Transcription, RNA Processing, Gene Regulation, and Epigenetics Flashcards
1
Q
Besides the change between T and U, the RNA product should be identical to the
A
DNA Coding strand
2
Q
The unit of prokaryotic RNA Polymerase that associates with the core enzyme to generate the holoenzyme
A
σ subunit
3
Q
What are the three stages of transcription?
A
- ) Initiation
- ) Elongation
- ) Termination
4
Q
Initiation is dependent on what two thing?
A
- ) σ subunit binding RNA polymerase (making holoenzyme)
2. ) Holo-RNA Polymerase binds promoter
5
Q
What is the principal site for regulation of transcription?
A
Initiation
6
Q
What initiates the elongation step?
A
σ dissociates from RNA polymerase and the promoter
7
Q
Transcript is lengthened by the addition of nucleotides to the 3’ end of the RNA strand located in the active site of RNA Polymerase II
A
Elongation
8
Q
What happens during termination?
A
- ) RNA synthesis stops
- ) RNA transcript is released
- ) Dissociation of RNA polymerase from DNA template
9
Q
What are the two sequences within the prokaryotic promoter region that are recognized by the RNA polymerase holoenzyme?
A
-35 sequence (TTGACA) and Pribnow box (TATAAT)
10
Q
Located ~7 base pairs upstream of the start of transcription
A
Pribnow box (TATAAT)
11
Q
What is the distance between the -35 sequence and the Pribnow box?
A
19 bp
12
Q
Can make DNA from RNA
A
Reverse transcriptase
13
Q
Is translation reversible?
A
No
14
Q
How many different RNA polymerases are there for
- ) Prokaryotic transcription
- ) Eukaryotic transcription
A
- ) One
2. ) Three (with a 4th that functions in mitochondrial transcription)
15
Q
The structural and catalytic component of ribosomes
A
rRNA
16
Q
rRNA comprises about
A
80% of cellular RNA
17
Q
The ribosome includes four different rRNAs that are typically designated by their sedimentation coefficients. For example, in human cells, there is
A
5S, 5.8S, 18S, and 28S
18
Q
Functions as an “adaptor” molecule that delivers amino acids to the ribosome
-about 15% of cellular RNA
A
t-RNA
19
Q
The template for protein synthesis, and is heterogeneous in size, varying according to the length of the encoded protein
-typically represents less than 5% of total RNA content
A
mRNA
20
Q
small nuclear RNAs (snRNAs) are involved in
A
DNA splicing
21
Q
Small nucleolar RNAs (snoRNAs) function in
A
rRNA processing
22
Q
microRNAs play important roles in
A
Regulation of gene expression
23
Q
A cluster of genes encoding proteins involved in sugar utilization
A
lac operon
24
Q
What type of reaction is the polymerization that forms RNA?
A
Nucleophilic attack by 3’ OH of growing strand on α-phosphate of incoming NTP (leaving group is pyrophosphate [PPi])
25
In contrast to DNA replication, RNA transcription does not require a
Primer
26
The core enzyme RNA polymerase is catalytically active but unable to recognize specific promoter DNA sequences until
σ subunit binds
27
Although they have only one core RNA polymerase, bacteria have several
σ subunits
28
What gets rid of the positive supercoils generated in the DNA by RNA polymerase during transcription?
DNA gyrase
29
What gets rid of the negative supercoils generated in DNA by RNA polymerase during transcription?
Topoisomerase I
30
What is the actual site of transcription initiation?
+1
31
Transcription is regulated primarily at the level of
Initiation
32
Either recruit RNA polymerase to the promoter, or stabilize its binding to promoter DNA
Transcriptional activators
33
Block RNA polymerases interaction with DNA
Transcriptional repressors
34
Transcription termination signals are present at the ends of genes, but function at the
RNA level
35
What are the two classes of bacterial terminators?
