Genes(GP) Flashcards
Where is the glycosidic bond found?
The covalent bond attaching the deoxyribose/ribose sugar to the base
Which bases are complementary to each other and what class of molecule is each pair?
Adenine and Guanine - Purines
Cytosine and Thymine - Pyrimidines
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?
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)
What is the 3D structure of DNA and what 4 interactions stabilise this?
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
Which form of DNA is usually seen in nature?
B form - right handed - bases on inside, backbone on outside
Why is RNA less stable than DNA, why is this NOT an issue, and how can RNA form double helices?
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
What is DNA replication in simple terms and how is it semiconservative and bidirectional?
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
Which pair of bases are weaker and why?
A-T is weaker than G-C, as A-T only has 2 hydrogen bonds, whereas G-C has 3 hydrogen bonds
What does dNTP stand for?
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
What 2 things does DNA polymerase require to start synthesising the new DNA strand and in what direction does it synthesise the new strand?
- 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
What is a pyrophosphate group?
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)
What are the 2 problems and solutions associated with replication?
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
What does DNA polymerase 1 do (in terms of the primer)?
Deletes the RNA Primer fragment from the newly synthesised DNA strand.
(Has 5 prime to 3 prime exonuclease activity)
What is the evidence for the lagging strand in DNA replication?
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
What are nucleases and where do they target exactly?
Class of enzymes that digest DNA/RNA by hydrolysing phosphodiester bonds
What are the 3 types of nucleases?
- 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
What are the 3 other enzymes (not DNA ligase or polymerase) needed during DNA replication?
- 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
How are errors in replication spotted and corrected? (2)
- 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
What 2 things are mutations caused by and what are the 3 effects mutations have?
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
What is an example of a disease caused when DNA damage repair goes wrong?
Werner’s syndrome
Defect in one of the many DNA repair enzymes
Causes premature ageing and cancers
What is an example of DNA damage and the enzyme that is used to detect and repair this damage?
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
Why is the intermediate RNA (mRNA) step needed? (4)
- 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)
What is the key enzyme of transcription and what 2 things does it require?
RNA polymerase (II)
Requires one DNA template strand and RNA nucleotides (ATP, GTP, UTP, CTP - nucleoside triphosphates (no d, as not deoxy))
In what 4 ways does RNA polymerase differ to DNA polymerase? (How is the formation of the new strand in transcription different to translation)
- 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
What is the sigma factor of RNA polymerase and what are the start + stop signals for RNA polymerase on the gene?
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
What are the 3 modifications made to mRNA after transcription has taken place, and describe what each of them?
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
How is the mRNA destroyed after it has been used in protein synthesis?
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)
What are the 3 possible reading frames and how can we tell which one is used for a particular Open Reading Frame (ORF)?
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
How can we tell which codon is the start codon?
How can we tell which codon is the stop codon?
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
What are the 2 main types of substitution and what are the affects of each of them? (3 +1)
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
What molecule carries out translation, what do tRNA molecules contain, and how are amino acids attached onto tRNA molecules?
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
What happens when a stop codon is reached in translation?
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.
What are the 3 stages of isolating nucleic acids (DNA) from cells, and how do these things happen?
- Lyse the cell (break the cell apart) with detergents like SDS
- Remove other cell components (e.g. ribosomes) via purification
- 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.
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?
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
How can gel electrophoresis be used to visualise DNA fragments (nucleotides)?
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.
How is a specific fragment identified in the mixture after gel electrophoresis has been used? (2 steps)
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
What is multiplex PCR?
2 different primer pairs amplify 2 genes at the same time
How is RNA visualised?
How can RNA be copied into DNA to allow DNA techniques to be used?
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)
