Lecture 4 - 6 (Ch 8) Flashcards
1
Q
The problem of linear DNA replication is ___
A
stranded RNA primer at the end of the lagging strand after RNA primer removal
2
Q
Without telomere activity, this overhang will be ___, leading to ___
A
removed, leading to the lagging ends of the daughter strand to be shorter than the parent
3
Q
Telomeres are ___; they are synthesized by
A
repetitive DNA sequence end of chromosome, synthesized by telomerase
4
Q
With the telomeres, the chromosome (is/isn’t) shortened; how?
A
is; lack of telomerase means the telomeres won’t be remade! However, it will not effect vital genome
5
Q
Telomerase creates telomeres in 4 steps:
A
- Telomerase (containing repetitive RNA sequence) attach to the 3’ overhang (longer parent sequence) 2. Telomerase RNA acts as the template for DNA elongation of the 3’ overhang (in the 5’->3’ direction) 3. Telomere translocates down the overhang 4. Step 2 repeats, causing DNA elongation 5. DNA polymerase fills the resulting “gap” in the daughter sequence*
6
Q
Telomerase RNA sequence runs in ___ direction
A
3’->5’
7
Q
3 Steps of PCR are:
A
- Denaturation of dsDNA to ssDNA via heat 2. RNA Primer annealing at a lower T 3. Primer extension via Taq DNA Polymerase and dNTP
8
Q
PCR is carried out in (one/both) DNA strands
A
both
9
Q
In PCR, helicase, SSB, primase, ligase, and sliding clamp is not needed because:
A
Helicase and SSB: heat creates ssDNA, so neither are needed Primase: RNA primer of interest provided manually Ligase: No Okazaki fragments created, because ssDNA rather than dsDNA opening up Sliding Clamp: Taq has ++ efficiency, and sliding clamp is not heat stable
10
Q
Deoxynucleotide DNA Sequencing: DNA v. dDNA?
A
DNA has 5-carbon ring with 5’-Phosphate and 3’-OH dDNA has 5-carbon ring with 5’-Phosphate and 3’-H Therefore dDNA cannot form P-d bonds
11
Q
ddNTP DNA Sequencing: 3 key ingredients & their functions are ___
A
- 5’ primer labeled with radioactive 32P (for later visualization on gel) 2. ddNTP (one base per condition) 3. dNTP (all 4 bases in all conditions)
12
Q
ddNTP DNA Sequencing: DNA fragment stops are by ___
A
chance
13
Q
ddNTP DNA Sequencing: After performing all 4 conditions, describe the gel and its results. The sequenced strand is the ____ strand. To read the original strand of interest, ____
A
4 lanes for 4 bases of ddNTP. Furthest is the smallest (closest to 5’ primer). Read the sequenced strand bottom-up, creating 5’->3’ complement; complement; original strand is 3’->5’ complement of the sequenced strand
14
Q
Next Gen DNA Sequencing has 3 characteristics:
A
- smaller DNA sample 2. sequence by synthesis - each time a new nucloetide is added, the whole seunce stops; the base is recorded via flourescence, and ddNTP exchanged with dTP 3. Sequence of many targets
15
Q
mRNA in ____ were located through the pulse-chase experiment, in which ___
A
Eukaryotes; radiactivity of uracil was tracked from the nucleus to the cytosol
16
Q
Commanalities between DNA and RNA synthesis (3):
A
5’->3’; dependent on DNA template; P-P elimated with each P-d bond
17
Q
Conservation between bacterial and euakryote transcription is ____, but not its ___.
A
RNA polymerase structure, but not its units
18
Q
RNA polymerase structure that is analog in bacteria and in eukaryote are: ___; non-conserved structures are: ___
A
5 subunits (alpha I, alpha II, beta, beta’, omega); sigma
19
Q
In bacteria, the sigma subunit ___; in eukarya, the sigma subunit ____.
A
changes for the type of promotor it binds to; does not exist, but other additional subunits exist
20
Q
In bacteria, the subunit that gives RNA Polymerase specificity is the ___
A
sigma subunit
21
Q
In eukarya, the subunit that gives RNA Polymerase specificity ___
A
does not exist; rather, there are multiple RNA polymerases for different functions.
