Other Senester One

Question 1

DNA replication

Nucleotide =

Answer 1

• semi conservative
• 5’ to 3’
• antiparallel
• irreversible because of the breakdown of ppi

Deoxy nucleotide triphosphate

Question 2

Coupled reaction of DNA replication

Answer 2

Addition of dNTP to the strand

Breakdown of two phosphates

dNTP + strand = strand + dNMP + 2Pi

Question 3

Leading and lagging strand

Answer 3

Leading strand is continuous
Lagging is discontinuous

Okazaki fragments because it is made in the opposite direction to DNA helicase movement

3’ to 5’

Question 4

Primer for DNA synthesis

Answer 4

Extension of a short RNA primer

The primer is made by DNA primase and onto the template.

Question 5

Fragments

Answer 5

• all the fragments are made using primers
• DNA polymerase adds to the primers and makes the fragments
• the primer is erased by ribonuclease H
• it is replaced by DNA
• DNA Ligase seals the fragments by creating a covalent bond

Question 6

DNA ligase

Answer 6

• uses ATP to work
• two step catalytic reaction

P-P is broken and released creating E by pyrophosphatase

The AP remaining from ATP adds to the 5’ phosphate on the fragment making APP.
The OH on the 3’ attacks the APP and releases AP to leave the most favourable P-P backbone.

Question 7

DNA helicase

Answer 7

Used ATP to separate parental DNA strands at the replication fork

Wraps itself around one of the strands of the fork and spins around creating rotational force that is converted to foreword motion using ATP.

Question 8

DNA helicase mutations

Answer 8

Werner’s syndrome- premature ageing progeria
Autosomal recessive in RECQ helicase gene WRN

incomplete DNA replication because of no DNA helicase.

Bloom syndrome- rare cancer
Loss of function mutation in RECQ family DNA helicase which maintains genome integrity

Question 9

DNA polymerase processivity

Sliding clamp

Answer 9

Enhanced by sliding clamp

Once a DNA polymerase has begun to add a primer it is likely to continue to the end.

Because of the ATP sliding clamp that fixes the enzyme to the primer template junction

A clamp loader protein attaches the clamp to the primer template junction. The clamp loader dissociates before DNA polymerase begins.

Question 10

SSBPs

Answer 10

Single stranded binding proteins

Make single stranded DNA more available by keeping it straight

Stop folding on itself and binding incorrectly.

If DNA helicase is fast The single strands will be very long and more SSBPs are required

Question 11

What is highly conserved between E. coli and humans

Answer 11

Key components of the replication fork

Found in all organisms.

Question 12

Replication origin

Why only one replication per cycle

Answer 12

Yeast - autonomously replicating sequences ARS. best understanding

Rep origin is where DNA replication is initiated.

Humans- LMNB2. MYC. HBB. genes have been found to be origins of rep.

Only one replication per cycle and the selection of the rep origin is done in G1

The activation of the origin is in S phase so there can only be one per cycle.

Question 13

How replication origin is activated

Answer 13

The origin recognition complex binds to the replication origin

It recruits helicase loading proteins cdc6 and cdt1 which bind to the origin recognition complex

Helicase mcm27 binds and completes the formation of the pre replicative complex

The replication origin has been chosen now in G1.

Question 14

How is the origin of replication activated

Answer 14

In S phase.

High levels of cyclin dependant kinase activates the pre replicative complex and stops formation of new pre replicative complexes

Low levels of cyclin dependant kinase allows for pre replicative complex development.

Making sure the chromosomes are replicated once.

Question 15

Finishing DNA replication with the last primer

Answer 15

One primer left at the end and when it is removed it will leave a gap.
Every round of replication would have a shortening of the strand.

To stop this telomerase adds TTAGGG repeats to the end of the sequence to let that be lost instead of coding DNA.

Question 16

Telomerase structure and shuffle

Answer 16

Ribonucleoprotein complex with an intrinsic RNA component which is a template for telomere production.

AAUCCC complementary to telomere

Has an extra AAU on the end to make an extra TTA on the telomere. This is for telomerase to bind to next time.
Moves foreword six nucleotides.

1- telomerase binds to telomere 
2-telomerase adds end three TTA 
3-moves down six
4-bind to TTA on end and make the next six 
5-move down six

Question 17

Why does DNA have a negative charge ?

