Final Exam Flashcards
(107 cards)
1
Q
Describe the two types of eRNA generated by transcription at enhancers.
A
2D : No post modifications and in cis
1D : Further modified and in trans
2
Q
Theories of eRNA function
A
- Transcriptional Noise
- Transcription dependent effect (Aberrant transcription due to proximity of promoter and enhancer)
- Activity in Cis
- Activity in Trans
3
Q
Why is the GTP Cap important for RNA
A
Prevents degradation because ribonucleases will not recognize
4
Q
Mechanism of GTP Cap addition
A
- Initial transcript has 2-3 phosphates to prevent degredation.
- GTP added to 5’ end
- Methylation on Guanine base
- Methylation of 2’ ribose OH
5
Q
When does GTP Cap formation occur?
A
When the RNA pol pauses when Ser 5 phosphorylated. Complexes are already bound to CTD
6
Q
What is the Polyadenylation signal?
A
AAUAAA
7
Q
When do poly A proteins bind?
A
When Ser2 is phosphorylated to end pausing
8
Q
Mechanism of poly A tail formation
A
- Assembly of scaffolding
- Poly A polymerase adds a few A bases
- Poly A binding protein binds the short A tail
- This sends polymerase into hyperdrive and adds hundreds more A’s
9
Q
Structure of the Intron Product after splicing
A
Lariet Structure
10
Q
Specific structure of an Intron?
A
Bold are important
GU—– A —– (py rich) —-CAG
In general: Starting GU, branched A, pyrimidine rich region, ending AG
11
Q
Mechanism of self splicing intron?
A
- Branch A OH attacks beginning G phosphate
- That G OH attacks ending G phosphate
Basically breaking and reformation of phosphodiester bonds
12
Q
U1 RNP importance
A
Binding to 5’ splice site forms commitment complex.
U1 is escorted by an SR proteins (ser and arg rich)
13
Q
Group 1 Splicing mechanism
A
- RNA folds up in specific conformation allowing purine rich region to bp with 5’ splice site
- G binds
- 1st Transesterification puts G on 5’ end
- 5’ end attack 3’ splice site excising intron
- Purine rich region base pairs with 5’ splice site again
- 3’ G binds the splice site
14
Q
Technique to find size of an RNA fragment
A
Northern blot
15
Q
What do the RNA’s that bring together the spliceosome mimic
A
A group II self splicing intron
16
Q
Why spliceosome can work in trans
A
Spliceosome does not require a particular middle sequence in catalysis
17
Q
Given a mutation at the 5’ splice site. How can you restore splicing activity?
A
Get a compensating mutation in the Prp8 protein
18
Q
Main role of Prp8
A
The massive protein is involved in so many confomers of the spliceosome during the cycle. Involved in like everything
19
Q
What binding sites are opened after Brr2 unwinds U4 and U6
A
2 Magnesium ion binding sites. Coordinate Splice site and U6
20
Q
Conformational requirements for Spliceosome splicing
A
BP U2 (at 5’ splice site) and bulged A with stacking of G nucleotide of the GU splice site
21
Q
Role of Magnesium ions in splicing
A
Negates charge buildup with phosphodiester bonds
Makes hydroxyl group better nucleophile for attack
22
Q
Requirements to recruit metal ions
A
Unwinding of U4 from U6
Specific bases in U6
Base pairing with U2
Stem loop
23
Q
Importance of intron encoded proteins
A
Have similarities to Prp8 but not necessary for splicing. Proteins stabilize structures
24
Q
When is mRNA immediately released from spliceosome?
A
After helicase unwinds mRNA from U5
25
What proteins are important in determining chosen splice sites
The chaperon proteins of U1
26
What protein is required for U2 to bind
U2AF
27
Functions of the subunits of U2AF
65 recognizes poly pyrimidine
| 35 recognizes 3' splice site
28
mechanism for inhibition of splicing
hnRNP protein binds and oligomerizes along RNA blocking splice site and binding of U2AF. Removed by protein blocking full oligomerization
29
How is splicing generally regulated in different tissues
Tissue specific proteins
30
Draw the strucutre of dihidryl uridine and inosine
Will put something later
31
Importance of 2 step tRNA synthesis
Lowers error rate of wrong amino acid on wrong tRNA
32
Mechanism of tRNA synthesis
1. Amino acid + ATP --> Amino acid-AMP
| 2. Amino acid-AMP + tRNA --> Amino Acid-tRNA + AMP
33
Eukaryotic Translation Initiation
1. 40S subunit primed with IF3
2. Binding of Met-tRNA + eIF2
3. Binding of mRNA + eIF4F (cap structure)
4. Scans for start codon
5. Successful scan = 60S subunit binds.
34
What does full interactions with 4G of eIF4 create before elongation
A pseudo circularized mRNA
35
Importance of 4A in eIF4
Removes secondary structure that are upstream of start codon
36
Role of eIF2B
Catalyzes switching of GDP with GTP on eIF2 to continue cycling. Often a point of inhibition by phosphorylation.
