week 2 Flashcards
1
Q
Eukaryote vs prokaryote in transcription:
Transcription and translation are coupled or uncoupled in E vs P
A
E = uncoupled
P = coupled
2
Q
E vs P in transcription:
which has many processes (5’ end capping, polyadenylation and splicing)?
A
E = many processed
P = not as many
3
Q
E vs P in transcription: Which is monocistronic and which one is diff gene having the same promotor
A
E = mono
P = poly
4
Q
E vs P in transcription:
which one has 3 RNA polymerases?
A
E
4
Q
E vs P in transcription: Are promotors longer or shorter
A
E = longer
P = shorter
5
Q
E vs P in transcription: Which DNA is wrapped around histones?
A
E
6
Q
in eukaryotes, transcription is regulated not at the level of RNA polymerase recruitment to the ____, but at the level of its ___
A
promotor
activation
7
Q
Euchromatin and heterochromatin, explain differences in accessibility to TF
A
euchromatin = accessible for TF
heterochromatin = poorly accessible
8
Q
What is mRNA processing?
A
Alternative splicing as a regulatory mechanism of gene expression in eukaryotes.
9
Q
What are the 3 processes to mRNA splicing and which are there to protect from degradation?
A
- 5; end capping (yes)
- 3’end polyadenylation (yes)
- splicing
10
Q
Describe 5’ end capping?
A
attachment of modified guanosine onto 5’ part, protects 5’ end from being recognised by nucleases
11
Q
Describe 3’end polyadenylation
A
where multiple adenine is added to the end. sequence at 3’ end of gene during transcription is recognised by specific nuclease and is cleaved, adenines are added
12
Q
overtime mRNA gradually loses more and more As are added. what happens eventually?
A
nucleases completely degrade the whole mRNA molecule
13
Q
What is the significance of the cap-binding proteins
A
- protein binds polyA tail and 5’ end cap. these 2 complexes interact, forming a protective cap to the ends of the mRNA
- during translation, polyA tail becomes shorter due to nuclease getting access to it and gets degraded. As this happens, the protein no longer binds onto the polyA tail and protective power of cap is lost. mRNA is degraded.
14
Q
What is splicing?
A
the excision of introns by spliceosome
15
Q
What does alternative splicing result in?
A
different protein variants expressed from the same gene
16
Q
What does post translational modification of proteins include?
A
activation or inactivation of proteins by attaching groups to save energy
17
Q
What is the most common protein modification?
A
protein phosphorylation
18
Q
Describe the process of protein phosphorylation
A
Protein kinases are enzymes which transfer a phosphate group from ATP to serine, threonine or tyrosine on a protein.
19
Q
Why can’t other amino acids undergo phosphorylation?
A
Other amino acids cannot be phosphorylated as the phosphate group required an OH group as an acceptor.
20
Q
How does protein phosphprylatioin regulate protein-protein interactions? 2 ways…
A
- low affinity due to being too bulky and not fitting
- too much negative charge and repels
21
Q
Why is protein phosphorylation key to the cell cycle
A
- Eukaryotes express cell cycle dependent kinases with the activity specific to each stage of the cell cycle.
- These kinases activate the proteins required for a specific stage of the cell cycle. When these proteins are no longer needed, they are inactivated by phosphatases or degraded.
22
Q
How to limit protein presence In cell cycle to a short time? how are they degraded after accumulation?
A
ubiquitin-dependent degration
23
Q
describe how ubiquitin-dependent degration works?
A
ubiquitin marks proteins that needs to be graded. it attaches onto the ligase. the proteasome degrades protein (consists of multiple subunits where proteins are swallowed and gets shredded into pieces)
24
What is the parallel of ubiquitination in bacteria?
proteolysis
25
Explain how proteolysis works.
signalling peptides in target proteins recognised by different adaptors which bring the target proteins to different proteases for deflation
26
What are some advantages to using yeast as model organism?
- easy to grow and maintain in labs
- short generation times(only 1.5 hour per cell cycle)
- large number of offspring - proliferates exponentially
- well studied genome - one of the best in eukaryotes
27
In the cell budding cycle, there's an and alpha haploid cells which mate to make a/alpha diploid. how do a and alpha cells know to mate with eachother?
they have receptors to sense pheromones of the opposite sex
28
What is expressed in a cells?
TF that activates a specific genes but not alpha specific genes
29
What is expressed in alpha specific genes?
- a2 = repressor to inhibit a specific genes
- a2 = activator to activate alpha specific genes
30
explain how binding of alpha factor leads to mating and not budding
alpha factor binds to receptors where signal is transmitted to a scaffold protein which gathers 3 proteins that undergo phosphorylation in order of proximity. 2 extra proteins are phosphorylated one inhibits G1 to S transition (G1 cell cycle arrest) and the other activates genes for mating such as shmooding
31
why is G1 arrest during yeast mating important
because mating a cell in G1 and a cell in G2 is bad, causes a cell to have 3 copies of chromosome, triplet cell wont be able to go through meiosis no spores produced.