Where do things bind to DNA

What is a binding site

Answer 17

Phosphate backbone and so the binding proteins will be positive due to argenine or lysine.

Major groove and form hydrogen bonds with the sequence which will hold them for a while.

A section of bases that the TF can bind to (response element)

Question 18

Transcription factors can detect interactions between two bases.

Answer 18

They look for the order of hydrogen bond acceptor and donors, hydrogen atoms and methyl groups.

Question 19

Rox1 and consensus

Answer 19

TF in yeast

Binds to eight sites across 3 genes

Three on HEM1, four on ANB1 and one on rox1

The different sites will have a different affinity for the rox1 protein.

A consensus can be used to overlap the binding site sequences and see how they are similar.

Y = C or T. H = A C or T

It can be used to find more genes that it could bind to if they also have a similar sequence

Question 20

Why isn’t TF binding needed to be strong

Answer 20

The cell doesn’t want a perfect binding site so the interaction is only transient

This is to stop over expression and allow control.

Question 21

Conservation of introns and Exons

Answer 21

Exons are very well conserved.

Introns are able to change rapidly during evolution

The introns that are conserved must be very important regulatory sequences where proteins need to bind.

Question 22

Helix turn helix DNA binding motif

Answer 22

The recognition helix inserts into the major groove to form hydrogen bonds.

The other helix stabilises the position on the backbone.

They are a dimer in a head to head format.

The site they bind to has to be palindromic.
AACAC

Question 23

Zinc finger DNA binding motif

Answer 23

Four amino acids hold a Zn atom in place

An alpha helix interacts with the major groove and recognises two bases.

There are usually many zinc fingers in a row

They use argenine and histine to interact with bases.

Question 24

Leucine zipper DNA binding motif

Answer 24

Two alpha helixes held together by hydrophobic amino acid leucine.

Chopsticks shape and it interacts with two parts of the major groove.

Homodimers - both helixes have the recognition site. And will bind to DNA that has two identical sequences after eachother.

Heterodimers - helixes have different recognition sites.

They can have complex regulation of genes due to the variety of binding sites.

Question 25

Helix loop helix

Answer 25

Modification of leucine zipper The loop allows more flexibility in the positioning of the protein. Homo or Hetero dimers. They bind as dimers to increase binding strength.

Question 26

Identifying DNA binding proteins EMSA

Answer 26

Electrophoretic mobility shift assay Radioactively label one end of a specific DNA sequence with p32 Incubate it with the cell extract or purified protein containing the binding protein. Load onto a gel for electrophoresis. Small goes furthest so the radioactivity will be detected at the bottom because DNA is small. The DNA bound by the binding protein will be larger and will get stuck earlier on. Detect the bound radioactive DNA and purify the binding proteins on it to get the protein of interest.

Question 27

Identifying DNA binding proteins DNase 1 foot-printing.

Answer 27

Radioactivity label one end of the DNA with p32 to make a probe. Mix it with the cell extract or purified protein. Add DNase enzyme which will cut the DNA into small pieces. Not too much so the pieces aren’t too small. Electrophoresis to sort the pieces by size. The DNA binding protein will protect the DNA from the enzyme. So the long pieces of DNA will have a binding protein. If no DNA is protected then there will not be any long pieces of DNA.

Question 28

Types of TF

Answer 28

Permissive- general transcription factors. Required for all transcription. TATA binding protein. Specific/regulatory- can be activators that increase transcription or repressors that stop transcription. Some can be both. They interact with the RNA polymerase. They bind to other TFs They alter acetylation They bind anywhere around the gene.

Question 29

DNA looping

Answer 29

The DNA has to be able to wrap around the protein complex to interact with all sides. Chromatin does not bend easily and the binding sites have to be 500 bases apart. Or the protein could bind to directly neighbouring DNA. if the loops get too big then it will be harder for the DNA to find the protein because it will be so far away.

Question 30

Activator binding sites... Repressor binding sites... Insulators...

Answer 30

Enhancers Silencers. Enhancers are promiscuous and will work on all genes. Insulators block enhancers from activating all genes. And only allow them to do certain ones.

