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1. MOLECULAR CELL BIO > patrick (L12-13) > Flashcards

patrick (L12-13) Flashcards

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Biology 101 flashcards
1
Q

define the central dogma

A 
dna
(rna synthesis - transcription)
rna
(protein synthesis - translation)
protein
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2
Q

steps at which gene expression can be controlled

A

LOOK AT L12 S4-5

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3
Q

why is gene regulation important?

A

major control point for tissue/site specific expression

determines the phenotypes and so determines the species

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4
Q

precision of gene regulation

A

Depending on development stage and tissue, specific combinations of genes are expressed, others are not. Even different abundances/quantities of transcripts are produced
very important that genes are expressed and how many mRNA transcripts are produced at different times

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5
Q

what determines the transcription of genes

A

chromatin structure
rna polymerase binding
additional binding and activation factors

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6
Q

regulation of chromatin structure

A

expressed genes are found in open chromatin

genes within highly packed heterochromatin are usually not expressed

the histone code hypothesis proposes that specific combinations of chemical modifications to histones and dna of chromating help determine chromatin configuration and influence transcription

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7
Q

types of histone modifications

A

ACETYLATION - acetyl groups are attached to positively charged lysines in histones tails. this process loosens chromatin structure, thereby promoting the initiation of transcription

METHYLATION - the addition of methyl groups can condense chromatin

PHOSPHORYLATION - the addition of phosphate groups next to a methylated amino acid can loosen chromatin

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8
Q

define the histone code

A

modifications of each histone protein regulate the activity of that section of dna
(forming a heterochromatin will silence genes)

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9
Q

transcribed regions moving in the nucleus

A

they move to different positions to be transcribed

chromatin can move within the nucleus to alter gene expression. active regions are central to the nucleus / heterochromatic regions are close to the nuclear membrane

Loops move in the nuclear neighborhood for gene expression for the dna to be transcribed
Loops move to nuclear neighborhood for gene silcencing and will not be transcribed (silenced DNA regions)

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10
Q

eukaryotic rna polymerases structure

A

LOOK AT L12 S16

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11
Q

types of eukaryotic rna polymerases and their individual functions

A

RNA POL I - ribosomal rna
RNA POL II - all protein coding genes, transcribes all proteins
RNA POL III - small rnase that dont encode for any proteins (very important for rna degradation), has tRNA genes

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12
Q

RNA polymerase II

A

requires many additional proteins GENERAL TRANSCRIPTION FACTORS

Zinc magnesium - give stability and they have to catalyse rna pol activity

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13
Q

gene promoter for RNA POL II

A
  • elements influencing transcription can be upstream or downstream of the transcriptional start site
  • may be many kilobases distant from the gene
    TATA box is most important for sequence that signals the start of transcription

We cannot predict where the promoter ends. Can predict where the TATAA boxes end/start, and where promoter starts

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14
Q

TATA box

A

it is a consensus sequence
Pol II promoters have a TATA box at -25 relative to the transcriptional start
(( prokaryotes TATAAT at -10 ))

eukaryotic promoters are more complex and organisation of eukaryotic genome in nucleosomes –> eukaryotic rna pol II depends on general transcription factors

–>Very conserved so must be used in transcription

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15
Q

rna pol II initiation complex

A

it consists of rna polymerase II plus general transcription factors
the TATA box is the site for nucleation of initiation complex assembly

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16
Q

functions of different general transcription factors in RNA POL II

A

TFB subunit recognises TATA box

TAF subunit regulates dna binding

TFIIB positions rna polymerase

TFIIF stabilises rna polymerase

TFIIE attracts and regulates TFIIH

TFIIH unwainds dna and phosphorylates Ser5, and releases rna pol from the promoter

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17
Q

INCORPORATION OF RNA POL II

A

LOOK AT MECHANISM DIAGRAM IN L12 S22

TFIID (transcription factor 2D) and TBP (tata box binding protein) bind to the tata box
TFIIB is guided by TFIID to build the foundation for the RNA to come into and bind

