Genes(GP)

Question 1

Where is the glycosidic bond found?

Answer 1

The covalent bond attaching the deoxyribose/ribose sugar to the base

Question 2

Which bases are complementary to each other and what class of molecule is each pair?

Answer 2

Adenine and Guanine - Purines
Cytosine and Thymine - Pyrimidines

Question 3

What type of bond are nucleic acids joined by and what is the backbone called? Why is the backbone called this? What does it look like roughly?

Answer 3

Phosphodiester linkage
Hydrophilic pentose phosphate backbone - hydrophilic as it is negatively charged, and so likes water
P with 2 negative Os and then 2 other Os, one attached to each nucleotide (see summary sheet for diagram)

Question 4

What is the 3D structure of DNA and what 4 interactions stabilise this?

Answer 4

Antiparallel, right-handed double helix

Stabilised by:
- Hydrogen bonds between base pairs
- Hydrophobic affect
- Van der Waals between adjacent bases
- Hydrophilic interactions of polar phosphate groups (phosphate backbone) with the outside aqueous environment

Question 5

Which form of DNA is usually seen in nature?

Answer 5

B form - right handed - bases on inside, backbone on outside

Question 6

Why is RNA less stable than DNA, why is this NOT an issue, and how can RNA form double helices?

Answer 6

Less stable than DNA due to having an OH in the ribose sugar instead of the H. OH groups of adjacent ribose sugars will repulse each other slightly, making the RNA weaker

Doesn’t need to be as stable as it is just an intermediate for DNA to form it’s protein

RNA can form double helices by folding over itself via intramolecular interactions

Question 7

What is DNA replication in simple terms and how is it semiconservative and bidirectional?

Answer 7

Replication = copying DNA, where each DNA strand acts as the template for the new strands to synthesise

Semiconservative = Each new (daughter) DNA helix has one old and one new chain

Bidirectional = starts replication at a specific site in the DNA and replicates in both directions away from that site

Question 8

Which pair of bases are weaker and why?

Answer 8

A-T is weaker than G-C, as A-T only has 2 hydrogen bonds, whereas G-C has 3 hydrogen bonds

Question 9

What does dNTP stand for?

Answer 9

The nucleotide triphosphates are referred to as dNTP, where N is any base (A,C, G, or T), d is deoxy and TP is triphosphate

e.g. dATP = deoxyadenosine triphosphate

Question 10

What 2 things does DNA polymerase require to start synthesising the new DNA strand and in what direction does it synthesise the new strand?

Answer 10

• Template strand to replicate the new strand using complementary base pairing
• The 4 dNTPs (dATP, dTTP, dCTP, dGTP)

Synthesises DNA in a 5 prime to 3 prime direction by adding nucleotides onto the 3 prime end of the new, growing strand

Question 11

What is a pyrophosphate group?

Answer 11

The 2 phosphates that are released once the dNTP nucleotide has attached to the end of the strand (only one phosphate is kept for the backbone)

Question 12

What are the 2 problems and solutions associated with replication?

Answer 12

Problem 1 - Synthesising a new strand only works in the 5 prime to 3 prime direction, so only works for one side of the replication fork

Solution = The lower strand is synthesised as a lagging strand (Replicated discontinuously and backwards). The gaps of the lagging strand are sealed by DNA ligase

Problem 2 - DNA polymerase can only extend an existing strand, can’t make a strand from scratch

Solution = RNA polymerase can make a new strand, so makes an RNA primer first, from which DNA polymerase can extend from this short RNA chain

Question 13

What does DNA polymerase 1 do (in terms of the primer)?

Answer 13

Deletes the RNA Primer fragment from the newly synthesised DNA strand.
(Has 5 prime to 3 prime exonuclease activity)

Question 14

What is the evidence for the lagging strand in DNA replication?

Answer 14

Prescence of short DNA fragments in DNA extracted from actively growing cells. Short fragments were not found after DNA synthesis was stopped by inhibitory drugs, suggests that they are apart of DNA synthesis

Question 15

What are nucleases and where do they target exactly?

Answer 15

Class of enzymes that digest DNA/RNA by hydrolysing phosphodiester bonds

Question 16

What are the 3 types of nucleases?

Answer 16

• 5 prime to 3 prime exonuclease (digests DNA from end of chain in the forward direction)
• 3 prime to 5 prime exonuclease (Cleaves DNA from end of chain in the backwards direction)
• Endonuclease - cleaves in the middle of the chain to form 2 smaller chains

Question 17

What are the 3 other enzymes (not DNA ligase or polymerase) needed during DNA replication?