1. ) Rho-dependent
| 2. ) Rho-independent
36
Binds a specific RNA sequence as a hexameric protein and contacts RNA polymerase, signalling the polymerase to terminate transcription and dissociate from the DNA template
Rho
37
RNA hairpin structures generated by palindromic repeats followed by a U rich region
Rho independent terminator
38
In the case of rho-independent termination, a specific structure (stem and loop) forms in the RNA transcript, signalling
Termination
39
Binds the β subunit of bacterial RNA polymerase and inhibits initiation
Rifampicin
40
Rifampicin is an effective antibiotic for treatment of certain
Bacterial infections (ex: tuberculosis)
41
Binds the DNA template, intercalating between neighboring base pairs
Actinomycin
42
Actinomycin blocks
Transcript elongation
43
The DNA structure that binds actinomycin is conserved between prokaryotes and eukaryotes. As a result, actinomycin serves as an effective therapeutic for treatment of some
Cancers
44
Actinomycin binds the
DNA phenoxazone ring
45
The core promoter region of eukaryotic class II genes typically (but not always) contains a
-located about 25 bp upstream of the transcription start site (+1)
TATA box
46
The “nucleation site” for assembly of a transcription complex that includes a set of general transcription factors (GTFs) and RNA polymerase II and is functionally comparable to the –10 and –35 regions of a bacterial promoter.
TATA box
47
Transcriptional stimulatory sequences are generally located upstream of the core promoter in eukaryotes and include
Promoter proximal elements and enhancer sequences
48
Promoter proximal elements are usually located within 120 bp of the transcription start site and serve as binding sites for transcriptional activators of
-Ex: enzymes involved in metabolism
Ubiquitously expressed genes ("housekeeping genes")
49
The most well characterized activator that functions at a proximal promoter is called
SP1
50
Found at variable distances from the core promoter and are DNA sequences that bind specific transcriptional activators
Enhancers
51
Binding sites for transcriptional activators that either make direct contact components of the RNA polymerase II transcriptional machinery, or recruit chromatin remodeling complexes to the regulatory promoter regions
Enhancers
52
What do RNA Polymerases I, II, and III transcribe?
RNA Polymerase I = rRNA
RNA Polymerase II = mRNA
RNA Polymerase III = 5s component of ribosome, tRNA, and certain snRNAs
53
Eukaryotic RNA polymerases do not recognize Specific DNA sequences on their own. Instead, promoter recognition requires
General transcription factors (GTFs)
54
A general transcription factor which includes TATA binding protein
TFIID
55
Required to position RNA Polymerase II at the promoter
GTFs
56
Structurally similar to prokaryotic RNA polymerase
Eukaryotic RNA polymerase II
57
As a subunit of the larger TFIID complex, binds the TATA element and confers a sharp bend in the promoter DNA.
TATA binding protein (TBP)
58
Explain the process of RNA polymerase II binding
TBP (as TFIID) binds the TATA box, enabling TFIIB to bind, which enables RNA polymerase II and TFIIF to bind
59
Once RNA polymerase II is bound to DNA, enable RNA polymerase to start transcription
TFIIE and TFIIH
60
Stimulate transcription by binding GTFs, either to recruit or stabilize their binding to the core promoter
Transcriptional activators
61
There are two notable features of eukaryotic RNA pol II transcription that do not occur either in bacteria or by RNA pol I or RNA pol III. These are
Addition of 5' cap and 3'-poly(A) tail
62
A 7-methylguanosine “CAP” is covalently linked to the 5’-end of mRNA by a unique 5’ → 5’ triphosphate linkage. The CAP is not encoded by the DNA template. Instead, CAP is added to the mRNA shortly after
Initiation
63
Consistent with it being a unique feature of mRNA, the CAP is recognized by components of the translational machinery, thereby facilitating
Protein Synthesis
64
Transcription termination by RNA polymerase II involves the addition of a
3'-pol(A) tail
65
Termination is specified by distinct sequences in the DNA template and involves which two enzymatic reactions?
