - form basic structure of ribosome and catalyse PS (71%)

3) transfer tRNA - act as adaptors between mRNA and AAs (15%)


When extract RNA from human tissue, mRNA is only a small % of the total RNA in a cell, but it has a big job - to code for all cell’s proteins

6

What are the features of mature mRNA?

1) initiation codon: where the first AA (methionine) will be coded

2) termination codon: end of coding sequence
ORF in between 1&2 = codon triplets which code for a peptide

3) polyadenylation signal: in the 3’ UTR where here are signals - 95% of the time it’s AAUAAA

4) poly A tail: lots of adenosine nucleotides added to increase stability

- untranslated regions in both ends all copied from DNA (5’ and 3’ UTR)

7

What do RNA polymerases (multi-subunit enzymes) do?

Make RNA from DNA.

1) RNA pol I synthesises rRNA
2) RNA pol II synthesises mRNA
3) RNA pol III synthesises tRNA, 5S rRNA, snRNAs

- These enzymes were purified by elution at different salt concentrations
- Each has a different sensitivity to the fungal toxin a-amanitin (inhibits RNA polymerase)
- RNA pol II cannot transcribe mRNA from DNA on its own - needs many helpers

8

How is transcription initiated?

To transcribe a gene, a pre-T initiation complex has to assemble upstream of the start site which helps RNA polymerase II to get started

- A number of proteins come together in a specific order to help RNA pol II transcribe genes = GTFs

- GTFs position RNA pol II at promoter resulting in a PIC
- Polymerase then copies DNA into RNA
- Major regulatory point in Eu cells

9

What are regulatory molecules?

Regulatory molecules: trans - not on the same molecule as DNA

- proteins which control T initiation by binding to DNA
- made in cytoplasm and go back into nucleus and bind to DNA
- can diffuse through cell from site of synthesis to bind to the regulatory sequence (such as a promoter) and affect T
- usually have 2 domains, a DNA binding domain and a T activation domain
- eg GTFs needed for T initiation


10

What are regulatory sequences?

Cis - usually very close to the start site of the gene

- short sequences of DNA which control T initiation
- usually found in non-coding (no protein being made) regions of genome
- these regions have a high affinity to GTFs
- interact with DNA binding proteins eg. TFs resulting in induction or repression of TFs resulting in induction or repression of T initiation
- called “cis” bc they influence genes on the same DNA molecule
- eg. TATA box, HREs (hormone responsive elements)

11

What is the link between cis acting regulatory sequences and trans acting regulatory sequences?

1) DNA > mRNA > Protein (TAF)
2) TAF goes back into the nucleus where it binds to a different bit of DNA which regulates something (binds to CAS)
3) RNA pol II comes to copy


GTF (TAF) > Promoter eg TATA > RNA pol II > Copy

12

What is the TATA box?

- the most common promoter
- highly conserved (across different life forms) promoter in many Eu genes

- located ~25-35bp upstream from the start site of transcription

- involved in positioning of the RNA pol II (via GTFs) for correct T initiation (large % of gene had the TATA box motif)

13

How is gene expression regulated?

- expressed selectively under certain conditions/specific cells
- eg. Cyclins required for cell division expressed at a specific stage in cell cycle OR a gene expressed only in skin cells

14

What are promoters?

- short sequence found upstream of a coding region
- important in initiation of transcription
- site where PIC is assembled
- eg the TATA box

15

What are TFs?

- Proteins which ensure that genes are correctly expressed at a specific time and in response to internal or external stimuli

- Can bind specific DNA sequences and specific proteins eg promoter or RNA pol II

16

What are the 2 main groups of TFs?

1) GTFs aka basal TFs: proteins which can bind DNA and are needed to form a preinitiation complex and to recruit RNA pol II

2) Sequence-specific DNA binding TFs: these facilitate or prevent T initiation of specific promoters

17

What are GTFs needed for?

What is the core and proximal promoter?

For all RNA pol II transcribed genes.

Core = where RNA pol II and GTFs will bind eg TATA box

Proximal = where other TFs will bind (if there was a sequence specific TF)

18

What can a 5’ end of a regulated gene contain?

Many regulatory cis elements (short DNA sequences which are conserved)

- along with the promoter, the 5’ upstream region of a gene can contain other regulatory sequences
- enhancers can be found upstream or far away from the promoter and can lead to increased expression
- upstream promoters (CCAAT or SP1) can bind to specific TFs which control whether a gene is switched on or off
- reg elements that can bind hormones or other signals which control whether a gene is switched on or not (eg. GRE glucocorticoid response element)

19

What does RNA pol II do?

- copies DNA into mRNA by catalysing an enzymatic reaction (5’ to 3’) which results in synthesis of RNA using a DNA template and dNTPs

- large multi-subunit protein (8-12 subunits) ~500 KDa

20

What are GTFs?

- group of proteins which are needed for the PIC to be formed
- can bind to promoters
- help RNA pol to locate the start of genes
- required for T of most Eu genes

- multi-meric and highly conserved proteins
- aka basal T factors
- assemble in a specific order in vitro
- made up of more than one subunit

21

What do TFs control?

- development
- cellular process eg cell cycle progression, differentiation and signal transduction

- metabolism
- response to stimuli
- immune system and reproduction

22

What is the normal and mutated consequence of TF binding?

Normal:
TF binds to DNA > correct mRNA expression of target genes > correct expression of proteins > optimal reg of pathways

1) mutation in coding sequence of TF:
Doesn’t bind properly to DNA > abnormal mRNA expression of target genes > abnormal expression of proteins (altered AAs) > altered regulation of pathways

2) mutations in cis sequence (TF binding site) upstream of DNA:
Abnormal mRNA + protein expression

23

How is gene expression regulated by hormones?

- signals to switch genes on/off by binding to receptors in cell membrane and start a signalling cascade (binds to cis sequences upstream on 5’ end = influences T to turn on or off)

- eg insulin or thyroid hormone: produced in the pancreas of beta cells and controls sugar uptake & thyroid produced in thyroid but affects metabolism in the entire body = SIGNALS produced in one part of the body which then travel to a different part

- some hormones enter the cell and then bind to a receptor, the receptor-hormone complex moves to the nucleus where it interacts with DNA + stimulates T of target genes (T initiation) to bring about a physiological change in the cell

24

What are hormones?

Produced by endo system, are exogenous chemical released into circulation which can travel and affect target tissues.

The effect of hormones can stimulate synthesis of large amounts of protein in certain tissues (target tissues).

The endo system can reg and co-ordinate changes in GE of many systems.