MGC - Genes

What indicates a promoter region in a DNA sequence?

TATA box and -35 element

Where is the TATA box located?

Position -10 (e.g. 10 bases upstream of the start of RNA sequence)

What position does the RNA start being made from?

\+1

What is the sequence in a TATA box?

TATAAT

What is the sequence in the -35 element?

TTGACA

Where are the STOP signal sequences contained?

Terminator region

How do differentiated cells vary in terms of RNA production?

• Synthesise different proteins

• Transcribe only required genes

• Different patterns of gene expression

What is the RNA ‘life cycle’ after transcription?

1. RNA processing

2. Nuclear export

3. Protein synthesis

4. RNA degradation

What 3 things happen during RNA processing?

Capping, polyadenylation, splicing

What is the purpose of RNA capping?

(Me)Gppp at 5’ end. Protection and label for beginning

What is RNA polyadenylation?

Poly(A) tail → AAAAA… at 3’ end. Protection, increased stability and marker of RNA age

What is the RNA cap?

(Me)Gppp at 5’ end (7-Methyl guanosine)

Which enzyme is responsible for polyadenylation of RNA?

Poly(A) polymerase

What is a non-coding region of DNA called?

Intron

What is a coding region of DNA called?

Exon

What happens during RNA splicing?

Introns are cut out and Exons joined together

What causes RNA to be degraded?

Poly(A) tail is shortened until an enzyme can cut off the cap

What degrades RNA?

Ribonucleases (RNases)

What do start codons mark?

Start of an Open Reading Frame (ORF)

What do all proteins begin with?

Methionine

Where is the start codon normally located?

First AUG downstream of mRNA cap and the Shine-Dalgarno sequence (AGGAGG)

Where is the STOP codon located on mRNA?

First stop IN FRAME signals end of ORF

What is an Indel mutation?

Insertions and deletions of one or more nucleotides

What is a nonsense mutation?

Base substitution leading to a STOP codon

What is a missense mutation?

Base substitution leading to a different amino acid

What can an Indel mutation lead to?

Frameshift mutation

What human beta-globin genetic mutations lead to anaemia?

Haemoglobinopathies

What is thalassaemia?

Imbalances in alpha and beta haemoglobin subunit ratios

What causes sickle cell anaemia?

Haemoglobin protein variants

What is the large subunit of the ribosome made up of?

49 proteins and 3 rRNAs

What is the small subunit of the ribosome made up of?

33 proteins and 1 rRNA

What end of tRNA attaches to the amino acid?

3’

How are tRNAs specific to different amino acids?

Anticodon complementary to mRNA sequence

How are amino acids attached to tRNA?

Ester bond

Which enzyme attaches the amino acid to the tRNA?

Aminoacyl tRNA synthetases specific to each tRNA/amino acid combo

How many tRNAs can bind to a ribosome at once?

3 (3 docking sites, E, P and A)

Where does the aminoacyl-tRNA enter the ribosome?

A site

Where does the tRNA leave the ribosome?

E site (for ‘empty’ tRNA)

What tRNA docking site is the amino acid attached to the chain in?

P

What enzyme synthesises the new peptide bond in the ribosome?

Peptidyl transferase

What attaches to the STOP sequence of mRNA in the ribosome?

Release factor

How is DNA isolated?

• Cell lysing (SDS)

• Removal of other cell components (purification)

• Precipitating DNA

How can DNA be precipitated?

Lowering solvation (adding ethanol or salt)

How do ethanol and/or salt lower solvation?

Remove the water molecules associated with the DNA

What wavelength do bases absorb radiation at?

260nm (UV)

How can the H bonds between bases be broken without disrupting the backbone?

Heating, raising pH, chaotropic agents

What is a chaotropic agent?

Very strong H bonds (e.g. formamide)

How can reannealing be prevented?

Ice bath

What is the hyperchromic effect?

Double stranded DNA absorbs less light than single stranded

What causes the hyperchromic effect?

Stacked bases in double stranded absorb less light

What is TM?

