Lectures 5-13 Flashcards by Iqraa Sajjad

Components of all human genes

DNA
Promoter
Transcription start + stop signal
Exons + introns
Upstream regulatory regions

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What are transcription factors (activators)?

Proteins that regulate the transcription of genes

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How is a gene ‘switched on’?

Binding of transcription factors in and around promoter region of gene

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What is RNA polymerase II?

Enzyme involved in transcription. Synthesises complementary strand of RNA

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In regards to 3’ + 5’, which way does RNA polymerase build a complementary RNA strand?

5’ —> 3’

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What are upstream enhancer sequences?

Short regions on distal end of DNA where activators (proteins) bind to increase the likelihood of transcription

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What is RNA polymerase I responsible for?

The production of large ribosomal RNA

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What is RNA polymerase II responsible for?

The production of mRNA

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What is RNA polymerase III responsible for?

The production of tRNA and the small ribosomal RNA molecules

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What is the ‘promoter’ of a gene?

Region of the gene that initiates transcription

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How does RNA polymerase initiate transcription?

1) Collides with DNA in nucleus + binds to promoter
2) Opens up section of DNA helix + exposes DNA on each strand
3) One strand = template

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What is a transcriptional repressor?

Blocks the attachment of RNA polymerase and the promoter OR block function of activators

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What is the Wilm’s tumour protein?

Transcriptional repressor

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How does the Wilm’s tumour protein work?

Binds to promoter region of EGR-1 gene (transcriptional activator) in developing kidney + so gene is not expressed

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What would happen if the gene coding for Wilm’s tumour protein was mutated?

Uncontrolled expression of EGR-1 –> leads to kidney tumours in early life

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What is the process of 5’ capping?

1) RNA triphosphatase hydrolyses 5’ triphosphate group to make diphosphate-RNA
2) Addition of GMP by guanylyl transferase  produces guanosine cap
3) Addition of methyl group to guanosine cap by methyl transferase

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What is the poly-A tail?

Long chain of adenine nucleotides

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What is the process of polyadenylation?

1) 3’ end cleaved to free a 3’ hydroxyl molecule

2) Poly-A polymerase adds poly-A tail to RNA molecule

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What is RNA splicing?

Removal of introns on pre-mRNA molecule to make m-RNA

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How many amino acids in total?

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What are the START codons?

AUG

GUG

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What are the STOP codons?

UAA
UAG
UGA

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What is translation and what are the 3 steps?

Process in which mRNA is decoded in ribosome to produce specific amino acid and folds into active protein

initiation
elongation
termination

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What is the process of translation initiation?

1) Initiator tRNA carrying MET associates with ribosome
2) Ribosome recognises 5’ end of mRNA + scans mRNA for start codon
3) MET binds to mRNA

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What is in association with ribosome to mediate binding of tRNA to it?

eIF2

Which 2 initiation factors is mRNA capped with for ribosome to recognise?

eIF4G | eIF4E

What is the process of translation elongation?

1) tRNA brought into ribosome A site + matched with codon 2) Hydrolysis occurs to fix it in place 3) Peptidyltransferase - bond between peptide (AA) + tRNA broken in P site + bond made between peptide + new amino acid (the AA in the A site) 4) Used tRNA ejected fro E site

What is the process of translation termination?

1) Ribosome comes across stop codon 2) Cytoplasmic release factors bind to stop codon 3) Carboxyl end of polypeptide chain = freed

What are gene isoforms?

mRNAs produced from same locus (on chromosome) but have different transcription start sites/coding DNA sequences etc.

What are the components of DNA?

- Nitrogenous base - Pentose sugar (deoxyribose) - Phosphate group

What is the bond between the nitrogenous base + sugar?

Glycosidic bond

What is the DNA structure of adenosine?

Nucleoside (sugar + base)

What is the DNA structure of AMP?

Nucleoside monophosphate

What is the DNA structure of ADP?

Nucleoside diphosphate

What is the DNA structure of ATP?

Nucleoside triphosphate

Which 2 base pairs have the strongest interaction?