1. ) Endonucleolytic cleavage of nascent transcript
| 2. ) Addition of poly(A) tail
66
The poly(A) tail is not encoded in the DNA template and is a unique feature of eukaryotic RNA pol II transcription. The poly(A) tail is involved in
mRNA stabilization, transport of mRNA from nucleus to cytoplasm, and efficient translation
67
Unlike DNA replication, which is semi-conservative, trancription is
Conservative
68
In eukaryotic transcription, the core and regulatory elements can be far apart, but in prokaryotes, they must be
Adjacent
69
In prokaryotes, translation of the mRNA begins
Prior to transcription termination
70
In ekaryotes, transcription takes place in the nucleus and translation in the
Cytoplasm
71
In prokaryotes, The 5' end in the DNA sequence of a gene is directly proportional to the
N-terminus of the protein
72
In most eukaryotic genes, the coding information is
Discontinuous (introns and exons)
73
Nucleic acid sequences that are transcribed and retained in the corresponding mature mRNA
Exons
74
Nucleic acid sequences that are transcribed but spliced from the primary transcript to yield the mature mRNA
Introns
75
A gene that is divided into 3 exons and 2 introns
- The ATG start codon is found in exon 1
- The TAA stop codon is contained in exon 3
β-globin
76
Why must the translation start and stop codons be within exons?
Because introns are spliced out either co- or post-transcriptionally, but PRIOR to translation
77
When the intron sequences are removed, the adjacent exons are
Spliced together
78
pre-mRNA splicing is very important because a single nucleotide error in the splice point would have what consequence?
The reading frame would be shifted, resulting in an entirely different amino acid sequence
79
The base sequence of an intron
1. ) Begins with?
2. ) Ends with?
-these sequences are invarient (never change)
1. ) GU
| 2. ) AG
80
A conserved sequence found 20-50 nucleotides from the 3' end of the intron
The "branch" site
81
Intron sequences other than the 5' and 3' splice sites, and the "branch site", are unimportant in determining
Location of splicing
82
The length of an intron varies from
Less than 100 nucleotides to several thousand nucleotides
83
Intron splicing occurs by two
Transesterification reactions
84
The first transesterification reaction involves
Cleaving the phosphodiester bond between exon 1 and intron 1, and using the 2' OH of an adenylate residue at the branch site to form a phosphodiester bond with the 5' end of the intron
85
In the second transesterification, the 3' OH of exon 1 cleaves the phosphodiester bond between the intron and exon 2. As a result,
Exon 1 and 2 are now bound together, and the intron is released as a "lariat" structure
86
An assembly of ribonucleoprotein particles (SNURPs) that recognize the 5' splice sit, the 3' splice site, and the branch site
-Catalyzes the splicing reaction
Spliceosome
87
The spliceosome assembles de novo from its constituent SNURPs on the precursor RNA in a process that requires
ATP
88
Required for the spliceosome assembly, but not for either of the ensuing transesterification reactions
ATP hydrolysis
89
Which spliceosome protein(s) bind the
1. ) GU 5' splice site
2. ) Branch site A
1. ) U1
| 2. ) U2
90
Approximately 15% of mutations that cause genetic disease affect
pre-mRNA splicing
91
A group of hereditary anemias characterized by defective synthesis of hemoglobin
Thalassemias
92
One source of Thalassemias is due to point mutations that occur in either of the two β-globin gene
Introns
93
Point mutations in β-globin gene introns create
-results in either shorter or longer forms of abnormal β-globin
Splicing defects
94
What are three DNA sequence elements in eukaryotic transcription?
1. ) Enhancers (>250)
2. ) Proximal promoter (CpG: -120 to -60)
3. ) Core promoter (TATA: -40 to +40)
95
In eukaryotes, contains the transcription start site (+1), the TATA box (-25), and down stream promoter element (DPE: +30)
Core promoter
96
The proximal promoter is the binding site for certain
Activator proteins
97
What are the core promoter elements of RNA polymerase II?