22
Q
The two differences between RNA Polymerase in bacteria and in eukarya are:
A
- one RNA polymerase modified by sigma subunit in bacteria, and multiple RNA polymerase in eukarya (to recognize different promotors) 2. Eukarya requires RNA complex
23
Q
Eukarya have (less/more) diverse promotor sequences and consensus sequences that bacteria
A
more
24
Q
Eukarya: 3 RNA Polymerases are:
A
- RNA Pol I 2. RNA Pol II 3. RNA Pol III
25
Eukarya: Explain the functions of 3 RNA Polymerases:
1. RNA Pol I: transcribes 3 rRNA genes 2. RNA Pol II: transcribes protein coding genes and most snRNA (used in RNA processing) 3. RNA Pol III: transcribes tRNA, 1 snRNA, and 1 rRNA
26
Promoter are identified by \_\_\_
ID'ing protein-coding genes and comparing shared DNA; for example, the TATA box
27
TATA box are promoters with the sequence \_\_\_\_, ___ nucleotides away from +1 position
5' TATAAA 3'; -25
28
Consensus sequences were proven to have function in an experiment of 3 conditions. What were the 3 conditions? What was the indicator that the promotor had a function?
inserting cloned plasmid 1. without promotor; 2. with a promotor; 3. with a point mutation promotor into the cell, and looking for colored product of LacZ
29
In Myers LacZ experiment, was the unmutated promotor found to be necessary and sufficient for gene expression?
No-- point mutation still indicated LacZ activity, but in smaller amounts
30
If an ori was introduced to a plasmid, the plasmid will \_\_\_
replicate
31
In Myers LacZ experiment, promotor was added to ___ w/n the plasmid; this is because it contains many \_\_\_
Multiple Cloning Sites; restriction enzyme sites
32
In Myers LacZ experiment, point mutation was mapped out, and consensus sequences were elucidated-- how?
consensus sequences were visualized as clusters of nucleotides that lowered expression when point mutation was introduced
33
The relationship between RNA Pol II, Transcription Factor II, and promotor
RNA Pol II recognizes and binds to promoter sequence with the aid of proteins called TFII (the II refers to RNA Pol II)
34
\_\_\_\_ is the principle binding site during promotor recognition, which is highly conserved in many/most protein encoding genes
TATA
35
In Myers LacZ experiment, certain parts of the gene was protected from mutation by \_\_\_. Success of this assay was done through (1) \_\_\_, which separated DNA by \_\_\_. An alternative confirmation can be done by (2) \_\_\_, which \_\_\_
A protein covering it; (1) Gel shift assay; weight (protein-DNA \> DNA) (2) Chip assay, which adds antibody to known protein
36
What are the four steps to RNA Pol II attaching to the promotor?
1. TFIID (TAF+TBP) bind to TATA box 2. Minimal initiation complex forms with the addition of TFIIA, TFIIB, RNA Pol II, and TFIIF 3. Pre-initiation complex forms with the addition of TFIIE and TFIIH 4. RNA Pol II is released from the GTF's to begin transcription
37
TFIID contains two things: \_\_\_
TBF (TATA-box Binding Protein) & TAF (TBF-Associated Factor)
38
The minimal initiation complex contains five things: \_\_\_. Out of these, only ___ was present on the DNA from the previous step
TFIIA, TFIIB, TFIID, TFIIF, and RNA Pol II; TFIID
39
The pre-initiation complex contains seven things: \_\_\_. Out of these, only ___ were added during its formation.
TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA Pol II; TFIIE, TFIIH (The D comes first; ABFR; Ero H)
40
The 6 TFII are also known as General Transcription Factors (GTF) because \_\_\_. Is it necessary and sufficient for gene expression?
they're found in all cells; necessary for gene expression, but not sufficient because most DNA are wrppaed in proteins and must be unwrapped to expose it to TFII.
41
Pol II begins transcription at the ___ site
+1
42
Silencers and enhancers are located ___ of the gene
upstream or downstream
43
When either a silencer or an enhancer is located upstream of a gene, how does it affect the RNA Pol II?
DNA bends; a protein bridge with activator and coactivator protein attached to RNA Pol II binds to it
44
Is the +1 site of transcription ATG?
No, because of 5' UTR
45
Gene SHH is expressed differently in different tissues in eukaryotes due to \_\_\_
Tissue-specific Enhancers and tissue-specific TF
46
Enhancers were discovered in \_\_\_; it can be ___ away from the promotor
Virus; 150 kbp
47
Is the TF necessary for function of enhancer and silencers?
yes
48
TF are synthesized universally -- T/F?
