molec of gene Flashcards
(75 cards)
1
Q
RNA pol II is ______ to bind promoter
A
unable
2
Q
General transcription factors: 1. 2. 3. and are analogous to __
A
- recruit RNA pol II to promoter 2. melt DNA at promoter 3. allow RNA pol II to start RNA synthesis to sigma factor
3
Q
GTFs + RNA pol at promoter=
A
pre-initiation complex
4
Q
pre-initiation complex=
A
GTFs + RNA pol at promoter
5
Q
The 6 GTFs are TFII_
A
ABDEFH
6
Q
Human TFIIF has _____ subunits
A
2
7
Q
GTFs Work in a Step-Wise Fashion TFIID: TBP: TFIIA: TFIIB
A
recognizes TATA box
binds and distorts TATA
box stabilizes TFIID binding
recruits Pol II
8
Q
TFIID in initiation
A
recognizes TATA box
9
Q
TBP
A
binds and distorts TATA box
10
Q
TFIIA
A
stabalizes TFIID binding
11
Q
TFIIB
A
recruits RNA Pol II
12
Q
TBP _______, using a beta- sheet inserted into the minor groove, then TFIIB can bind a b-sheet on TBP
A
bends DNA,
13
Q
Draw Step wise binging of GTFs to promoter
A
14
Q
Action of TFIIE
A
involved in transition of closed to open transcription complex
- recruits TFIIH
- modulates IIH activities
- promoter escape
15
Q
Action of TFIIH
A
- promoter melting
- promoter escape
- helicase activities!
- associated kinase activity
- essential for NER
16
Q
Pol II CTD “Tail” composed of
A
composed of a series of 7-amino acid repeats
17
Q
phosphorylation of CTD tail on ____ leads to ___
A
Ser residues, leads to release of GTFs and promoter escape
18
Q
The TATA box is recognized by
A
TFIID
19
Q
____is the primary point of regulation of gene expression.
A
Initiation of transcription
20
Q
Transitioning from initiation to elongation requires______
A
phosphorylation of the Pol II CTD
21
Q
TFIIH controls the _______
A
ATP dependent transition into open complex
22
Q
TFIIH mediates _____
A
promoter melting
23
Q
2 transcription factors that transtion it from open to closed
A
TFIIE and H
24
Q
CTD tail is part of
A
RNA pol
25
CTD tail is phosphorylated by __ at \_\_\_\_\_\_in order to \_\_\_\_\_\_
TFIIH, at serine residues, to initiate promoter esacpe,
26
Factors that stimulate elongation
TFIIS and SPT5
27
TFIIS and SPT5
stimulate elongation
28
TFIIS
reduces pausing of RNA pol II to aid in elongation
29
SPT5
recruits and stimulates 5' capping enzyme
30
SPT5 and TFIIS are both recruited to
phosphorylated CTD tail of RNA pol II
31
draw basic elongation model
32
TFIIS activates \_\_\_\_\_\_\_\_within RNA polymerase to \_\_\_\_\_
RNAse activity, proofread during transcription
33
Capping the 5’ end of the RNA occurs
as soon as RNA emerges from RNA pol
34
How does the capping machinery disengage?
Dephosphorylation of CTD tail @ Ser5 disengages capping machinery.
35
Function of the 5’ Cap (3)
1. prevents degradation of pre-mRNA in nucleus
2. cap-binding protein (CBC) is needed for export
3. bound by small subunit of ribosomes
36
Termination and Polyadenylation factors
CPSF and CstF
37
CPSF
involved in termination and polyadenylation
cleavage and polyadneylation specific factor
38
CstF
cleavage stimulating factor
involved in termination and polyadenylation
39
Termination and Polyadenylation Steps
1. The poly-A signal sequence is transcribed.
2. CPSF and CstF bind the signal sequence in the RNA transcript.
3. CstF mediates RNA cleavage beyond the sequence and is released.
4. Poly-A polymerase (PAP) adds ~200 A’s to the 3’ end.
•does not use a template
5.Poly-A binding proteins (PABP) bind the poly-A tail.