C-G | --> 3 hydrogen bonds between them

Are nitrogenous bases hydrophobic or hydrophilic?

Hydrophobic

Is the sugar-phosphate backbone of DNA hydrophobic or hydrophilic?

Hydrophilic

What interactions do the stacked bases of the DNA helix have?

Van der Waals forces (electrostatic interactions)

In regards to 3' + 5', which end are nucleotides added to a polynucleotide chain in DNA?

3' end | Makes 3'-5' phosphodiester bond

In which way does the polynucleotide chain grow in DNA?

5' --> 3' 5' phosphate --> 3' OH

In which stage of the cell cycle does DNA replication take place?

S phase of INTERPHASE

What enzyme is used to break the base pairs in DNA?

DNA helicase

What is the leading strand?

A strand orientated 3' to 5'

What is the lagging strand?

A strand orientated 5' to 3'

What is the replication starting point in DNA replication of the leading strand?

Primer

What enzyme generates primers?

DNA primase

What is DNA polymerase a used for? (leading strand)

DNA replication | - binds to strand at primer + adds new complementary base pairs

Which enzyme takes over from DNA polymerase a and when? (leading strand)

DNA polymerase e | At approx. 20 base pairs

Which enzymes prevent the incorporation of incorrect nucleotides in DNA replication and how?

DNA polymerase a, e + delta | - proofreading 3' --> 5' exonuclease activity

What is the difference in regards to primers, in DNA replication of the lagging strand + leading strand?

Leading strand = one primer | Lagging strand = multiple RNA primers

What is DNA polymerase gamma used for? (lagging strand)

Generates complimentary DNA to the strand between RNA primers

What is significant about the replication of the lagging strand? (Okazaki fragments)

Replication is discontinuous as fragments between the RNA primers are not joined

What is the shared function of RNAse H and DNA pol gamma?

Degrade + fill RNA primers

What is the function of DNA ligase?

Joins any breaks in the leading and lagging strands to ensure DNA = continuous

What is a genome?

Total DNA in a cell

Do H3/H4 tetramers remain intact after synthesis?

Yes

What is epigenetics?

Heritable changes in phenotype/cell behaviour or gene expression in cells caused by changed other than in the DNA base sequence that control activity of genes

What is the function of epigenetic modifications?

To alter chromatic structure to control accessibility of transcription factors for gene transcription

What is PCR?

Polymerase chain reaction | - mimics DNA replication in vitro using DNA primers

How can exons provide variety in the proteins that are made?

Alternative splicing | - exons joined together differently during splicing

What can be the result of errors in gene expression?

- Uncommon disorders e.g. cystic fibrosis, muscular dystrophy/atrophy etc. - Can cause cancers

What gene error causes Duchene Muscular Dystrophy?

Mutation in DMD gene

What gene error causes Spinal Muscular Atrophy?

Mutation in SMN1 gene

What gene error causes Cystic Fibrosis?

Mutation in CFTR gene | OR mutation in exon 7 of SMN1 gene (exon 7 - skipped + protein is non-functional)

What gene error causes Pulmonary Arterial Hypertension?

Mutation in BMPR2 gene

What gene error causes breast cancer?

Mutation in BRCA1 / BRCA2 genes

What is haploinsufficiency?

When one copy of the gene is not enough

What is dominant mutation?

When a change in the gene sequence generates change in the protein that has dominance over the wild-type

How does gel electrophoresis work?

- Agarose gel mixed with buffer - Melted down - DNA loaded onto well in gel tank - DNA migrates from negative --> positive

Why does DNA migrate from negative electrode to positive electrode in gel electrophoresis?

DNA is negatively charged

Which technique is used to detect proteins?

Western blot

What are the steps in Western blotting?

1) Sample prep 2) Gel electrophoresis (different type) 3) Blotting 4) Blocking 5) Antibody probing 6) Detection

What is the Polymerase Chain Reaction?

Used to make multiple copies of segment of DNA | - large amount of copies from small sample

What are the 3 main steps of the PCR?

1) Denaturation 2) Annealing 3) Extension