False-- different cell types and different developmental stages have distinct array of TF
49
Availability of activated TF controlled through two ways \_\_; the main difference between the two are: \_\_
1. Synthesis control 2. Signal transduction pathways In (2), factors outside the cells ctrl gene expression, and TF are already made
50
Signal transduction pathway activates TF in 3 steps:
1. Receptor binds to ligand at the cell membrane 2 Janus Kinases (JAKs) phospohorylate the receptor and Signal Transducer and Transcription Factor (STATs) 3. STATs dimerize (activated) and move to nucleus to bind to promoters/enhancers of target gene
51
STATs stand for \_\_\_
Signal Transducer and Transcription Factor
52
RNA Pol I transcribes genes for ___ using a mechanism \_\_\_\_
rRNA; similar to RNA pol II transcription
53
rRNA genes are on \_\_\_, which is clustered together in \_\_\_. Ribosomes are assembled in \_\_\_.
chromosomal DNA; nucleolus; nucleoli
54
What are the analogus of enhancers/silencers, TATAA box, and TFIID in rRNA transcription?
upstream control element, core element, and UBF1
55
How does RNA pol I get recruited to the gene?
SL1 bind to both upstream control element and core element, which is then bound by UBF1
56
Difference between RNA pol I and RNA pol II transcription?
RNA pol I placed at a different position-- but still shows conservation
57
RNA pol III transcribe \_\_\_
tRNA, 1 rRNA, and 1 snRNA, and miRNA, siRNA
58
Where do snRNA, 5S rRNA, and tRNA have transcription control elements?
snRNA - 3 upstream elements 5S rRNA, tRNA - 2 downstream internal promotor elements
59
How does RNA pol III for tRNA and 5S rRNA transcription get recruited to the gene (3 steps)?
1. TFIIA & TFIIC bind to Box A and Box C w/n the internal promotor elements 2. TFIIB binds to TFIIA and C 3. RNA Pol III binds to TF and is positioned at +1.
60
Which RNA Pol do you expect has complex termination? Why?
RNA Pol II; because it produces proton-coding mRNA, and must be processed
61
How is RNA Pol I terminated?
Transcription Terminating Factor 1 (TTF1) binds to 17-bp consensus sequence, which physically stops the polymerse from moving; RNA is then cleaved 18 bp upstream of this binding site
62
How is RNA Pol III terminated?
long string of Us (with weak binding) causes Pol III & DNA template to fall off
63
3 Post-Transcriptional Processing are: __ This is done on ___ mRNA to make it into a \_\_\_
1. 5' capping 2. 3' polyadenlyation 3. intron splicing pre-mRNA to mature mRNA
64
5' capping is the \_\_\_\_.
addition of G to 5' end of pre-mRNA
65
5' capping: ___ adds G to __ end of pre-mRNA
guanylyl transferase; 5'
66
5' capping: the link catalyzed by guanylyl transferase is \_\_\_
5'-5' triphosphate linkage
67
5' capping: there are 3 PO4 in the 5' mRNA end and 3 PO4 in GTP-- which is conserved in the 5'-5' triphosphate linkage?
2 PO4 in the 5' mRNA end (only gamma removed) 1 PO4 in GTP (gamma and beta removed, like in all NTP addition rnxs)
68
5' capping: the last reaction of this process is \_\_\_
methylation of the 5' G
69
5' capping: 4 functions are:
1. protecting mRNA from deradation 2. facilitating transport of mRNA out of nucleus 3 facilitating subsequent intron splicing 4. enhancing translation efficiency by orienting the ribosome on mRNA
70
3' polyadenylation is the \_\_\_
REPLACEMENT of a section of 3' end of pre-mRNA with A
71
3' polyadenylation: visualize 3' UTR end of mRNA after the UAA codon
---[UAA] [AAUAA] [15-20 bp] [Cleavage Site] [U-rich region]-3'
72
3' polyadenylation: sequence removed in pre-mRNA is \_\_\_; it is recognized by \_\_\_
AAUAA; Cleavage & Polyadenylation Specificity Factor (CPSF)
73
CPSF stands for \_\_\_
Cleavage & Polyadenylation Specificity Factor
74
3' polyadenylation: outline 5 steps
1. CPSF binds to AAUAA, CStF binds to U-rich region and recruits PAP; the 3 factors and others "bend" the 3' UTR region
2. pre-MRNA cleaved, leaving PAP and CPSF at the 3' end
3 . 3' fragment is degraded, and CStF recycled in the nucleus
4. PAP adds new adenine to 3' end
5. PAPII molecules bind to increase rate of polyadenylation
75
3' polyadenylation: CPSF stands for\_\_, and binds to \_\_\_; its function is to \_\_\_
Cleavage & Polyadenylation Specificity Factor;
AAUAA (Polyadenylation signal sequence);
signal site of polyadenylation
76
3' polyadenylation: CStF stands for \_\_\_, adn binds to \_\_\_; its function is to
Cleaveage Stimulating Factor; U-rich region; recruits other factors, including PAP
77
3' polyadenylation: PAP stands for \_\_\_, and binds to \_\_\_; its function is \_\_\_
Poly Adenylate Polymerase; CStF; adds A to 3' end after clevage.
