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Flashcards in Unit 4 Deck (76):
1

difference between eukaryote and prokaryote DNA replication

-amount of DNA to be replicated (more in Eukaryotes)
-more complicated (more DNA and ori rep)
-takes longer in eukaryotes

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origins of replication

each one starts firing at the same time

3

eukaryots and prokaryotes similarities (DNA rep)

DNAP, helicase, primase, Beta clamp

4

DNAP difference

eukaryotes has leading and lagging DNAP

5

eukaryotes termination

circular in prokaryotes (e coli) vs linear in eukaryotes

6

termination in e coli vs eukaryotic humans

-primers removed in each
-e coli" DNA poly can fill in gap using free 3'OH
-DNAP can't fill in gaps because needs free 3' hydroxyl

7

linear DNA replication problem

lose a bit of DNA within each round of replication

8

how to fix problem with linear DNA rep

telomeres and telomerase

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telomeres

regions of the ends of linear DNA that was synthesized by telomerase

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telomerase

enzyme that can extend the ends of linear DNA

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3 key things with telomeres

-repetitive DNA sequence
-no protein coding info
-length doesn't matter
---will vary based on telomerase activity

12

aging

-telomerase activity goes down as cells get differentiated
-telomeres shrink without telomerase
-cells can't divide anymore (some will die-apoptosis)

13

Pot1

binds telomeres and inhibits telomerase activity
-as telomere gets longer and longer, more Pot1 protein (less telomerase activity)

14

transcription in eukaryotes

more complicated (more DNA)
-very small % of euk DNA is protein coding
-lots of transcription factors to help RNAP find DNA to transcribe

15

eukaryotic RNAP

-12 subunits
-RBP1
-binds to a promoter upstream of gene to transcribe

16

initiator

transcription start site

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RBPI

subunit with enzymatic activity (along with RBP2), make RNA polymer complementary to DNA template

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TATA box

-similar to -10/-35 region in prokaryotes, most common promoter

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TFIID

transcription factor that binds promoter (similar to sigma factor)

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RBPI tail

-tail must be phosphorylated to move past the promoter (breaks apart tight binding)

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elongation in eukaryotes

chromatin packaging- some chromatin more accessible than others

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prokaryote vs eukaryote transcription

pro- sigma and -10/35
euk- TFIID and TATA

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eukaryote general tf

-bind to promoter directly upstream of gene
-make direct contact with RNAPII

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eukaryote gene specific

-bind to DNA regions far away (thous nucleotides) from trans. start site
-need mediator

25

DNA regions

-enhancers: gene specific tf binds here to help RNAP
-silencers: Tf binds here to stop RNAP II

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mRNA prokaryotes

can get translated during transcription

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eukaryote mRNA

-gets transcribed in nucleus and translated in cytoplasm
-needs to last longer (be more stable)

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mRNA processing includes

-5' capping and 3' polyadenylation for splicing
-splicing
-termination

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3 proteins involved in adding 5' guanine methylated nucleotide cap

-RNA triphosphatase
-guanyl transferase
-methyltransferase

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RNA triphosphatase

remove one phosphate on 5' end

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guanyl transferase

attach a G nucleotide
-5' end of G added to 5' end of mRNA
-weird 5' to 5' bond can't be degraded by mRNA nucleases

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methyltransferase

-add methyl group to 5' cap
-help prevent degredation of nucleases

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function/role of 5' cap

-mRNA stability (avoid being degraded by nucleases)
-recruit splicing proteins
-assist transport to cytoplasm (5' cap recognized by transport proteins)
-helps mRNA bind ribosome (initiation factors for transl)

34

3' polyadenylation

-all Eukaryotic mRNA has = 250 A on 3' end
-poly A tail gets added on after transcription
---coupled to termination

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function/ role of poly A tail

same as 5' cap except
mRNA stability (3' end gets degraded by nucleases, takes a while)

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poly A tail added with 3 proteins

CPSF, CStF, PAP

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CPSF

cleavage and polyadenylation specificity factor, cuts mRNA

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CStF

cleavage stimulation factor

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PAP

polyA polymerase (adds A nucleotides to the new 3' end)