37
Why is there no protein folding during elongation of peptide chain?
Ribosome has a small exit channel that prevents this
38
Cycle of peptide elongation in translation
1. mRNA + tRNA in p site
2. tRNA + GTP + EF-Tu in A site
3. peptide bond forms in A site
4. Switching of sites (by EF-G)
5. EF-Ts (GEF)
39
Why is an incorrect tRNA ejected during accommodation
Base pairing of anticodon induces stress. Incorrect BP would induce too much stress and eject tRNA
40
Why is a correct tRNA not ejected during accommodation
Correct base pairing causes a shift in A bases in ribosomal RNA that H-bond with the t-RNA. A G base also stacks. All stabilize the interaction.
41
When A bases move to stabilize the codon-anticodon interaction. Where does the energy for this conformational change come from?
Proper base pairing of codon and anti-codon.
42
Why does paromomycin not affect eukaryotes
They do not posses the A base conformational change mechanism.
43
How EF-Tu regulates GTP hydrolysis
Requires a water molecule coordinated to a histidine to hydrolyze GTP. Interaction is prevented due to hydrophobic amino acid gate. The isoleucine is moved by helix 44
44
How is the hydrophobic gate of EF-Tu opened
the movement of the 2 stabilizing A bases causes a conformational change throughout helix 44 allowing h8/h14 to interact with switch 1
45
Reaction, location, and result of peptide elongation (sites)
Amion group in the A site attacks the carbonyl of the P site. This removes elongate chain in the P site to the A site
46
Puromycin
A peptide bond terminator due to lacking an ester after added to the growing chain
47
Orientation facilitation by the large subunit. (What interactions)
H-bonds with A site tRNA
H-bonds with P site tRNA
Directs towards each other for catalysis
Water molecule also aids in coordination
48
What happens immediately after peptide bond formation?
Rotation of the two ribosomal subunits relative to each other. Aids in movement of P and A sites to E and P sites
49
Importance of the L1 stalk
Stabilizes hybridized state of ribosome rotation
50
Formation of SiRNA
1. Cleavage of long double stranded RNA by Dicer
2. One strand is transferred over to the RISC complex
3. Splices the targetted sequence
51
Considering the RISC complex. What results if there is perfect complementarity and imperfect complementarity.
Perfect complementarity leads to cleavage
| Imperfect leads to removal of post modifications and then degredation
52
Product of DICER after cleavage
A stagger of 2 nucleotides gives a 3' overhang
53
Primary miRNA
Regions transcribed by Pol II and poly adenlyated. Many hairpin loops that get processed down into the individual miRNAs
54
Intron origin of miRNA
Drosha works in combination with another protein (complex called microprocessor) that cleaves into miRNAs
55
DROSHA/DGCR8
Recognizes sequences in the stem and loop. DROSHA recognizes sequences in the base of the hairpin
56
Product of DROSHA
Produces a staggered cut to produce the pre-miRNA. DICER binds the 3' overhang and produces the mature miRNA which gets transferred to RISC complex
57
Do all Ago proteins have cleavage activity?
No, determined by which ortholog of Ago is used
58
What happens with imperfect matching of the RISC complex?
1. May degrade RNA
| 2. May hold RNA temporarily in inactive form
59
How important is proper base pairing in RISC complex?
Not that important, NT 2-8 require perfect base pairing and that is it. Allows for many targets with a single miRNA
60
How precise cleavage occurs in RISC complex
Perfect complementarity in the seed region causes the cleavage domain to be in a specific spot
61
How the RISC complex represses translation
1. blocks eIF4F to bind CAP site
2. Could bind 60S subunit
3. Several others
In general, bound RISC complex is enough to prevent translation
62
GW182/TRBP
Can bind to the Ago protein as well as other locations such as poly A proteins or P-bodies.