Rna pol 2 - complex of diff protein subunits. Needs to be stabilised by TFIIF and helps with the assembly of the polymerase
TFIIE and TFIIH - help rna pol to bind solidly to dna. Tail of rna pol is now phosphorylated - the tail is now active to start transcription

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18
Q

transcription speed

A

20 nucleotides per second

>1000 transcripts (mRNA) can be synthesised from a single gene per hour

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19
Q

transcription complexes

A

eukaryotic rna polymerases cannot access dna selectively, they require additional factors that help to communicate to the rna pol that the this gene needs to be transcribed

(mediator - multi protein complex structure)
Mediates info from promoter to rna rol

additional factors mediate contact between upstream activators and basal factors
upstream activators enhance speed of assembly

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20
Q

roles of transcriptoon factors

A

TFs are gene regulatory proteins
to initiate transcription, eukaryotic rna polymerase requires the assistance of proteins called TFs
general transcription factors are essential for the transcription of all protein coding genes
in EUKARYOTES, high levels of transcription of particular genes depend on control elements interacting with specific transcription factors

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21
Q

ORGANIZATION OF A TYPICAL EUKARYOTIC GENE AND THE MECHANISM OF TFS

A

LOOK AT L12 SLIDES 27 TO 29

22
Q

define activator

A

it is a protein that binds to an enhancer and stimulates transcription of a gene
bound activators cause mediator proteins to interact with proteins at the promoter
activators are called SPECIAL TRANSCRIPTION FACTORS

The specific TF dont directly interact with rna pol - indirectly via mediator complex

Activators help that promoter region get in touch with mediator
Promoter regions may be very far away from where the transcription starts - so the promoter dna has to bend. Dna bending proteins

Help to recruit TFs. Rna pol can now fly in and bind to tata box

23
Q

combinatorial control of gene transcription

A

LOOK AT L12 S30

a particular combination of control elements can activate transcription only when the appropriate activator proteins are present

24
Q

LEUCINE ZIPPER TF

A

leucine zipper mediates both dna and protein binding
can bind to DNA as homodimers or heterodimers
expands potential regulatory repertoire

25
Q

how do TFs bind to specific sites on the DNA

A

Interact with dna
TF act specifically - not random. Recognise specific region in the promoter, Region they bind to is defined by the nucleotide sequence of the promoter (there are major and minor grooves)

Opposite the minor grooves, there is a major groove (and vice versa)
Nucleotides have specific signature - of H bond acceptor, H bond donor, hydrogen atoms and methyl group (shown in key)
–> These have specificity and recofnise structural features of the DNA

26
Q

how do dna binding domains bind to specific regions on the dna

A

they recognise specific promoter elements

gene regulatory proteins interact with several nucleotides (=sequence motifs)

27
Q

ZINC FINGER TF

A

Cys-Cys-His-His
Histidine and cysteine amino acid come together to form the fingers
Structure determined by the zinc atoms
Sequence of amino acid changes slightly to correspond to specific nucleotide sequence to bind and identify the promoter. Through the code.

If we know the sequence we can predict to which nucleotide the promoter will bind to

28
Q

how do different TFs work together

A

cooperative dna binding leads to considerable combinatorial complexity

The more TF can bind to the promoter sequence, the more variation in expression we can achieve and the more cell types we can regulate and manipulate the gene expression

Members of different TF families can come together (AP1 and NFAT bind onto dna to regulate a gene)

LOOK AT L12 S36

29
Q

how are eukaryotic TFs modular?