Answer 17

• Helicase = unwinds the 2 original strands
• SSBPs = protects the single stranded regions of DNA
• Topoisomerase = relieves tension in overwound regions of DNA by making temporary cuts

Question 18

How are errors in replication spotted and corrected? (2)

Answer 18

• DNA polymerase proofreads its own work in the 3 prime to 5 prime direction (goes backwards to check what its done is correct, and if it isn’t, then it fixes it’s own mistake)
• Any errors that get through are corrected after replication via enzymes that scan DNA for mismatches and corrects them

Question 19

What 2 things are mutations caused by and what are the 3 effects mutations have?

Answer 19

Causes:
- Mistakes during replication that are unspotted
- DNA damage - chemical changes to DNA structure that occur in everyday life e.g. UV light, free radicals etc.

Effects:
- Cancer
- Genetic diseases
- Genetic variation, evolution

Question 20

What is an example of a disease caused when DNA damage repair goes wrong?

Answer 20

Werner’s syndrome
Defect in one of the many DNA repair enzymes
Causes premature ageing and cancers

Question 21

What is an example of DNA damage and the enzyme that is used to detect and repair this damage?

Answer 21

Spontaneous Deamination (removal of amine group), leads C changing to the base U. After translation this base will then become T, so permanent mutation of C to T.
The enzymes Uracil-DNA glycosidase detects the alien U in the DNA and removes it + replaces it with a new cytosol before DNA replication occurs

Question 22

Why is the intermediate RNA (mRNA) step needed? (4)

Answer 22

• Can make multiple copies of mRNA and proteins from one copy of DNA (one gene) (amplifies the DNA)
• DNA is stored in the nucleus, but protein synthesis occurs in the cytoplasm, so need a transporter (mRNA) to transport the info to the ribosome
• Can control the “working copy” (RNA), without compromising the “master copy” (DNA) (original copy is still there in case anything goes wrong
• RNA was the original genetic material (Evolutionary remnant)

Question 23

What is the key enzyme of transcription and what 2 things does it require?

Answer 23

RNA polymerase (II)
Requires one DNA template strand and RNA nucleotides (ATP, GTP, UTP, CTP - nucleoside triphosphates (no d, as not deoxy))

Question 24

In what 4 ways does RNA polymerase differ to DNA polymerase? (How is the formation of the new strand in transcription different to translation)

Answer 24

• Doesn’t require a primer
• No proof-reading activity
• new strand doesn’t remain hydrogen bonded to the DNA template strand
• Not all of the DNA is transcribed - only genes (protein coding sequences) are transcribed

Question 25

What is the sigma factor of RNA polymerase and what are the start + stop signals for RNA polymerase on the gene?

Answer 25

Sigma factor = part of RNA polymerase that recognises the start site in DNA for transcription. It is released once transcription starts, and rebinds to RNA polymerase after it's past the stop site. Once the stop site is completed, the mRNA chain is also released Start site = promoter region, contains start signals for RNA polymerase Terminator region = contains stop signal sequences at the stop site to get RNA polymerase ready to stop transcribing and detach from the gene

Question 26

What are the 3 modifications made to mRNA after transcription has taken place, and describe what each of them?

Answer 26

Capping = Molecule at the 5 prime end of the chain, which doesn't contain nucleotides, to stop exonucleases chewing off the end of the strand and acts as a label for the beginning of protein synthesis. (e.g. MeGppp) Polyadenylation = poly(A) polymerase adds on 100s of As to the 3 prime end of the chain to form a poly(A) tail. Protects the 3 primes end from extra nucleotides being added on and stabilises the mRNA Splicing = introns (non-coding regions of DNA) are cut out and the exons (coding regions) are spliced together to make the mature mRNA transcript

Question 27

How is the mRNA destroyed after it has been used in protein synthesis?

Answer 27

Exonucleases start chewing off the poly(A) tail and it gets shorter. The shortened poly (A) tail allows another exonuclease to chop off the cap after translation. The RNA left is then degraded by ribonucleases called RNases (endonucleases). RNA degradation controls the amount of protein produced (protein synthesis continues until mRNA is destroyed)

Question 28

What are the 3 possible reading frames and how can we tell which one is used for a particular Open Reading Frame (ORF)?

Answer 28

Start reading from the first, second, or third base First codon is identified (start codon) to mark the beginning of the ORF. Start codon = AUG or ATG, which is the code for met, so all proteins start with a Met

Question 29

How can we tell which codon is the start codon? How can we tell which codon is the stop codon?