1. ) Initiator
2. ) Down stream promoter element (DPE)
3. ) TATA Box
4. ) TFIIB Recognition element
98
Nucleates the assembly of the transcription preinitiation complex
-TBP + 14 TBP-associated factors (TAFs)
TFIID
99
How does TFIID work?
TBP binds TATA box, TAF1 and TAF2 bind initiator, and TAF 6 and TAF 9 bind the DPE
100
Binds tightly to RNA Polymerase II and inhibits the elongation stage of transcription
-Eukaryotic version of Rifampicin
α-amanitin
101
Can produce multiple, related proteins from a single gene
Alternative splicing
102
What is the Donor splice site?
5' end of intron
103
What is the Acceptor splice site?
3' end of intron
104
Many hormones (e.g. epinephrine, insulin, epidermal growth factor) activate transcription by activating
Cell-surface receptors
105
Directly stimulate gene transcription as hormone-receptor complexes
Steroid hormones
106
Humans contain two copies (one from the mother and one from the father) of each
Chromosome
107
Soluble proteins that function as transcriptional activators in response to hormone binding
Steroid hormone receptors
108
In come cases, hormone binding exposes a nuclear localization signal, allowing the receptor-hormone complex to enter the nucleus, where it binds
Promoter DNA (in a sequence specific manner)
109
In other cases, the hormone receptor is already located in the nucleus. In this case, hormone binding does one of what two things?
1. ) Stimulates receptor-DNA binding
| 2. ) induces conformational change in the receptor-DNA complex
110
Regardless of the specific mechanism, the DNA-hormone-receptor complex then interacts with a "coactivator" complex to stimulate
RNA Polymerase II transcriptional machinery
111
Steroid hormones receptors, like other transcriptional activators, consist of discrete functional domains. What are they?
DNA-bining domain, hormone binding domain, dimerization domain, and activation domain
112
Bind hormone receptors and stimulate receptor activity (gene expression)
Ex: Anabolic steroids
Agonists
113
Bind hormone receptors and block receptor activity (repress gene expression)
Ex: tamoxifen
Antagonists
114
Recognizes specific enhancer elements
DNA binding domain
115
Upon hormone binding, Steroid receptors typically undergo a conformational change to create a transcriptional activation domain that
Binds a "coactivator" complex
116
These DNA sequences are enhancers for steroid responsive genes
-bind hormone receptor complexes, resulting in stimulation of gene expression
Steroid response elements (SREs)
117
Typically palindromic, and are related to one another by subtle, but important sequence differences
SRE's
118
Steroid hormone-receptor complexes are potent activators of transcription. The mechanism of activation involves
Binding of the hormone-receptor complex to the SRE (cognate regulatory element) and subsequent binding to a transcriptional coactivator that in turn stimulates transcription
119
Some co-activators interact with both activators and the core transcriptional machinery (RNA Poly II and the GTFs), where they essentially
Bridge the two components
120
Other coactivators do not interact with the core machinery, but instead target
Chromatin (either displacing or modifying nucleosomes)
121
Normally inhibit transcription
Nucleosomes
122
Some work by recruiting or stabilizing RNA polymerase II/GTP to the core promoter, and other "clear a path" for RNA polymerase II by altering the DNA template
Coactivators
123
DNA in eukaryotic chromosomes is not naked, but is tightly associated with proteins to form
Chromatin
124
Steroid hormone receptors ultimately activate gene expression by altering
-Allows RNA polymerase II to bind the promoter
Chromatin Structure
125
In summary, a sequence specific enhancer element (DNA) binds the hormone receptor-hormone complex (transcriptional activator), which in turns binds a coactivator complex (chromatin modifier) to
Displace or disrupt nucleosomes
126
Coactivators include two classes of chromatin modifiers. What are the two classes?