Question 31

Tryptophan

Answer 31

The protein represses the gene required for synthesis. Low tryptophan means there isn’t a lot there to repress transcription. So more is made. High tryptophan means there is lots there to repress transcription. So less is made.

Question 32

How to regulate TFs functioning.

Answer 32

- regulate the synthesis of the TF - ligand binds to TF and activate or repress it. - TF can be phosphorylated and this will activate or repress it. - another subunit could bind and a/r it - phosphorylation can cause an inhibitory protein to leave. The inhibitory protein could stop the TF entering the nucleus. - TFs can be held to a membrane and have to be cleaved off.

Question 33

How TFs interact with each other.

Answer 33

Synergistically- help each other increase transcription. One TF can make 1/2 units if transcription Two TFs can make 100 units of transcription They bind to form dimers. So they are less likely to fall off the DNA. The binding of one TF to the DNA could unwind it and allow the binding of another TF. positive or negative feedback loop. By activating or repressing their own expression. Two TFs can inhibit eachother in a flip flop loop. Feed foreward loop. Two TFs activating the same other TF.

Question 34

Boveri and Sutton Morgan

Answer 34

First spotted chromosomes in a light microscope Speculated the genes are on chromosomes as they watched them divide. Structural rearrangements-of chromosomes are correlated with phenotypic changes in fruit flys.

Question 35

Functions of proteins in DNA

Answer 35

Packaging and unfolding DNA in the nucleus Controlling replication DNA repair Genetic recombination Maintain integrity Mitochondria and chloroplasts contain DNA

Question 36

When is the best time to see a chromosome What is a metaphase spread. Chromosome painting and abnormalities

Answer 36

Metaphase - Most fully condensed, has been replicated and is ready to attach to the spindle. Smashing dividing cells onto a microscope slide to rupture the nucleus and splay our the chromosomes Distinguishes the chromosomes from one another. Labelling DNA sequences and hybridising them. The chromosomes are being identified by their ability to hybridise to the chromosome paint. Translocations shown by chromosomes that are half one colour and half the other.

Question 37

Each chromosome pair differs in Karyotype

Answer 37

Size and DNA content. 1 is the biggest. Organised representation of all chromosomes in a cell at metaphase

Question 38

Organised interphase nucleus

Answer 38

Each chromosome occupies a distinct 3D space in interphase nuclei. All the active genes are pushed to the centre of the nucleus. The nucleolus is not always active. Chromosome paints can follow the movement of chromosomes from the periphery to the nucleus.

Question 39

Chromosome structure Fibre

Answer 39

Highly coiled fibre of chromatin 10nm fibre- a single molecule of DNA coiled around histone. Under the microscope interphase chromatin resembles beads on a string. The beads are nucleosomes. It can be supercoiled to make the 30nm fibre when it is condensed.

Question 40

Linked histone H1 What happens after remodelling

Answer 40

Changes chromosomes condensation. Many amino acids for a positive charge so they can interact with DNA. straps DNA into histones and stops it moving to stabilise the creation of the 30nm fibre and keeps the DNA condensed Clips the entry strand of DNA to the exit strand at the histone. The DNA can be histone free with no H1 and very accessible to proteins. Chromatin is very flexible.

Question 41

Replication origins

Answer 41

More complex organisms have more of them. Specific DNA sequences that are where DNA replication is initiated

Question 42

Telomeres

Answer 42

Short repeated DNA sequences | TTAGGG

Question 43

Centromeres Kinetochore

Answer 43

Repeated DNA sequences. Repeats of repeats called higher order repeats or alpha satellite repeats. Form very condensed chromatin. Kinetochore binds here- The K inner plate are proteins bound to the alpha satellite DNA The outer k plate are proteins bound to the mitotic spindle. The alpha satellite has half of it interacting with the kinetochore and half not. The half that is interacting with the kinetochore has a centromere specific histone H3. (CENPA) The other half contains a normal H3 variant that is dimethylated at lysine 4. (H3-K4me)

Question 44

Yeast kinetochore

Answer 44

The kinetochore is a basket that links a single nucleosome centromeric chromatin to a single microtubule.

Question 45

Eukaryotic genomes %

Answer 45

1.5% codes for cellular proteins 20% introns 30% non repetitive DNA that aren’t introns or Exons. They are transcription regulation elements. 50% repeated DNA sequence elements. More complex organisms have more protein coding genes and more non protein coding genes. This is required for regulating the access to the protein coding genes.