32
What is MAP kinase pathway? how does it amplify signals?
MAP kinase stands for Mitogen-Activated Protein kinase. - Their role is to
transmit a molecular signal into a response.
33
How does signal amplify exponentially?
cascade phosphorylation allows fast responses to signals as every activated molecule activates multiple kinase molecules so cascade amplifies signal for greater affect
34
Explain how spores can ensure mating pairs are formed?
mother can switch mating type but daughters can't to ensure diploids are formed to survive unfavourable conditions during sporulation
35
How is mating type switching inhibited in daughter cells?
daughters can’t switch because the ASH1 gene is present which represses the gene switch. when daughter becomes mother, mRNA is transported into daughter so the rule is coordinated. ensures there are both a and alpha cells to mate.
36
What is epigenetic?
the study of heritable changes in gene expression and gene function that cannot be explained by changes in DNA sequence.
37
Epigenetic changes can boost or interfere with _____ of specific genes
transcription
38
epigenome =
all epigenetic markers attached onto a gene
39
how is epigenome heritable?
can be carried on through meiosis and mitosis
40
What is DNA methylation?
transfer of a methyl group on C nucleotides often at opposite strands so both strands are methylated at same position.
41
DNA methylation is established by ___ and removed by ____ recognised by ___
- writer enzymes
- eraser proteins
- Reader proteins
42
methylation in gene promotor region ___ binding of TF
inhibits
43
How is DNA methylation maintained during DNA replication?
At CG sites specific “readers/writers” can recognise hemi-methylated DNA and methylate the other strand
semi-cobservative nature of DNA replication allows this.
1. initially, methylation is only present on the old strand used as template
2. writer enzymes are recruited by readers and deposit methyl group onto the new strands
3. mitotic inheritance of epigenetic markers
44
Histone modification: describe
DNA is packaged around histone proteins which forms a nucleosome unit. Histone residues (in tail regions) can be acetylated, methylated, phosphorylated, and ubiquitylated by writers
45
H3K9ac - what does it do
acetylation by writers neutralises positive charge of DNA
- losoer DNA so promotes transcription
46
H3K27me3 - what does it do
condensation of histones leads to silencing (others can lead to promotion)
47
How is histone modification maintained during DNA replication?
parental histone partition equally between daughter cells, which means histones get diluted (4 nucleosome on dna stretch→ 2). to compensate, new histones are added (in purple) but they lack modification. marks on nearby parental histone are recognised by readers in yellow and associated writers deposit marks (red marks) on new histones (restoring original epigenetics state)
48
How is histone modification maintained during meiosis?
- Epigenetic marks removed by eraser proteins in animal gametes. Removal mostly absent in plants
- If erasure not complete or marks re-established after erasure = transgenerational inheritance
49
How is regulation of flowering time achieved via epigenetic?
- this plants germinates in autumn and initially flowering is repressed due to gene expression of FLC (flowering locus C)
- prolonged exposure to cold activates writer protein and modifies histone near FLC locus and represses FLC expression
- during spring when its no longer cold, the FLC is re-expressed from memory
- summer triggers flowering
- epigenetic markers reset for each generation (not inherited)
50
What determines the difference between a queen and a worker female?
environmental factors: - larvae are initially fed by worker bees with royal jelly
- at day 3 of age, larvae is either switched to a worker jelly diet or royal jelly diet
- individual that gets put in either jelly becomes either a queen or worker
- since there is a difference in diet composition, queens usually is bigger in size and live longer
51
Is it the (continued) exposure to royal jelly or the lack of exposure to worker jelly that really matters? which dietary regime will produce a queen
lack of exposure to worker jelly maintains queen-specific gene expression. being fed ‘ worker jelly’ prevents queen identity development. deliberate case of “food poisoning”
51
Honey is rich in phenolic compounds which leads to...
higher exposure to DNA methylation
52
Give examples of cases where sex is determined by epigenetic
- environmental conditions (temp) of many reptiles
- some crustaceans species change sex during their lifetime
53
What are the 3 components of nucleotides?
phosphate, sugar, base
54
What is the diff between DNA and RNA
DNA = deoxyribose/T
DNA - ribose/U
55
How is structure of ATP similar to that of DNA
ribonuclotide with 3 phosphate instead of one, adenosine as base
56
Explain how a sugar phosphate backbone is made referring to C1,3,5
C1 = interacts with bas e
C3 and C5 = interacts with phosphate
57
TO extend DNA or RNA strands, nucleotides are always added from ____
3' end
58
Explain how energy is provided for polymerisation of DNA
Enzyme DNA polymerase synthesises DNA strands. New nucleotides to be added comes in the triphosphate form. This is because DNA synthesis consumes energy so phosphate bonds are broken to provide the energy. Di-phosphate released in the reaction.
59
What does DNA polymerase 3 do?
adds nucleotides
60
What does exonuclease do?
opposite of DNA polymerase, removes wrong base
61
What does topoisomerase do?