63
RISC competes with several processes in cells
Yes
64
How GW182/TRBP can inhibit ribosome scanning
GW182 may interact with the protein NOT1 leading to inhibition of the helicase activity of eIF4A. Buildup of secondary structure prevents scanning
65
How the RISC complex promotes degredation of mRNA
Proteins like GW182 and NOT1 recruit the deadenylation complex as well as the decapping complex
66
Role of P-bodies and the RISC complex
P-bodies house many proteins for deadenylation and decapping.
May also house temporarily inactive mRNA bound to RISC
67
Why the ends of DNA (telomeres) do not interact
t-loop + shelterin protein complex protects the DNA end
68
How an equilibrium is established regarding telomere length?
Proteins bind the repeated telomere sequence until eventually enough proteins present to inhibit telomerase. Over time, telomere degrades, less proteins present, telomerase activates
69
Why do telomeres shorten?
In the lagging strand, cannot replicate the last okazaki fragment
70
What activity does DNA polymerase alpha do
Primase activity
71
What is pol III*
Everything except the beta clamp. Consists of alpha, epsilon, and delta subunits
72
What does DNA polymerase delta do
Synthesizes lagging strand
73
What does DNA polymerase epsilon do
Synthesizes leading strand
74
Where does DNA polymerase stop on the okazaki fragment
Before primer
At a gap
At a nick
75
What happens to PolII (Pol* + beta clamp) after replication
PolIII* dissociates, beta clamp remains
76
Phosphorylation of beta clamp
2 sites can be phosphorylated in free beta clamp. Masked when bound to pol III*
77
Why does ORC bind during G2 and M phases
DNA is most accessible. G2 is generally used for DNA repairs
78
Pre-replicative complex (licensing complex) mechanism
1. ORC already bound to DNA
2. ORC, CdtI, and Cdc6 recruit Mcm
3. McM is activated by recruiting Cdc45 and GINS
79
Why can you find e-coli cells defective in replication with temperature
Signals that heat shock proteins could be mutated which prevents some stabilization
80
Role of topoisomerase in DNA replication
detangles the DNA. Undoes positive supercoiling
81
What is the initiation site of Ecoli for replication
the 13-mers and 9-mers
82
Role of DnaA
Regulates initiation of prokaryote replication, many copies bind to 9-mers, creates torsional stress
83
role of HU
Histone protein in ecoli that stabilize during replication
84
Role of the 13-mers
Torsionally strained, produces strand separation
85
Role of DnaB
A helicase, maintains strand separation
86
DnaC
Delivers Dna B
87
DnaG
Primase, DNA Pol III requires RNA primer
88
Termination sequences and Tus
Exists permissive and non permissive sides
Tus binds to short termination sequences, can halt pol III causing it to dissociate. Helicase blocks Pol II on non permissive side
89
Role of DNA Pol I
For repair, fixes unreplicated DNA regions
90
Trombone model for lagging strand
Helicase opens DNA
Primase adds primer
Lagging strand is pushed through alpha
Synthesizes okazki fragment till it hits the previous
91
Bacterial polymerase alpha subunit
DNA polymerase
92
Bacterial polymerase epsilon subunit
3' ---> 5' exonuclease
93
Bacterial polymerase Tau subunit subunit
Dimerizes core
| Binds gamma complex
94
Bacterial Polymerase gamma subunit
Binds ATP
95
Bacterial polymerase sigma subunit
Stimulates epsilon subunit
96
Start of DNA replication. What does polymerase do?
Starts with alpha primase activity then switches to delta (lagging strand)
97
Role of Geminin
Keeps Cdt I inactive when bound
98
Role of Cdt I
Recruits McM
| Becomes incorporated into the McM
99
Requirement for loading of Cdc45
Phosphorylation
100
What is a very common recruiter of other proteins for DNA replication
PCNA (beta clamp)
101
What inactivates Cdc6 and Cdt1
Phosphorylation
102
Mechanisms to prevent re-initiation during S phase
1. Kinases
2. Inhibitors
3. Compartmentalization
103
Is Pol alpha recruited by the beta clamp?
No, everything else is
104
Importance of Tau
Has massive affinity for alpha subunit of Polymerase after completion of replication. Separates beta clamp and Polymerase due to outcompeting beta clamp
105
How the clamp loading works
1. binds beta clamp
2. breaks 1 connection of beta clamp using ATP (opens it up)
3. Binds RNA,DNA duplex
4. Only the single strand DNA is fed through the beta clamp
106
How the beta clamp is removed
The delta subunit of the clamp loader exists in excess. Responsible for removal of the beta clamp
107
What does the beta clamp recruit?
Pol I and DNA ligase