Question 46

What are transposons Types

Answer 46

Repeated sequences DNA transposons Retro viral transposons Non retro viral polyA transposons

Question 47

description of types of transposons

Answer 47

DNA - cut and paste mechanism and no self duplication. The enzyme transposase cuts out a section and puts it into another place. Discovered by mcclintock while studying maize. Retro viral- Behave like retro viruses. They exist as integrated DNA copies. They replicate via RNA intermediates and produce new DNA copies which will integrate into new locations. Reverse transcriptase is used to make RNA and then the DNA can be integrated. Non retro viral polyA- Replicate via RNA intermediates using its own reverse transcriptase in a copy and paste way. A section of integrated DNA in the genome is converted to RNA. This is used as a template for reverse transcriptase to create DNA which can then be reinserted.

Question 48

Bad transposon insertions Alu

Answer 48

Can cause heamophilia 1 million copies of alu gene which all evolved from a single transposon.

Question 49

Ribosome structure and stuff

Answer 49

-50 ribosomal proteins and several ribosomal RNAs -two subunits. Large- catalyses the polymerisation of the amino acids. Small- facilitates tRNA to mRNA interaction The subunits come together to begin translation and separate at a stop codon. Two amino acids a second. 3’ direction EPA can each hold a tRNA but only two can be in the ribosome at a time. Charged tRNAs enter the ribosome at the A site and peptidyl transferase adds the next amino acid and the tRNA is moved to the P site because of a conformational change. The small subunit moves along the mRNA three places and the tRNA is removed.

Question 50

Elongation factors

Answer 50

Once an anticodon is bound, EF1 causes two delays before peptidyl transferase can act The amino acid bond to the tRNA cannot be broken before a GTP has been hydrolysed to GDP. Then GDP and EF1 will dissociate from the tRNA and peptidyl transferase can act. EF1 slows it down so that there is time for incorrect binding to be released and without this many errors occur. The hydrolysis of GTP will occur more rapidly if the codon and anticodon are correctly matched.

Question 51

Ribozymes

Answer 51

The riboproteins lie on the ribosomes surface. Peptidyl transferase works inside the ribosome so it’s not a protein enzyme. It’s a ribozyme which is RNA that catalyses reactions.

Question 52

Initiating translation

Answer 52

RNA leaves the nucleus with its polyA tail and cap stuck together by EIFs The small subunit recognises the RNA and met tRNA binds to the cap with EIF2. Met scans along the mRNA using ATP and settles on the first start codon AUG it finds. EIF2 is released and the large subunit binds and translation begins.

Question 53

Polysome

Answer 53

Multiple ribosomes bind to one mRNA. They are spaced 80 nucleotides apart. Polysome is where a strand has many ribosomes.

Question 54

Stop codon recognition

Answer 54

Stop codons are recognised by release factors which look like charges tRNAs. Molecular mimicry Bind to the codon just like tRNA would. They cause dissociation of the ribosome from the mRNA.

Question 55

Protein folding

Answer 55

Folding begins immediately after leaving the ribosome. Hydrophobic side chains go to the middle. Molten globule- initial folding which is roughly correct. The amino acid sequence has thought to have evolved to help the molten globule form. Correct folding is a multi step process. Missfolded proteins have exposed hydrophobic regions and can aggregate.

Question 56

Hsp 60

Answer 56

Molecular chaperone Heat shock protein Missfolded proteins into isolation. The hydrophobic entrance of the chamber binds to the protein and unfolds it. GroES cap attaches for 15 seconds. Protein refolds and is released. 30% is not successful.

Question 57

Hsp 70

Answer 57

Molecular chaperone Heat shock protein Works directly on proteins as they exit the ribosome and binds to exposed hydrophobic amino acids. Protects them so the protein can fold without aggregating.

Question 58

Irreversible miss folded proteins. Aggregation causes

Answer 58

Digested by proteases. Marked by uniquitination and digested by the proteosome. Aggregation leads to disease. CJD Huntington’s. Alzheimer’s. Aggregates are large and proteosome resistant. They can cause other proteins to missfold. Prions convert good proteins to prions which are miss folded. Prions stick together to form plaques. These form cross beta filaments.