- topoisomerases removes supercoiling
- mechanistic stress is generated due to helical nature of DNA
- DNA is unwound by topoisomerase to remove mechanistic stress
62
Describe the process of DNA replication on leading strand.
Replication fork is where replication takes place. Replicative helicase unwinds 2 parental strands and DNA pol 3 synthesises the new strands. Leading strand is synthesised in the same direction as the unwinding happens. another enzyme, called Primase synthesises a short RNA primer, to which Pol III then adds DNA nucleotides.
62
Describe the process of DNA replication on lagging strand.
Because DNA can be extended only at the 3’ end, one of the two strands (lagging) has to be synthesised in the direction opposite to the direction of replication. This dictates the synthesis in short stretches, called Okazaki fragments.
- As Pol III elongates Okazaki fragments up to the next primer, the RNA primers remain on DNA after Pol III has finished. The RNA primers need to be removed. DNA Pol I degrades them and fills the gap by elongating the 3’ end of the Okazaki fragment next to the primer, leaving a nick.
- Nick is a break in the sugar-phosphate backbone, with no bases missing
- DNA Ligase seals the nick by connecting a phosphate on the 5’ end to the ribose on the 3’end. The removal of RNA primers and ligation of Okazaki fragments completes the replication of the lagging strand.
63
describe each of the enzymes and their function:
DNA Pol I
DNA Pol III
Helicase
Ligase
Primase
Topoisomerase
- removes RNA primers and adds newly synthesised DNA
- addes nucleotides
- opens DNA by breaking hydrogen bonds
- seals faps between fragment
- synthesises RNA primers to start replication
- helps relieve stress when unwinding DNA
64
What are the 3 differences in replication fork between eukaryote and prokaryote
1. leading and lagging strand in eukaryote is synthesised by different polymerases
2. primer needs to be extended by polymerase in eukaryote before pold synthesises fragments. in prokaryotes Pol1 does this
3. The replicative helicase which separates parental strands encircles the lagging strand template in prokaryotes, while in eukaryotes, it travels along the leading strand template.
65
How is the bacteria genome replicated? circular dna
- Each circular DNA has a single replication start site, called an origin or replication.
- Replication starts at the origin and proceeds in both directions around the circle, until it terminates on the opposite side from the origin.
66
Why does eukaryotic chromosomes require multiple origins? how?
- A mammalian genome is approximately 1,000 times bigger than a bacterial genome. Therefore, it needs to be replicated from multiple origins in parallel.
- Eukaryotic chromosomes are linear and every chromosome contains multiple origins. They don’t fire all simultaneously. The origins situated in euchromatin (actively transcribed open chromatin) are fired first.
67
What is a replicon? Compare E vs U
DNA region replicated from a single origin
- each DNA molecule is a replicon in prokaryotes
- in eukaryotes, there are multiple replicons (number of origins = number of replicons)
68
How is cell cycle controlled in terms of replication?
Replication origins are fired only once per cell division cycle
68
What happens in each part of the cell cycle in terms of DNA replication?
M = ARS recognition by ORC
G1 = licensing
S = activates helices and makes it ready for replication. brings all relevant enzymes to origin
69
What is the end replication problem in linear chromosomes?
- RNA primers don’t lead until the very tip and even if it was, the RNA would be removed
- with every replication cycle, the lagging strand will shorten and chromosomes will shorten
70
What are telomeres and their functions
- Telomeres consist of non-coding DNA which is highly repetitive and G-C rich. All vertebrates have the same telomeric repeat sequence TTAGGG.
- There are proteins which recognise these telomeric DNA repeats and bind them. The protein complex binding telomeres in human cells is called Shelterin. Shelterin bound to hundreds of telomeric repeats forms a protective protein cap at the chromosome ends. This capping protects the DNA from being degraded.
70
How are telomeres extended?
telomerase:
- enzyme that extends telomere caps
- it is a reverse transcriptidase because it uses RNA templates to extend telomeres
- add 6 nucleotides at a time to extend telomere and okazaki frangement is used to synthesise the complementary strands.
71
Where is DNA replication origins in bacteria vs eukaryotes
bacteria = oriC
eukaryote = ARSes
72
What are unused origins called?
dormant
73
origin firing starts with ___ at the locus.
melting A-T rich DNA
74
in E.coli DnaA protein binding at Orin leads to ___.
local DNA melting
75
in eukaryotes, describe origin firing as a complex process
- ARS binding by ORC protein complex in late M
- replicative helices MCM binding in G1 (licensing)
- recruitment of other proteins In early S through phosphorylation by CDk
76
Which end is usually marked by an arrowhead
3'
77
How does helicase team up with ssDNA specific nucleases
helices unwinds dsDNA which creates a substrate for ssDNA specific nucleases. one particular strand is degraded leaving one strand (used widely in DNA repair)
78
all nucleotides are added in which direction
5' to 3'
79
Why are polymerases more pocessive in vivo?
clump (PCNA) is a closed ring which lies and guides the polymerase
