Gene expression

Gene expression

• The information encoded in a gene is converted into a protein

The genetic information is made available to the cell

• Phases of gene expression

1. Transcription

2. Translation

3. Protein folding

Functional protein

1. DNA’s information is copied into messanger RNA (mRNA) molecule in transcription

2. mRNA directs synthesis of a protein with amino acid sequence determined by the base sequence of the codons in mRNA

Translation

Unfolded

Folded

3. Correct folding of a protein is needed to achieve functional activity

Transcription:• a mRNA copy of a DNA sequence is produced

• RNA polymerases make RNAs

• a template strand

• mRNA copy has one strand

• Beside the coding area also other sequences are added to mRNA molecule

• Sequence is complementary for DNA • Ts are replaced with uracils, U

• mRNA is produced and processed in the nucleus:

1.Introns are cutted off2.Methyl cap is added to 5’

end3.Poly A tail is added to 3’

end4. Alternative splicing

•The ready mRNA molecule is transported to the cytoplasm

Nucleus Cytoplasm

From a mRNA to a protein…

• Decoding mRNAs is dependent on transfer RNAs (tRNA)

• All tRNAs have similar structure•amino acid part•anticodon part

• Anticodon part base pairs with it’s anticodon structure in mRNA

• Amino acid part carries correct amino acid

tRNAs are needed for recognition and transport

Amino acid

mRNA

Protein synthesis

• Happens in ribosomes in cytoplasm

• Ribomes recognize the initiation codon from mRNA

• Elongation of a protein chain includes three steps main steps

Step 0.mRNA arrives to the ribosome and the ribosome starts to ”read” mRNAs code

Step 1.tRNA forms a pair with the corresponding codon in mRNA

Step 2. A bond is formed by ribosome between the adjacent amino acids

Step 3. The ribosome translocates to the next mRNA codon and the ”used” tRNA is discharged from the ribosome

• Previous steps are repeated until the ribosome arrives to the stop codon

Step 4.Termination is carried out with the help

of termination factors

the nascent protein is released ribosome dissociates

mRNA is released

Step 5. Nascent proteins are folded and chemically modificated

• Proteins folding is dictated by it’s amino acid sequence

3D structure can be predicted from the aa- sequence

• Correct folding is needed for the protein to achieve proper functional properties

• “Chaperone-proteins” help in folding process

•The function of a protein can be predicted from it’s structure

Protein folding

Protein folding…

Expression control

• The action of a cell is dependent on it’s proteins

• Amount of the proteins are determined by:

1. Concentration of the RNA 2. Frequency at which the RNA in translated to

the protein3. Stability of the protein

• Only a small portion of the genes in a cell are expressed

Depends on the cell type, developmental stage, environmental factors…

• Regulation at every stage of gene expression

• Control of the transcription initiation is the most important

• Different kind of control elements are found

• In eukaryotes, control elements are found both inside and outside the gene area

• Most important control element is the promoter

Initiation factors regocnize Initiation place Directs binding of the enzymes needed to produce RNA

Control of the initiation of transcription

• 5’ regulatory sequences control the site of transcription initiation The promoter

• RNA polymerase can`t recognise transcription start sites

• Start sites are positioned 25 bp to 3’ direction from a nucleotide sequence motif called the TATA BOX

• General transcription factors guide RNA polymerase to the start site

TFIID-protein binds to TATA BOX Directs the binding of the RNA

polymerase

• Other transcription factors are also needed TFIIA, TFIIB, TFIIE and TFIIH bind close to the start site

• Some transcription factors bind to the RNA polymerase • Critical properties are brought by transcription factor

needed for example to unwind the DNA

• Also enhancer are needed for activation of transcription Are found through out the genome

Binding sites for activators

Studying the Gene Expression

• All the proteins in a living cell form a complicate, but functional system

• Before “one gene – one experiment” –approach

• DNA micro arrays Arrays give possibility to monitor the activities of thousands of genes simultaneously

• Monitoring the gene expression is central for a wide variety of biological research projects

DNA micro arrays:

• You compare the gene expression of two or more sample groups

• An array provides a medium for matching known and unknown DNA samples Based on complementary pairing!

Phases of the Micro array experiment:

1. The study is designed carefully

2. Appropriate gene chip for the study is chosen

3. Chips are fabricated/ordered

Making of a DNA chip:Probes, with known identities are attached to the base.Probes are either cDNA probes or oligonucleotide probes.Probes are assortment of the genes of the study’s subject.

4. RNA is isolated from the samples and converted to cDNA

5. Samples are labelled with marker chemical

6. Sample cDNAs are let to form base pairs with probes (complementary paring)

7. The amount of the labelled sample associated with each probe is monitored

With this data it is possible to calculate the amount of each mRNA species represented in the sample

Because the identities of the probes are known, identification of the sample RNAs is possible

Micro array analysis give a huge amount of data, so the helpof the computer experts is really needed!

Sample cells

Total RNA

Samples are labelled with different fluorescence dues

Samples are combinedand added to the chip

• If the sample contains cDNA with the complementary sequence to the probe in a spot, the sample cDNA will attach to it

•After the hybridization array is scanned to find out which probes have bound cDNA the amount of the sample is measured

• The amount of the bound cDNA is due to the intensity of the fluorescence label attached to the probe and scanned

• different fluorescence colours were used in the labelling the samples

Samples can be separated from the plate

• Usually green and red colour are used

• The end product of an array is a scanned plate with colourful spots

Interpretation of the results:

•If the sample nro1 was labelled with green dye and the sample nro2 with red dye…

1. A Red spot means that the sample nro2 has more cDNA of the gene represented in this spot compared to nro1

2. A Green spot means that the nro 1 has more cDNA of the gene represented in this spot compared to nro2

3. A yellow spot means that the both samples have the same amount of the cDNA of the gene represented in this spot (red + green Yellow)

• The intensities got from the array image are quantified by measuring the average intensity of the spots

• The ratio of the florescent intensities for a spot is interpreted as the ratio of concentrations for it’s corresponding mRNA in the two samples

• Interpreting the array data is challenging Fluorescence noise Non specific hybridization Changes in the detection efficiency Errors in the laboratory work

• A lot of interesting can be obtained quite ”easily”…