A

in both prokaryotes and eukaryotes, transcriptional activators and repressors are modular proteins, composed of distinct functional domains:

  • dna binding domain
  • activation/repression domain: interacts with other proteins or another subunit of the same protein

Some TF interact with mediator complex and inhibit transcription - acetylation etc
They don’t only activate transcription, they can also interact with the mediator complex and inhibit transcription

30
Q

regulatory proteins assemble into complexes on dna

A

Specific TF can interact with coactivators and corepressors which would allow interaction with the mediator

LOOK AT L12 S38

31
Q

eukaryotic gene control region regulate transcription

A

LOOK AT L12 S39

32
Q

how to predict a phenotype from a genotype?

A 
USING FORWARD GENETICS
phenotype/mutant
gene
protein
genotype
indentify gene (function) based on a phenotype
33
Q

explain the genetics of hypersensitivity to uv radiation

A

the Xerooderma Pigmentosum (XPD) gene codes for a component of TFIIH (one of the 9 subunits)
TFIIH also functions in dna repair
XPD sufferers - hypersensitive to UV radiation and suffer extensice skin damage

34
Q

how to predict a phenotype from gene expression analyses?

A

alternative approach - REVERSE GENETICS

genotype to protein to gene to phenotype/mutant

Genotype is defined by its transcriptome (which gives us clues on improving the phenotype)
transcriptome analysis will analyse all genes at the same time (not just one)
To understand the phenotype

35
Q

how is the genesis predicted

A

must understand the function of cells
and the behaviour of multicellular complex organisms

This is very complex if you deal with multicellular organisms because of diff cell types with diff transcriptomes

zygote -> differentiate -> multicellular organism

36
Q

gene expression (transcription) - what do we need to know?

A

We want to know: where is the gene expressed (specific tissue, cell type), when is it expressed (specific treatment) and why is it expressed (promoters that induce or repress the gene)
Allows us to come up with therapies and treatments (for cancer for example)

37
Q

signalling pathway in a cell

A

Receptor on surface membrane
Signalling cascade in cell
Activate TFs which bind to promoters and regulate genes
Then at the end, there is a response and a phenotype

38
Q

why analyse gene expression?

A

to understand the where, when and why

analyse the expression of a single gene

  • reverse transcriptase polymerase chain reaction (RT-PCR) // where when
  • live cell imaging (CLSM) // where when
  • promoter studies (identify elements in silico and in vivo // where when why

analyse the expression of all genes

  • microarrays analysis (affymetric, agilent, tiling) // where when why
  • RNA sequencing (using next generation sequencing // where when why
39
Q

define the steps of RT-PCR

A

LOOK AT L13 S12
1. cDNA synthesis
(( You harvest a tissue
Extract the mrna (which is very unstable)
Reverse transcribe the mrna (produce a complementary dna template)
Cdna is more stable, easy to handle
Transcribe the gene of interest ))
2. PCR amplification
3. gel electrophoresis
(( Separate the new amplicons that you amplified with the PCR
Determine if the gene is true, induced or repressed by the thickness of the band ))

it is quick and easy but can only be done one gene at a time

40
Q

define live cell imaging

A

use the microscope to analyse gene activity and localisation
L13 S13-14

You want to analyse WHERE the gene is expressed
Take promoter
Fuse to reporter gene
Reporter gene could be the lacZ (will give blue cells) or green fluorescent protein
Mrna is expressed in the cell

41
Q

analysis of transcription factor binidng sites

A

add reporter gene to promoter constructu - encodes an easily assayed enzyme (like B galactosidase)

transfect reporter DNA construct to cultured cells

  • allow time for expression, dna makes rna make protein
  • extract the protein
  • assay for reporter enzyme activity

[[ Which motifs contribute to the expression of genes
If we know which TF binds to this region, then we know which TF is involved in the expression of the specific gene

We take the promoter of the gene and we fuse it to a green fluorescent protein
The cells in which the construct is expressed are transformed and show a green colour ]]

42
Q

how to test promoter function by transfection

A

modify reporter construct and repeat experiment (transfection, assay)