1. ) Coactivators that catalyze covalent modifications to histones
2. ) ATP-dependent remodeling complexes
127
The most well characterized covalent histone modification is acetylation of histone lysine residues in the N-terminal tails of histones H3 and H4, catalyzed by
Histone acetyltransferases (HATs)
128
Histone modifications occur primarily at the
N-terminal tails of histones
129
These complexes do not catalyze covalent histone modification, but instead use the energy of ATP hydrolysis to remodel nucleosomes, thereby allowing other transcription factors to bind DNA and ultimately promote gene transcription.
ATP-dependent chromatin remodeling complexes (coactivators)
130
Steroid hormones include estrogens, androgens, glucocorticoids and mineralocorticoids. These cholesterol derivatives, each slightly different in structure, bind specific hormone receptors to
Activate different sets of genes
131
Target the oviduct to stimulate expression of genes whose encoded proteins participate in the ovarian cycle
Estogen
132
Target the liver (and other tissues) to promote expression of genes required for gluconeogenesis, glycogen synthesis, degredation of fat, and inhibition of the inflammatory response
Glucocorticoids
133
A DNA virus that causes breast epithelial cell carcinoma. It's genome includes a DNA sequence that is identical to the estrogen enhancer
Mouse mammary tumor virus (MMTV)
134
In MMTV, the estrogen/estrogen receptor complex binds the MMTV enhancer and stimulates wnt-1 gene expression, which results in
Uncontrolled cell proliferation
135
Anti-cancer drug that binds the estrogen receptor and prevents coactivator binding
Tamoxifen
136
Despite being a "general transcription factor" required for the expression of all protein genes, TFIIH also plys a very important role in the
Coupling of transcription with DNA damage repair
137
Altered forms of TFIIH have been identified in what three diseases?
-These diseases are not due to defects in transcription initiation, but rather to defects in DNA repair
1. ) Xeroderma pigmentosum (XP)
2. ) Trichothio-dystrophy (TDD)
3. ) Cockayne syndrome (CS)
138
Will move 3' to 5' on the DNA template to generate a new 5' to 3' strand
RNA transcription
139
The sequences indicating the -35 and TATAAT box are not exact, but rather consensus sequence. All six of the consensus nucleotides are never actually seen in the same gene because
Sigma would bind too tightly
140
The σ subunit binds the -35 and TATAAT sequences
Simultaneously
141
Which sigma unit recognizes E coli promoters?
σ 70
142
A double stranded DNA sequence that the σ subunit binds to
Promoter
143
What are the 5 catalytic subunits of RNA polymerase II?
2α, β, β', and ω
144
Found in the crab claw of RNA polymerase II and help make up the active site
Two magnesium ions
145
The core promoter spans about
80 base pairs
146
Not a promoter in the sense that it does not bind
RNA polymerase. Binds proteins that are regulatory proteins for "housekeeping" genes (genes encoding proteins that every cell needs to have)
Proximal Promoter
147
Core promoter, proximal promoter, and enhancers are all
Double-stranded DNA
148
Which GTF binds to -35?
TFIIB
149
The only place a 5' to 5' triphosphate linkage occurs is on the
-prevents degredation of mRNA and facilitates the initiation of translation
5' cap
150
The code to terminate transcription is in the
| DNA template and has the consensus sequence
AAUAAA
151
Transcription factors that bind to double stranded DNA
Steroid receptors
152
What is a common structural motif in transcription factors?
Zinc finger
153
Has 1.74 turns of double stranded DNA and about 147 bps
-an impediment to RNA polymerase
Nucleosomes
154
Rely on estrogen-mediated pathways for cell proliferation
-Why tomoxifin is a good anti-cancer
Breast tumors
155
Zinc atoms associate with domains of steroid receptor protein domains to form zince fingers that
Bind DNA