* protect the transcript from degradation
* allow for export
40
draw diagram of termination and capping steps of transcription
41
Draw structure of a gene
promoter, exon, intron, etc
42
gene with longest coding region
TITIN
43
titin bps
280,000
44
titin aas and location
38000 and c'some 2
45
longest human gene, bps, aas, and its location
dystophin at 2.4 mil bps and 3500 AAs, on the X
46
average interon
3300 bps
47
average exon
1500 bps
48
spicosome structure
150 proteins
5snRNAs and 5snRNPs
49
snRNA types
U 1,2,4,5,6
50
purpose of snRNA
1. recognition of mRNA splice sites via complementary bps
2. recognize eachother via bps
3. catalytic activities
51
3 critical sequences at in pre-mRNA
1 and 2. short conserved sequences at each (5 and 3) splice junction
3. conserved sequence within intron, containing branch site
52
BASIC mechanism of mRNA splicing
1. 5' splice site is cut
- upstream exon is held in place by the spicosome
- 5 intron is attached to the branch point
2. 3' splice site is cut
- the up and dwn stream exons are joined by phosphodiester bond
- releasing the lariet from the sequence
53
\_\_\_\_\_\_\_is required to assemble and operate the splicing machinery
ATP
54
Detailed mRNA splicing mechanism
1. U1 binds complementary to and then cleaves at 5' junction (AG|GU)
2. U2AF attaches at 3' end, then BPP binds, then U2 binds, connecting att snRNPs and forming the splicosome
3. BPP and U2AF leave
4. U5/U6 attach to splicosome
5. transesterification attaches 5' G to OH of branch site A to form lariet loop
6. U1 leaves
7. 3' end is cleaved, and exons are joined
8. splicosome releases taking lariet loop with it
55
How is INAPPROPRIATE SPLICING prevented?
Active site formation is a late step!
Only occurs after 5’ and 3’ splice sites + branch point are recognized by multiple components of spliceosome
All spliceosome components are successfully assembled.
56
Potential Errors During Splicing
1. 5’ splice site is recognized, but the nearest 3’ splice site is missed
2. A sequence similar to splice site is treated as one (“pseudo” splice sites).
57
What mechanisms exist to ensure ACCURATE SPLICING? (2)
RNA pol II and ESEs
58
RNA pol II role in splicing accuracy
* ‘cotranscriptional’ loading
* IMMEDIATELY deposits splicing machinery at the 5’ splice site
* insures interaction with FIRST 3’ splice site (avoids competition from downstream sites!)
59
ESEs
Exonic Splicing Enhancers
## Footnote
* sequences spread throughout exons, and bound by special proteins!
* rich in Ser-Arg these proteins (SR proteins) interact with early spliceosome subunits
60
Function of ESEs in Splice Site Selection
SR proteins bind to ESEs
* interact with U2AF and deposit at 3’ splice site
* interact with U1 and deposit at 5’ splice site
61
3 classes of splicing
nuclear pre-mRNA
Group I and II introns
62
Nuclear pre-mRNA splicing
2 tranesterifications, branch site A, major and minor splicososomes
most common
63
group II splicing
rare 2 transest, branch A, RNA enzyme
64
group II splice
rare, 2 transest, branch G, ribozyme
65
splice diagrams comparing
66
•Nuclear pre-mRNA splicing may have evolved from \_\_\_
Group II
67
How does ALTERNATIVE SPLICING
occur?
SR proteins are expressed differentially:
1. different varieties in different cell types
2. different varieties during development
varying levels of SR proteins
controlled by physiological signals
68
Additional Ways Alternative Splicing Occurs
•alternative promoters
•
•alternative poly(A)sites
•
•
•trans-splicing
69
Mechanisms Ensure Mutually Exclusive Splicing
1. steric hindrance
2. major and minor splice site combinations
3. nonsense-mediated decay (NMD)
70
Human Disorders Result From Defects in Splicing
15% of diseases that are caused by point mutations alter splice sites!
b-thalassemia
TTGGT → TTAGT
familial isolated growth hormone def type II
Frasier syndrome
impaired kidney/gonad development
dementia
defective cytoskeleton protein
cystic fibrosis
71
Some Human Disorders Result From Defects in Splicing
Splicing machinery defects
retinitis pigmentosa
retinal degeneration, blindness
spinal muscular atrophy
loss of spinal neurons
Cancer
72
why are there introns?
1. alternitive splicing
2. formation of new genes via exon shuffling
73
Exon Shuffling
The rearrangement of exons by RECOMBINATION, resulting in NEW GENES
Some supporting evidence:
1. Exon boundaries often coincide with protein DOMAINS.
2. Genes show evidence of exon DUPLICATION and DIVERGENCE.
3. Related exons appear in unrelated genes.
74
Reshuffling of Exons, how and favorability?
Recombination IN INTRONS may lead to reshuffling of exons.
Recombination IN EXONS may lead to their disruption.
SO: short exons, long introns favors…?
Conservation of splice sites between all genes allows interchangeability.
Alternative splicing may allow the original and reshuffled message to be spliced!
75
