Lecture 7 Slides Flashcards Preview

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Flashcards in Lecture 7 Slides Deck (63):
1

What does DNA polymerase require at chromosome ends

A primer

2

At what end of DNA does replication end? What is incomplete at end of replication?

End of replication of DNA is at 5' end. At the end of replication, replication of lagging strand is incomplete.

3

Without telomeres, what would happen to 5' end of DNA?

It would continue to shorten over successive rounds of DNA replication

4

Telomerase has homology to what?

Reverse transcriptase

5

How does telomerase work?

It synthesizes DNA strand (5' to 3') from RNA template that is part of the enzyme

6

What does DNA polymerase do after telomerase?

It elongates opposite strand

7

What is human telomere sequence?

T2AG3

8

Steps of telomerase function

1. Telomerase binds to incomplete lagging strand at end of replication
2. Binds to parental strand at 3' and adds to it.
3. Extends three end with RNA templates DNA synthesis (G strand)
4. DNA polymerase completes lagging strand (C strand)

9

What is shelterin complex

Protects telomere DNA and recruits telomere at S phase

10

What does shelterin complex regulate

Formation of t-loop

11

Which shelterin complex proteins bind single strand DNA

TPP1 and POT1

12

Which shelterin complex proteins bind double strand DNA

RAP1, TIN2, TRF2, TRF1

13

What is the difference, at end of DNA replication,between parent and lagging strands?

3' overhang

14

How is t loop formed

Overhang loops above parent strand and links into it

15

In what cells is telomere active

In germ cells and some stem cells

16

Where does telomere DNA gradually shorten? What does that do to its activity?

In somatic cells
Activity is low

17

What do humans with with mutation in one copy of telomerase gene display?

Premature aging

18

When do primary cultures of animal somatic cells cease to divide?

After ~50 cell cycles; telomerase shortens at each cycle

19

What is function of DNA surveillance system

Detects chromosome defects, inhibits cell cycle or promotes cell death

20

If telomerase is active in cells, do they proliferate

Yes, indefinitely

21

What allows transformed cell lines and cancer cells to divide indefinitely

Active telomerase

22

What enzyme is used to make rna from DNA?

Rna polymerase

23

Difference between ribose and deoxyribose

Second carbon in deoxyribose has H i/o OH

24

Difference between uracil and thymine

Uracil carbon double bond has no methylated carbons, but thymine has one methyl attached to carbon

25

Two types of dna dependent rna polymerases

Multi-subunit polymerases and single-subunit polymerases

26

Bacteria multisubunit polymerase

-one polymerase synthesizes all RNAs
alphaI, alphaII, beta, beta', + sigma
-polymerase binds DNA via alpha subunits, sigma factors
-beta subunit has polymerase activity
-homology of all five bacterial proteins are found in archaea and eukaryotes

27

Archaea multisubunit polymerases

One polymerase; 11-13 subunits;
Closely related to eukaryotic polymerases

28

Eukaryotes multisubunit polymerases

Three polymerases; Pol I - 14 subunits; Pol II-12 subunits; Pol III - 17 subunits
Pol IV and pol V - roles in plant siRNA function

29

Single subunit polymerases

Bacteriophage T7 RNA polymerase
Mitochondrial RNA polymerases
Some chloroplast RNA polymerases

30

Where can one find RNA dependent RNA polymerases

In some viruses (like polio virus)
In eukaryotes, play a role in mi-RNA and si-RNA pathways

31

A-amantin sensitivity of RNA polymerase I. Genes transcribed

Insensitive
5.8S, 18S, rRNA genes

32

A-amanitin sensitivity of rna polymerase II? Genes transcribed.

Very sensitive
Protein coding genes, miRNA, siRNA genes

33

A-amanitin sensitivity of RNA polymerase III. Genes transcribed.

Intermediate sensitivity
TRNA genes, 5s rRNA genes, other small RNA genes

34

Which dna strand is used as template

Either. It depends on promoter orientation.

35

Can there be multiple rna polymerases transcribing

Yes

36

What controls rate of transcript accumulation

Rate of initiation

37

Steps in transcription cycle of RNA polymerase II

1. Pre-initiation complex (PIC) formation
2. Pol II recruitment
3. Transition to initiating pol II
4. Transition to elongating pol II

38

What happens during PIC formation?