Answer 29

Start codon is always the first AUG downstream from the shine-Dalgarno sequence - AGGAGG. The shine-Dalgarno sequence is present in the mRNA, but not in the final protein, as it is part of the mom-coding sequence (see diagram on summary) Stop codon = the first IN FRAME stop codon (In frame = the bases are lined up with the amino acids) (see diagram). Stop codons = UAA, UGA, UAG

Question 30

What are the 2 main types of substitution and what are the affects of each of them? (3 +1)

Answer 30

Substitution (point mutations) - one base substituted for another Indels - Insertions/deletions of one or more nucleotides Affects of substitution: - Missense = one amino acid is swapped for another - Nonsense = an amino acid is changed to a stop codon - shorter protein is produced - Silent = amino acid doesn't change (lucky, new codon codes for same amino acid as old one) Affects of indels = frameshift, all amino acids after the mutation are read wrong

Question 31

What molecule carries out translation, what do tRNA molecules contain, and how are amino acids attached onto tRNA molecules?

Answer 31

Ribosomes Amino acid on the 3 prime end Anticodon at the bottom which is complementary to the mRNA codon The anticodon is also complementary for the amino acid attached Amino acids attach via their specific enzyme - e.g. tryptophan is attached onto it's tRNA molecule via tryptophanyl tRNA synthetase

Question 32

What happens when a stop codon is reached in translation?

Answer 32

Release factor binds to site A in the ribosome. A water molecules then attaches to the polypeptide chain to form the COOH end of the polypeptide, and the release factor and last tRNA molecule then leave the ribosome.

Question 33

What are the 3 stages of isolating nucleic acids (DNA) from cells, and how do these things happen?

Answer 33

1. Lyse the cell (break the cell apart) with detergents like SDS 2. Remove other cell components (e.g. ribosomes) via purification 3. Precipitate the DNA from the solution Precipitation - DNA is surrounded by water molecules, which keeps it dissolved in solution. The DNA can be precipitated by lowering the solvation (adding ethanol and salt). The salt will convert into ion, which are negatively charged and so will attract the water molecules surrounding the DNA, pulling them away from the DNA, precipitating the DNA out of solution.

Question 34

What wavelength of light does DNA absorb? How can we tell if the DNA is denatured or not using this? (hyperchromic effect) What is the Tm (melting temp) and what does the melting temp of DNA depend on?

Answer 34

260nm (UV) Single stranded DNA absorbs more UV light at 260nm than double stranded DNA, so if the absorbance of DNA increases, this shows that the double stranded DNA has been denatured into single strands of DNA Tm = temp at which half of the DNA is denatured Tm depends on the base composition of DNA -> G-C has 3 hydrogen bonds, A-T has 2 hydrogen bonds, so more GC bases in the DNA = higher Tm

Question 35

How can gel electrophoresis be used to visualise DNA fragments (nucleotides)?

Answer 35

Bind fluorescent dyes to the DNA, before undergoing gel electrophoresis. This allows the positions of DNA fragments (nucleotides) to be visualised under a UV light Restriction endonucleases are used to cut the DNA into smaller DNA fragments, that are more manageable for using in gel electrophoresis. The sizes (base pairs long) of the fragments are characteristic for the particular DNA molecule Fragments show up as a bar code pattern.

Question 36

How is a specific fragment identified in the mixture after gel electrophoresis has been used? (2 steps)

Answer 36

Southern blotting - complementary probes are used to attach onto the fragment of DNA we are looking for and help identify it PCR - selectively amplifying the desired fragment, amplified fragment will be brighter than all the other fragments, and so will be easily viewed on the gel PCR steps: - DNA heated to separate strands - DNA cooled to allow primers to bind to target sequences of DNA - DNA heated slightly and heat tolerant DNA polymerase is used to replicate the target sequence of DNA Results in double the amount of DNA being produced with each cycle

Question 37

What is multiplex PCR?

Answer 37

2 different primer pairs amplify 2 genes at the same time

Question 38

How is RNA visualised? How can RNA be copied into DNA to allow DNA techniques to be used?

Answer 38

Can use gel electrophoresis, but need to use formaldehyde to denature any tertiary structure (folding over itself) Northern blotting is used - same as southern blotting, but for RNA Can use RNA-dependant DNA polymerase from RNA viruses to make a "complementary DNA" copy (cDNA)