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eukaryotic termination

new 5' end (after mRNA cut for poly) gets recognized by XRN2
----degrade mRNA and bumps into RNAP so it falls off DNA

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mRNA since nucleus in eukaryotes

mRNA needs to get transported and be more stable

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exons

contain protein coding info

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introns

don't have protein coding info and get spliced out

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spliceosome

group of proteins (and RNA seq) that coordinate splicing

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alternative splicing

happens in about 50% of human genes and creates functional protein

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splicing mistakes

splicing gets messed up and leads to a non-functional protein

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examples of splicing mistakes

-intron gets left in the mRNA, doesn't get spliced
-too much RNA gets spliced out (intron and some exon info)

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intron gets left in the mRNA, doesn't get spliced out

-intron nucleotides can be translated
-extra amino acids disrupt protein structure and function
-might contain stop codon--> end translation early

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too much RNA gets spliced out

-lost some amino acids
-frameshift mutation

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frameshift mutation

change which 3 nucleotides are getting read

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order of eukaryotic mRNA processing

occurs while transcription occurs

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C terminal domain (CTD) of RBPI subunit of RNAPII steps

5' cap
splicing
termination
polyadenylation

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initiation of transcription CTD gets phosphorylated which

breaks protein interactions with TF and RNAPII

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CTD steps

-CTD tail on mRNA with RNAP
-CTD has phosphorylations
-5' capping enzymes get recruited to P CTD
-after capping is done, lost couple P groups
-splicesome gets recruited
-change in P of CTD
-polyadenylation machinery gets recruited to CTD

55

translation in eukaryotes vs prokaryotes

euk- transcription in nucleus, mRNA transported to cytoplasm, mRNA needs to find ribosomes

pro- translation and transcrip occur simultaneously

56

translation initiation steps

5' cap and poly A tail
-each feature is bound by proteins that help find ribosome

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proteins that help find ribosome

-poly A binding protein (PABP) --> binds to poly A tail
-eIF4F binds to 5' cap

PABP and eIF4F bind to each other

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mRNA circular for translation in euk

increase translation efficiency, easier for ribosome to reinitiate and multiple ribosomes translate 1 mRNA

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new for euk translation initiation

-eIF4F binds 5' cap also binds small subunit
-scanning: small subunit finds start codon

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similarities among euk/prok

-initiation factors bind E and A sites of small subunit
-everything assembles on small subunit, then large subunit binds

61

for elongation and termination with euk and prok

no differences

62

regulation of translation in prok

tmRNA, results in protein degraded

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regulation of translation in euk

mutation- no stop codon
mutation- early stop codon

64

no stop codon

ribosome gets stuck at end of mRNA (poly A tail AAA, codon for lysine)
----mutated protein will have a string of Lys at end
-two proteins recruited..1 degrades mRNA and 2 degrades protein

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early stop codon

before the final exon junctions
-after translation, mRNA still has exon junction proteins

66

exon junction proteins

recruit other proteins
-remove ribosome from mRNA
-degrade the mutated mRNA
-mutated protein doesn't get degraded

67

RNA silencing

mechanism to regulate mRNA levels
-post transcrip petunia

68

1st key details of RNA silencing

-dsDNA looks foreign and dangerous to eukaryotic cells
-micro RNA gets transcribed from DNA but doesn't get translated

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miRNA

transcribed and processed in nucleous

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miRNA steps

-transported to cytoplasm and binds RISC
-mature mRNA binding to RISC
---can base pair with target mRNA
-target mRNA gets degraded

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RISC

RNA
Induced
Silencing
Complex

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2 ways to degrade mRNA target

1) perfect/near perfect between miRNA and mRNA, then RISC degrades mRNA
2) not perfect base pairing, then translation repressed

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siRNA

-application of miRNA process
-quick/easy/inexpensive way to temporarily knock down protein expression

74

example of siRNA

protein of interest= helicase
-want to get rid of helicase protein expression

75

siRNA steps

1) look at mRNA seq of helicase
2) use mRNA seq to design siRNA
3) insert siRNA into cell/animal of interest
4) RISC will use siRNA to silence complementary mRNA

76

CRISPR vs siRNA

CRISPR- permanent because modifies DNA
siRNA- temporarily (modify RNA)