78
3' polyadenylation: When does CPSF, CStF, and PAP bind to the mRNA? When does it leave?
CPSF, CStF, PAP: bind simultaneously
CStF leaves first when cleavage occurs
CPSF and PAP does not leave until poly-A tail is complete
79
3' polyadenylation: 3 functions?
1. facilitate transport of mature mRNA across nuclear membrane
2. protect mRNA from degradation
3. enhancing translation by enabling ribosomal recognition of mRNA
80
3' polyadenylation: All eukaryotic genes undergo this (T/F)?
F. Histone genes do not.
81
Common functions of 5' and 3' processing? Different functions?
Both protect mRNA from degradation, facilitate transport of mRNA out of nucleus, and enhance translation (thru different means)
5' G cap facilitate further intron splicing
82
The presence of introns can be demonstrated by R-looping, which hybridizes ___ and \_\_\_. It's called "looping" because \_\_\_
DNA template and mature mRNA;
intron will not have an analogue and "loop" out
83
Splicing: Visualize the splicing signal sequences of Intron A between Exon 1 and Exon 2. Remember to indicate the key bp:
[Exon 1] {[AU...(5' Splice Site)]...[Branch Site with A][(3' Splice Site) ...AG)]} [Exon 2]
84
Splicing: Outline 6 steps
1. snRNP U1 binds to 5' splice site, and snRNP U2 to branch site
2. snRNP U4, U5, U6 bind to complex and form inactive splicesome
3. U4 dissociate to form the active splicesome
4. 5' cleavage between Exon 1 and Intron A; 2'-5' P-d bond between 5' site and branch site A
5. 3' end of intron cleaved
6. Exon 1 -Pd- Exon 2
85
Splicing: SnRNP stands for \_\_\_; its subunits are \_\_\_
Small nuclear Nuclear Ribonuclear Proteins;
U1, 2, 4, 5, 6
86
Splicing: snRNP U1 binds to \_\_\_
5' Splice site
87
Splicing: snRNP U2 binds to...
branch site near the 3' end
88
Splicing: snRNP U4, U5, U6 bind to \_\_\_; their function is to \_\_\_
U1 and U2 to form the inactive splicesome
89
Splicing: special function of snRNP U4 is \_\_\_
that it is an inhibitor of the splicesome
90
Splicing: what bonds are broken? what bonds are formed?
1. Exon 1 - 5' Splice site (GU...) breaks
2. 2'-5' P-d bond between 5' G and branch point A forms
3. Exon 2 - 3' splice site (...AG) breaks
91
Introns are removed (one by one/simultaneously) (in 5'-\>3' order/randomly)
one by one; randomly
92
The three steps of pre-mRNA processing is tightly coupled (T/F)
T
93
Where is the assembly platform and regulator of pre-mRNA processing?
Carbonyl Terminal Domain (CTD) of RNA Pol II
94
In CTD of RNA Pol II, 4 factors exist. Together, they are called \_\_\_. The factors' name and function are:
PreInitiation Complex (PIC) inhibits splicing before transcription starts
1 TF initiates transcription
2. CAP makes 5' G cap
3. Splicing Factor (SF) help the splicesome
4. pA (polyAdenylation) help 3' poly A
95
Order when 5' cap, 3' poly-A tail, and intron splicing occurs
5' cap first, then intron splicing while elongation continues, and 3' poly-A tail at end
96
With the same DNA template, different mature mRNA can be created with alternative pre-mRNA processing. The 3 are:
1. alternative splicing patterns
2. alternative promoters initiate transcription at different points
3. alternative localization of poly-A produce different mRNA