We can quantify how many green fluorescent proteins there are by calculating the number of green fluorescent cells show up??
At a certain point, we lose the expression of GFP
this tells us that there are certain regions in the promoter that are very crucial for the expression of gfp

and if we know which transcription factors bind to the DNA then we know which transcription factor contributes to this phenotype
we need to single cell systems to quantify this

43
Q

Chromatin Immunoprecipitation ChIP

A

identify the dna binding sites of TFs
how many binding sites are there?
what is the sequence recognition preference?
do the sites of binding change druing development, disease or environmental responses?
next generation sequencing allows the identification of millions of fragments
can identify every bindin site for a selected transcription factor

LOOK AT DIAGRAM AND EXPLANATION ON L13 S17

44
Q

how to identify the TF binding site by ChIP an next generation sequencing (ChIPseq)

A

We can lead the TF onto the DNA then sequence it
Left and right of TF binding site, we get sequences based on the sequencing approach
We cant sequence what the TF is binding to
Once we have this info (using the chip sequencing method), because we can do it on a whole transcriptome level, we can figure out if we can find this sequence (that TF binds to) on other promoters of other genes

45
Q

define phylogenetic fingerprinting

A

evolutionary conservation of DNA sequence in essential regions

We can find other sequences on the DNA that are highly conserved
–> we can do this using a computer by my lining up our sequence with different strains
then we could find other structures of dnase and promoters that are highly conserved
this will tell us there are other DNA’s with the same sequence that the same TF binds to

46
Q

what are the 3 key regulatory genes in budding yeast that control genes

A

Matɑ1 + Mcm1
activate red labelled genes

Matɑ2 + Mcm1
repress blue labelled genes

Matɑ2 + Matɑ1
repress green labelled genes

47
Q

what is automation and what was it used for?

A

automation allowed scientists to measure expression of thousands of genes at one time using DNA microarray assays
(studying the expression of interaction groups of genes)

DNA microarray assays compare patterns of gene expression in different tissues, at different times or under different conditions

48
Q

how do microarrays work?

A

On the microscope light
Each spot represents one gene
Not the whole mRNA is spotted on these slides
You pick a portion of the mrna that is unique for this mrna (for no crosslinking with other genes)
You only have one strand of the mrna present on the slide
This is because you prepared how you extract the mrna and transform it in complimentary cDNA
Which is also single stranded
Once we have the cDNA, we can hydrolyse it

To detect this, the mrna is labelled so we can do a red and green labelling (red for control // green for treatment)
hydrolyse this and get a mixer of as mRNA
On each spot, you have each strands of mrna in high abundances (1000s of copies)
so when you hybridise your sample mrna or complementary cdna you get a colour code

colour code:

  • darker green, the more the gene is expressed
  • the more the red, the more the gene is not expressed)
49
Q

expression analysis by next generation sequencing

A

arrays are no longer used

Extract all of the mrna from a tissue (1000s of no long)
Fragment new mrna (100-200 bases long)
generate cDNA because it is more stable
then ligate adaptors to both ends, which are important because that is where sequencing primers bind to
Sequencing means you read all the cdna that you find in the sample
The more mrna you produced, the more cDNA is present and the more of the respective gene i will find in my sample

get the sequence
Align the sequence to the genome
And count them
how many transcripts do you have of the gene?
Y axis is the gene expressed level (number of reads)

50
Q

what is RNAseq show

A

shows which exons are transcribed in sample
count the number of RNAs per exons that you have sequenced in your sample to calculate the expression level

RNASEQ ALLOWS THE IDENTIFICATION OF GENE SPLICING VARIANTS

51
Q

how to enhance food security

A

crop productivity is improved by

  • enhancing root stress resistance (organisation of root immunity)
  • root growth (yield)
52
Q

ORGANISATION OF ROOT IMMUNITY

A

LOOK AT DIAGRAMS IN L13 S31 TO S38

LECTURE CAPTURE THESE SLIDES THO…. CANT BE BOTHERED TO RIGHT NOW…. SOZ

1. MOLECULAR CELL BIO (6 decks)

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