Temperature at which 50% of DNA is denatured

What affects TM?

GC bonds require more energy to denature than AT

What can a TM measurement be used for?

Characterising GC content

What dyes can be used to see DNA on a gel electrophoresis plate?

Ethidium bromide, SYBR green or gold

How do dyes allow DNA to be seen on a gel electrophoresis plate?

Intercalates between the stacked bases

What does oligo- mean?

Few

What is a ‘hot’ copy?

Radioactive

What can hot DNA copies be used for?

Autoradiography

What can an autoradiograph of an agarose gel be used for?

Identification of very small amounts of DNA (e.g. forensics)

What is the upper size limit of gel electrophoresis?

20,000 base pairs

How is DNA cut into fragments for gel electrophoresis?

Restriction endonucleases

Where do restriction endonucleases cut?

Palindromic recognition sites

How can specific fragments (e.g. genes) be identified in a mixture>

• Probes complementary to the fragment (Southern blotting)

• Selective amplification of the fragment (PCR)

What is the process of Southern blotting?

• Run sample on gel

• Denature DNA (alkali)

• Transfer to membrane

• Add probe

• Wash

• Expose to a film

• Develop film

What are the basic steps of PCR (3)?

• Heat to denature 94C

• Cool to anneal primers 55C

• Polymerisation 72C

What is needed when running gel electrophoresis of RNA due to it being single stranded?

Formaldehyde (denatures any tertiary structure)

How is Northern blotting different to Southern?

Uses RNA

What enzymes are needed to make DNA from RNA?

Reverse transcriptases from viruses

What is formed when making DNA from RNA?

Complementary DNA (cDNA)

What is the sense strand in DNA?

Top, copying strand

What is the antisense strand in DNA?

Bottom, non-coding, template strand

Which DNA strand ‘becomes’ the copy?

Sense strand. RNA is made based on the antisense strand so sense strand is the same as mRNA except for T/U swap.

How many base pairs are there in a transcription bubble?

17

Is transcription or DNA replication quicker?

DNA replication

What is an ‘activated precursor’ needed for RNA polymerase to work?

Nucleoside triphosphates

What is the error rate of RNA synthesis?

1 in 10^4 (no proof reading)

How are sugars joined in DNA?

5’ 3’ phosphodiester linkage

Which end of the DNA chain is the start?

5’

What rule dictates A-T and G-C with them in a 1:1 ratio to each other?

Chargraff

What is the percentage of G (and so C) in human DNA?

20

What is the percentage of A (and so T) in human DNA?

30

What ensures damage to a DNA strand does not lead to information loss?

Redundancy

What increases the stability of the double helix?

• H bonds

• Hydrophobic effect (no water in centre)

• vdW forces

• Hydrophilic interactions b/n phosphate and water

How many chromosomes in human DNA?

23

How is the DNA sequence usually written?

Top strand only, 5’ to 3’

How is uracil different to thymine?

Missing a methyl group

Who demonstrated semi-conservative replication?

Meselson and Stahl

What forms when DNA replication begins?

Bubble

What are the end points of the DNA replication bubble called?

Replication fork

What marks a DNA origin of replication in the amino acid sequence?

Enrichment of T and A (easier to break 2 H bonds)

How is the process of DNA replication sped up?

Multiple replication origins

Which DNA polymerase is the major replication enzyme?

DNA polymerase III

Which end does DNA polymerase III build DNA in?

5’ → 3’ (nucleotides attached to 3’ end)

What reaction is catalysed by DNA polymerase III?

Elimination of 2 phosphates by breaking covalent bond

What is the problem with replicating the top and bottom DNA strand?

Polymerase only works 5’ → 3’. Bottom strand is opposite direction

How is the leading (top) DNA strand replicated?

Continuously

How is the lagging (bottom) DNA strand replicated?

Discontinuously

How is the lagging strand replicated?

Okazaki fragments formed and joined by DNA ligase

Why can’t polymerase III begin replication alone?

Cannot initiate new strand synthesis

How is DNA replication started?

RNA primer formed (10 bases long)