Assembly of TFIID
TATA-box binding protein (TBP) + 13 other proteins at TATA box

39

What ahopens at pol II recruitment?

Assembly of additional general transcription factors and RNA pol II

40

Transition to initiating POl II

Helicase activity in TFIIH pries apart dna helix
Protein kinase activity in TFIIH phosphorylates residues in c terminal domain (CTD). CTD is unique to POL II largest subunit; repeated heptapeptide:
Tyr-Ser-pro-Ser-pro-Ser
Binds RNA processing factors

41

What happens during transition to elongate pol II

Pol II is released from initiation factors, elongation begins

42

What does preinitiation complex in vivo include

General transcription factors
Pol II
Mediator- a structurally flexible of about 25 proteins that links transcription factors (activators or repressors) to TNA pol II
Proteins for modifying chromatin structure

43

Steps in eukaryotes RNA processing

-DNA is in nucleus
-DNA has introns and exons
-rna is transcribed from transcription unit into primary rna transcript
-primary rna transcript gets 5' capping, RNA splicing, 3'polyadenylation
-becomes mRNA, which is then exported from nucleus
-mRNA is then translated into protein outside of nucleus

44

Prokaryotic rna processing pathway

DNA is in cytosol ( no nucleus)
Transcribed directly to mRNA
Translated in cytosol

45

Processing enzymes carried by C-Terminal domain

Capping factors at 5' end of mRNA
Splicing proteins
5' cap
3' end processing proteins

46

What makes up eukaryotic mRNA cap?

5'-to-5' triphosphate bridge
7-methylguanosine

47

Difference between polycistronic and monocistronic mRNA?

Poly has coding for multiple proteins.
Mono has coding just for one protein

48

How is 5' end cap added

Phosphatase takes phosphate
Guanyltransferase add guanosine to end
Methyl transferase adds methyl group to base
On so e caps, add methyl group to ribose

49

Purpose of intron splicing

Facilitates evolution of new proteins
Provides opportunity for regulation of gene expression
Alternative splicing allows numerous proteins from a single gene

50

Splicing step 1

1. U1 snRNP forms base pairs with the 5' splice junction
2. BBP (branch point binding protein) and U2AF (U2 auxiliary factor) recognize the branch point site

51

Splicing step 2

U2 snRNP displaces BBP and U2AF and forms base pairs with branch point site consensus sequence.

52

Splicing step three

The U4/U6.U5 "triple" snRNP enters reaction. U4 and U6 snRNAs are held firmly together by base pair interactions. Subsequently, rearrangements break apart the U4/U6 base pairs, allowing U6 to displace U1 at the 59 splice junction. This creates the active site that catalyzes the first phosphoryl-transferase reaction.

53

Splicing step four

Additional RNA-RNA arrangements create the active site for the second phosphoryl-transferase reaction, which then completes the splice

54

Processing at 3' end of transcript

1. Cleavage and poly-A signals encoded in DNA
2. Cleavage stimulation factor (CSF) and cleavage and polyadenylation specificity factor (CPSF) are attached to RNA polymerase
3. RNA is cleaved
4. Poly-A polymerase enters. Rna polymerase eventually terminates
5. Poly-Abonding proteins enter process
6. Poly-A length is regulated

55

What is polyadenylation signal

AAUAAA

56

At what sequence is there cleavage?shat is added at cleavage?

CA. Add hydroxyl group.

57

Where does gene rich dna lie in nucleus

In euchromatic regions

58

FISH stands for

Fluorescein in situ hybridization

59

Where are alu sequences found with FISH

In gene rich dna

60

What does fluorophore link to in fish

Antibodies

61

Transcription factories

Contains 8-10 RNA POL II molecules

62

Nuclear export of pol II transcripts

"Export-ready DNA exits through nuclear pore complex into cytoplasm
-on RNA there is a nuclear export receptor, hnRNP proteins, and SR proteins
-5' end exits first
-in cytoplasm, there are initiation factors for protein synthesis (eIF4G and eIF4E)
-factors binds to 5' end
- prepare for translation

63

What is the main purpose of a telomere?

To stabilize chromosome ends